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The effect of general anesthesia on canine lymphocyte function
Veterinary Immunology and Immunopathology, 13 (1986) 63--70 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
63
THE EFFECT OF GENERAL ANESTHESIA ON CANINE LYMPHOCYTEFUNCTION PETER J. FELSBURG, LYNETTE L. KEYES, DONALDR. KRAWIEC, and STANLEY I. RUBIN Clinical Immunology Laboratory, Department of Veterina~ Pathobiology, College of Veterinary Medicine, University of I l l i n o i s , Urbana, IL 61801, U.S.A. (Accepted 12 December 1985) ABSTRACT Felsburg, P.J., Keyes, L.L., Krawiec, D.R., and Rubin, S . I . , 1986. The effect of general anesthesia on canine l~tmphocyte function. Vet. Immunol. Immunopathol., 13: 63-70. The in v i t r o response of canine peripheral blood lymphocytes to mitogenic stimulat~n was 'evaluated following general anesthesia for a r e l a t i v e l y minor diagnostic procedure. A marked suppression in the blastogenic response to phytohemagglutinin and concanavalin A was observed 4 hours postinduction which persisted through the f i r s t 24 hours and was normal by 4 days. A mild suppression to stimulation with pokeweed mitogen was observed at 4 hours postinduction but the response was back to normal by 24 hours. These results suggest general anesthesia has a transient immunosuppressive effect on canine lymphocytes and T cells are more susceptible to the immunosuppressive effect than B cells. INTRODUCTION General anesthesia is used not only for surgical procedures, but also for various noninvasive diagnostic procedures. Situations exist where evaluation of a patient's immune competence is desired following a diagnosis which has involved the use of general anesthesia.
This prompts a frequently asked
question of the c l i n i c a l immunologistc-"Does general anesthesia have a suppressive effect on lymphocyte function tests and, i f so, how long does the suppression persist?". The few studies reported in humans (Park et a l . , 1971; Jubert et a l . , 1973; Berenbaum et a l . , 1973; Kanto et a l . , 1974; Cullen and Belle, 1975; Shade et a l . , 1975) and dogs (Kelly, 1980; Medleau et a l . , 1983) which have attempted to address this question have generally shown a depression in the in v i t r o blastogenic response of l~nnphocytes to mitogenic and antigenic stimulation. However, the results of these studies are confounded by the selection of patients studied and the various major surgical procedures which were performed in conjunction with the general anesthesia.
The question remains
whether the suppression was a result of the general anesthesia, the immune status of the patient, or the trauma and blood loss associated with major surgical procedures. 0165-2427/86/$03.50
© 1986 Elsevier Science Publishe~ B.V.
64 The purpose of this study was to evaluate the in v i t r o lymphocyte response to mitogenic stimulation in c l i n i c a l l y normal dogs following general anesthesia for a r e l a t i v e l y minor diagnostic procedure. MATERIALS AND METHODS Dogs Eight mixed-breed, random source, conditioned male dogs were used in this study.
All dogs were young adults and in good health at the time of the
study.
The dogs were routinely vaccinated and checked for internal parasites
prior to participation in the study. tained on a commercial diet.
The dogs were housed indoors and main-
These dogs were part of a project to determine
the effect of amphotericin B on renal function.
This study was performed in
the dogs prior to t h e i r receiving any drug therapy. Blood Blood was collected aseptically by venipuncture into syringes containing preservative-free heparin (20 units/ml of blood).
The heparinized blood was
immediately transferred into s t e r i l e plastic culture tubes. Culture Medium RPMI 1640 culture medium (M.A. Bioproducts, Walkersville, MD) supplemented with lO0 units/ml of p e n i c i l l i n , lO0 ug/ml of streptomycin, and 2 mM glutamine was used in a l l experiments. Mito~ens Phytohemagglutinin-P (PHA; Difco Laboratories, Detroit, MI), concanavalin A (Con A; Sigma Chemical Co., St. Louis, MO), and pokeweed mitogen (PWM; Grand Island Biological Co., Grand Island, NY) were reconstituted with s t e r i l e water, aliquoted in small volumes, and stored at -70 degrees C until
used.
For each experiment, aliquots were thawed and diluted to the desired concentrations in RPMI 1640. L~mphoc~te Transformation Studies The lymphocyte transformation tests were performed in quadruplicate using a micro whole blood technique (Felsburg et a l . , 1980, 1983). Five ul of heparinized whole blood was added to each well of round bottom microtiter plates containing lOO ul of RPMI 1640 with or without mitogens. Cultures were incubated at 37 degrees C in a humid, 5% CO2 atmosphere for 96 hours. Sixteen hours before harvesting, 0.5 uCi of 3H-thymidine (2.0 Ci/mmole; New England Nuclear, Boston, MA) was added to each well.
After incubation, the microcul-
tures were harvested onto glass f i l t e r strips by an automated cell harvester. The strips were washed extensively with d i s t i l l e d water, 3% acetic acid, and again with d i s t i l l e d water in order to lyse the red blood cells. After a i r drying, the f i l t e r discs were placed in s c i n t i l l a t i o n f l u i d and the
65 radioactivity measured in a liquid s c i n t i l l a t i o n spectrometer.
The results
are expressed as the counts per minute (CPM) in stimulated cultures minus the CPM in control cultures (Kristensen et a l . , 1982). Experimental Protocol Prior to anesthesia, the dogs received l mg acepromazine maleate intravenously and atropine (0.04 mg/kg) subcutaneously. Anesthesia was induced by the administration of thiamylal sodium (2.5 to 4 mg/kg) intravenously and f o l lowing intubation, anesthesia was maintained with methoxyflurane and oxygen. A small skin incision was made in the right flank, and a needle biopsy was collected from the right kidney using the keyhold technique (Osborne et a l . , 1974).
The incision was closed and the animal was allowed to recover from
anesthesia. Samples were collected for lymphocyte transformation prior to the induction of anesthesia, 4 hours postinduction, 24 hours postinduction, 4 days postinduction, and 7 days postinduction.
These sampling periods were selected
based upon the time intervals used in previous studies. Statistical Analysis Statistical analysis of the data was performed using a paired, two tailed, t test. RESULTS Table 1 i l l u s t r a t e s the blastogenic response of canine peripheral blood lymphocytes to PHA following general anesthesia. was observed 4 hours postinduction
A significant suppression
which persisted for at least 24 hours.
The response to PHA was back to normal by 4 days. The mitogenic response to Con A is shown in Table 2.
As with PHA, the
response 4 hours postinduction was suppressed by approximately 50%. However, by 24 hours postinduction, the blastogenic
response was recovering and was
back to normal by 4 days. Table 3 demonstrates the blastogenic
response to PWM. There was a 25%
suppression of the response 4 hours postinduction, however the response was back to normal levels by 24 hours. All
dogs exhibited a leukocytosis following general anesthesia which
reached i t s peak 24 hours postinduction.
The leukocytosis was characterized
by both an absolute neutrophilia and lymphocytosis.
66 The Effect of Anesthesia on the in v i t r o Blastogenic Response of TABLE I . Canine Peripheral Blood Lymphocytes to PHA. Do9 1 2 3 4 5 6 7 8 Mean SEM2 P (Paired t)
Hours (days) Postadministration of Anesthesia " ' Pre 4 hr. Day l Day 4 Day 7 168111 16034 5483 6577 4374 14204 I0781 5755
8191 5964 1642 2409 945 I0457 9034 1430
9827 6223 6589 I020 1907 3859 3645 I082
I0002 1825 ---
5009 1379 <0.005
4269 If04 <0.005
lO0 ---
44.5 8.2
49.6 12.0
13754 14116 9716 5391 5951 11906 13413 3887
18981 17939 4774 7293 4769 13067 I1644 5160
9767 1485 NS
I0454 2089 NS
Percent of Baseline: Mean SEM
105.1 13.3
lOI.9 3.7
IMean CPM of quadruplicate cultures. 2Standard error of the mean.
TABLE 2. The Effect of Anesthesia on the in v i t r o Blastogenic Response of Canine Peripheral Blood Lymphocytes to Con A. Do9 l 2 3 4 5 6 7 8 Mean SEM2 P (Paired t )
Hours (days) Postadministration of Anesthesia P~e 4 hr. Day 1 Day 4 Day 7 171291 15503 5555 4819 7126 16182 17807 4693
8223 5345 3340 2584 4371 8472 7335 3218
10696 6664 8204 2912 3530 10894 13061 3675
11102 2149 ---
5361 844 < 0.005
7454 1385 < 0.025
I00 ---
52.4 4.0
72.7 ll.6
I1951 13927 9864 7571 8874 17531 13844 4512
16077 17465 7359 7062 8222 21664 18917 5330
llOlO 1478 NS
12762 2290 NS
Percent of Baseline: Mean SEM
IMean CPM of quadruplicate cultures. 2Standard error of the mean.
I12.6 13.6
I19.3 6.1
6~ TABLE 3. The Effect of Anesthesia on the in v i t r o Blastogenic Response of Canine Peripheral Blood Lymphocytes to PWM. Hours ~a-y-s) PostadminTstration of Anesthesi~ Pre T-F~. ~._ .... -D=~-Da~7
Dog l 2 3 4 5 6 7 8
I03871 14150 4435 4900 8220 5112 9123 3976
Mean SEM2 P (Paired t)
8630 If510 3198 3363 5454 3849 7949 3005
13944 17334 8563 4288 4218 2762 5506 2108
7538 1280 ---
5870 I126 <0.005
I00 ---
76.2 4.6
7340 1982 NS
12853 18063 7499 6232 5986 6872 13354 4947
9986 14247 5740 4994 8284 7605 I1953 3638
9475 1676 < 0.025
8306 1289 NS
Percent of Baseline: Mean SEM
94.5 16.4
124.5 10.7
112.5 7.5
IMean CPM of quadruplicate cultures. 2Standard error of the mean. DISCUSSION This study describes a s i g n i f i c a n t suppression in the in v i t r o response of canine lymphocytes to mitogenic stimulation following general anesthesia for a r e l a t i v e l y minor, nontraumatic diagnostic procedure.
The suppression
to all three mitogens tested was most evident at 4 hours postinduction. response to PHA and Con A was s t i l l response to PWM was normal at this time.
The
suppressed by 24 hours, whereas the All responses were normalized by at
least 4 days postinduction. These results are in accord with most of the studies performed in human patients following general anesthesia for major surgery (Park et a l . , 1971; Jubert et a l . , 1973; Berenbaum et a l . , 1973; Cullen and Belle, 1975; Slade et a l . , 1975). The only human study to show no effect was that of Kanto et al. (1974); however, they only evaluated t h e i r patients 30 to 50 minutes postinduction of anesthesia.
In those studies examining the kinetics of the blas-
togenic response (Jubert et a l . , 1973; Berenbaum et a l . , 1973; Slade et a l . , 1975), similar findings were observed as those reported in this study.
Maxi-
mal suppression was observed between 2 to 6 hours postinduction with responses returning to normal between 4 to 7 days.
The d i f f i c u l t y with these human
studies is that i t is d i f f i c u l t to determine whether the suppression was an effect of the anesthesia or an effect of the surgical trauma. Cullen and
68 Belle (1975) and Slade et al. (1975) suggested that at least part of the suppression observed in t h e i r studies
could be attributed to the release of
corticosteroids induced by the surgical trauma. M a j o r surgical procedures cause a rise in plasma cortisol levels within 2 hours following surgery and return to normal by 24 hours, whereas, minor surgical procedures have l i t t l e effect on plasma cortisol
levels (Plumpton et
al.,
1968; Carter et
al.,
1970). The hemograms in these studies revealed a leukocytosis with a marked neutrophilia and lymphopenia similar to that observed following the administration of corticosteroids.
Although cortisol
levels were not measured in
this present study, the role of corticosteroids in the suppression of the blastogenic response was probably minimal since the animals experienced minimal surgical trauma and the leukocytosis
observed in these animals was not
typical of a steroid-induced leukocytosis
(Fauci et a l . , 1976; Nara et a l . ,
1979).
In addition, the anesthetics used in this study have been reported to
lower plasma cortisol levels (Oyama, 1973). Kelly (1980) reported a marked suppression in the PHA response in 4 dogs 4 hours postinduction of general anesthesia for laparotomies. was s t i l l
The response
suppressed at 24 hours and back to normal by 7 days.
The only
other study in the dog examined the blastogenic response to PHA during the first
24 hours following general anesthesia in 12 dogs undergoing routine
hysterectomies (Medleau et a l . , 1983). They reported a suppression in 4 of the 12 dogs at some time interval within that 24-hour period (all dogs were not tested at the same time intervals).
One of the potential problems with
this study was that almost half of the dogs had very poor responses to PHA before the induction of anesthesia. An interesting finding in this study was that the response to PHA appeared to be the most sensitive and the response to PWM the least sensitive to suppression following general anesthesia. man (Berenbaum et a l . ,
Similar results have been observed in
1973; Jubert et a l . ,
stimulate both B and T cells
1973).
PWM has been shown to
in man (Mellstedt, 1975; Weksler and Kuntz,
1976) and the dog (Wulff et a l . , 1982).
On the other hand, PHA is thought to
stimulate primarily T cells since human (Weksler and Kuntz, 1976) and canine (Wulff et a l . , 1982) B cells respond poorly to stimulation with PHA. Therefore, these findings suggest that the T cell population may be more susceptible to the suppressive effects of general anesthesia than the B cells.
The
response to Con A supports this suggestion since i t has been recently shown to activate both canine B and T cells (Wulff et a l . , 1982). In summary, i t
appears that general anesthesia does have a transient
immunosuppressive effect on lymphocyte function at least as evaluated in a
69 lymphocyte transformation test.
The results of this study suggest that one
should wait at least 4 days following general anesthesia before evaluating lymphocyte function in a patient using the lymphocyte transformation test. ACKNOWLEDGEMENTS This study was supported in part by the B i l l i e McFadden--American Boxer Club Research Fund. REFERENCES Berenbaum, M.C., Fluck, P.A., and Hurst, N.P., 1973. Depression of lymphocyte responses after surgical trauma. Br. J. Exp. Path. 54:597-607. Carter, M.E., and James, V.H.T., 1970. Pituitary-adrenal response to surgical stress in patients receiving corticotrophin treatment. Lancet 1:328-331. Cullen, B.F., and Belle, G.V., 1975. Lymphocyte transformation and changes in leukocyte count: effects of anesthesia and operation. Anesthesiology 43:563-569. Fauci, A.S., Dale, D.C., and Balow, J.E., 1976. Glucocorticoid therapy: mechanisms of action and c l i n i c a l considerations. Ann. Int. Med. 84:304315. Felsburg, P.J., Reilley, M.T., and Sinnigen, J.C., 1980. A new micro whole blood lymphocyte transformation test for evaluating the in v i t r o lymphocyte responsiveness of canine lymphocytes. Vet. Immun--o~.~unopath. 1:251-261. Felsburg, P.J., Serra, D.A., Mandato, V.N., and Jezyk, P.F., 1983. Potentiation of the canine lymphocyte blastogenic response by indomethacin. Vet. Immunol. Immunopath. 4:533-543. Jubert, A.V., Lee, E.T., Hersh, E.M., and McBride, C.M., 1973. Effects of surgery, anesthesia and intraoperative blood loss on immunocompetence. J. Surg. Res. 15:399-403. Kanto, J., Vapaavuori, M., and Viljanen, M.K., 1974. Mitogen-induced lymphocyte transformation after general anesthesia. B r i t . J. Anes. 46:733-735. Kelly, G.E., 1980. The effect of surgery in dogs on the response to concomitant distemper vaccination. Aust. Vet. J. 56:556-557. Kristensen, F., Kristensen, B., and Lazary, S., 1982. The lymphocyte stimulation test in veterinary immlmology. Vet. Immunol. Immunopath. 3:203-277. Medleau, L., Crowe, D.T., and Dawe, D.L., 1983. Effect of surgery on the in v i t r o response of canine peripheral blood lymphocytes to phytohemag~u~ . Am. J. Vet. Res. 44:859-860. Mellstedt, H., 1975. In v i t r o activation of human T and B lymphocytes by pokeweed mitogen. ~ln.~-6~'-E'xp. Immunol. 19:75-82. Nara, P.L., Krakowka, S., and Powers, T.E., 1979. Effects of prednisolone on the development of immune responses to canine distemper virus in Beagle pups. Am. J. Vet. Res. 40:1742-1747. Osborne, C.A., Stevens, J.B., and Perman, V., 1974. Kidney biopsy. Vet Clinics N.A. 4:351-365. Oyama, T., 1973. Endocrine responses to anesthetic agents. B r i t . J. Anesth. 45:276-281. Park, S.W., Brody, J . I . , Wallace, H.A., and Blakemore, W.S., 1971. Immunosuppressive effect of surgery. Lancet 1:53-55. Plumpton, F.S., Besser, G.M., and Cole, P.V., 1968. Corticosteroid treatment and surgery. I. An investigation of the indications for steroid cover. Anesthesia 24:3-9. Slade, M.S., Simmons, R.L., Yunis, E., and Greenberg, L.J., 1975. Immunodepression after major surgery in normal patients. Surgery 78:363-372.
70 Weksler, M.E., and Kuntz, M.M., 1976. Synergy between human T and B lymphocytes in their response to phytohemagglutinin and pokeweed mitogen. Immunology 31:273-281. Wulff, J.C., Tsoi, M.S., Aprile, J., Deeg, H.J., Durkopp, N., and Storb, R., 1982. Stimulation of canine lymphocyte subpopulations separated non. lytically by monoclonal anti-T and polyclonal anti-B cell antibodies. Blut 45:309-316.
63
THE EFFECT OF GENERAL ANESTHESIA ON CANINE LYMPHOCYTEFUNCTION PETER J. FELSBURG, LYNETTE L. KEYES, DONALDR. KRAWIEC, and STANLEY I. RUBIN Clinical Immunology Laboratory, Department of Veterina~ Pathobiology, College of Veterinary Medicine, University of I l l i n o i s , Urbana, IL 61801, U.S.A. (Accepted 12 December 1985) ABSTRACT Felsburg, P.J., Keyes, L.L., Krawiec, D.R., and Rubin, S . I . , 1986. The effect of general anesthesia on canine l~tmphocyte function. Vet. Immunol. Immunopathol., 13: 63-70. The in v i t r o response of canine peripheral blood lymphocytes to mitogenic stimulat~n was 'evaluated following general anesthesia for a r e l a t i v e l y minor diagnostic procedure. A marked suppression in the blastogenic response to phytohemagglutinin and concanavalin A was observed 4 hours postinduction which persisted through the f i r s t 24 hours and was normal by 4 days. A mild suppression to stimulation with pokeweed mitogen was observed at 4 hours postinduction but the response was back to normal by 24 hours. These results suggest general anesthesia has a transient immunosuppressive effect on canine lymphocytes and T cells are more susceptible to the immunosuppressive effect than B cells. INTRODUCTION General anesthesia is used not only for surgical procedures, but also for various noninvasive diagnostic procedures. Situations exist where evaluation of a patient's immune competence is desired following a diagnosis which has involved the use of general anesthesia.
This prompts a frequently asked
question of the c l i n i c a l immunologistc-"Does general anesthesia have a suppressive effect on lymphocyte function tests and, i f so, how long does the suppression persist?". The few studies reported in humans (Park et a l . , 1971; Jubert et a l . , 1973; Berenbaum et a l . , 1973; Kanto et a l . , 1974; Cullen and Belle, 1975; Shade et a l . , 1975) and dogs (Kelly, 1980; Medleau et a l . , 1983) which have attempted to address this question have generally shown a depression in the in v i t r o blastogenic response of l~nnphocytes to mitogenic and antigenic stimulation. However, the results of these studies are confounded by the selection of patients studied and the various major surgical procedures which were performed in conjunction with the general anesthesia.
The question remains
whether the suppression was a result of the general anesthesia, the immune status of the patient, or the trauma and blood loss associated with major surgical procedures. 0165-2427/86/$03.50
© 1986 Elsevier Science Publishe~ B.V.
64 The purpose of this study was to evaluate the in v i t r o lymphocyte response to mitogenic stimulation in c l i n i c a l l y normal dogs following general anesthesia for a r e l a t i v e l y minor diagnostic procedure. MATERIALS AND METHODS Dogs Eight mixed-breed, random source, conditioned male dogs were used in this study.
All dogs were young adults and in good health at the time of the
study.
The dogs were routinely vaccinated and checked for internal parasites
prior to participation in the study. tained on a commercial diet.
The dogs were housed indoors and main-
These dogs were part of a project to determine
the effect of amphotericin B on renal function.
This study was performed in
the dogs prior to t h e i r receiving any drug therapy. Blood Blood was collected aseptically by venipuncture into syringes containing preservative-free heparin (20 units/ml of blood).
The heparinized blood was
immediately transferred into s t e r i l e plastic culture tubes. Culture Medium RPMI 1640 culture medium (M.A. Bioproducts, Walkersville, MD) supplemented with lO0 units/ml of p e n i c i l l i n , lO0 ug/ml of streptomycin, and 2 mM glutamine was used in a l l experiments. Mito~ens Phytohemagglutinin-P (PHA; Difco Laboratories, Detroit, MI), concanavalin A (Con A; Sigma Chemical Co., St. Louis, MO), and pokeweed mitogen (PWM; Grand Island Biological Co., Grand Island, NY) were reconstituted with s t e r i l e water, aliquoted in small volumes, and stored at -70 degrees C until
used.
For each experiment, aliquots were thawed and diluted to the desired concentrations in RPMI 1640. L~mphoc~te Transformation Studies The lymphocyte transformation tests were performed in quadruplicate using a micro whole blood technique (Felsburg et a l . , 1980, 1983). Five ul of heparinized whole blood was added to each well of round bottom microtiter plates containing lOO ul of RPMI 1640 with or without mitogens. Cultures were incubated at 37 degrees C in a humid, 5% CO2 atmosphere for 96 hours. Sixteen hours before harvesting, 0.5 uCi of 3H-thymidine (2.0 Ci/mmole; New England Nuclear, Boston, MA) was added to each well.
After incubation, the microcul-
tures were harvested onto glass f i l t e r strips by an automated cell harvester. The strips were washed extensively with d i s t i l l e d water, 3% acetic acid, and again with d i s t i l l e d water in order to lyse the red blood cells. After a i r drying, the f i l t e r discs were placed in s c i n t i l l a t i o n f l u i d and the
65 radioactivity measured in a liquid s c i n t i l l a t i o n spectrometer.
The results
are expressed as the counts per minute (CPM) in stimulated cultures minus the CPM in control cultures (Kristensen et a l . , 1982). Experimental Protocol Prior to anesthesia, the dogs received l mg acepromazine maleate intravenously and atropine (0.04 mg/kg) subcutaneously. Anesthesia was induced by the administration of thiamylal sodium (2.5 to 4 mg/kg) intravenously and f o l lowing intubation, anesthesia was maintained with methoxyflurane and oxygen. A small skin incision was made in the right flank, and a needle biopsy was collected from the right kidney using the keyhold technique (Osborne et a l . , 1974).
The incision was closed and the animal was allowed to recover from
anesthesia. Samples were collected for lymphocyte transformation prior to the induction of anesthesia, 4 hours postinduction, 24 hours postinduction, 4 days postinduction, and 7 days postinduction.
These sampling periods were selected
based upon the time intervals used in previous studies. Statistical Analysis Statistical analysis of the data was performed using a paired, two tailed, t test. RESULTS Table 1 i l l u s t r a t e s the blastogenic response of canine peripheral blood lymphocytes to PHA following general anesthesia. was observed 4 hours postinduction
A significant suppression
which persisted for at least 24 hours.
The response to PHA was back to normal by 4 days. The mitogenic response to Con A is shown in Table 2.
As with PHA, the
response 4 hours postinduction was suppressed by approximately 50%. However, by 24 hours postinduction, the blastogenic
response was recovering and was
back to normal by 4 days. Table 3 demonstrates the blastogenic
response to PWM. There was a 25%
suppression of the response 4 hours postinduction, however the response was back to normal levels by 24 hours. All
dogs exhibited a leukocytosis following general anesthesia which
reached i t s peak 24 hours postinduction.
The leukocytosis was characterized
by both an absolute neutrophilia and lymphocytosis.
66 The Effect of Anesthesia on the in v i t r o Blastogenic Response of TABLE I . Canine Peripheral Blood Lymphocytes to PHA. Do9 1 2 3 4 5 6 7 8 Mean SEM2 P (Paired t)
Hours (days) Postadministration of Anesthesia " ' Pre 4 hr. Day l Day 4 Day 7 168111 16034 5483 6577 4374 14204 I0781 5755
8191 5964 1642 2409 945 I0457 9034 1430
9827 6223 6589 I020 1907 3859 3645 I082
I0002 1825 ---
5009 1379 <0.005
4269 If04 <0.005
lO0 ---
44.5 8.2
49.6 12.0
13754 14116 9716 5391 5951 11906 13413 3887
18981 17939 4774 7293 4769 13067 I1644 5160
9767 1485 NS
I0454 2089 NS
Percent of Baseline: Mean SEM
105.1 13.3
lOI.9 3.7
IMean CPM of quadruplicate cultures. 2Standard error of the mean.
TABLE 2. The Effect of Anesthesia on the in v i t r o Blastogenic Response of Canine Peripheral Blood Lymphocytes to Con A. Do9 l 2 3 4 5 6 7 8 Mean SEM2 P (Paired t )
Hours (days) Postadministration of Anesthesia P~e 4 hr. Day 1 Day 4 Day 7 171291 15503 5555 4819 7126 16182 17807 4693
8223 5345 3340 2584 4371 8472 7335 3218
10696 6664 8204 2912 3530 10894 13061 3675
11102 2149 ---
5361 844 < 0.005
7454 1385 < 0.025
I00 ---
52.4 4.0
72.7 ll.6
I1951 13927 9864 7571 8874 17531 13844 4512
16077 17465 7359 7062 8222 21664 18917 5330
llOlO 1478 NS
12762 2290 NS
Percent of Baseline: Mean SEM
IMean CPM of quadruplicate cultures. 2Standard error of the mean.
I12.6 13.6
I19.3 6.1
6~ TABLE 3. The Effect of Anesthesia on the in v i t r o Blastogenic Response of Canine Peripheral Blood Lymphocytes to PWM. Hours ~a-y-s) PostadminTstration of Anesthesi~ Pre T-F~. ~._ .... -D=~-Da~7
Dog l 2 3 4 5 6 7 8
I03871 14150 4435 4900 8220 5112 9123 3976
Mean SEM2 P (Paired t)
8630 If510 3198 3363 5454 3849 7949 3005
13944 17334 8563 4288 4218 2762 5506 2108
7538 1280 ---
5870 I126 <0.005
I00 ---
76.2 4.6
7340 1982 NS
12853 18063 7499 6232 5986 6872 13354 4947
9986 14247 5740 4994 8284 7605 I1953 3638
9475 1676 < 0.025
8306 1289 NS
Percent of Baseline: Mean SEM
94.5 16.4
124.5 10.7
112.5 7.5
IMean CPM of quadruplicate cultures. 2Standard error of the mean. DISCUSSION This study describes a s i g n i f i c a n t suppression in the in v i t r o response of canine lymphocytes to mitogenic stimulation following general anesthesia for a r e l a t i v e l y minor, nontraumatic diagnostic procedure.
The suppression
to all three mitogens tested was most evident at 4 hours postinduction. response to PHA and Con A was s t i l l response to PWM was normal at this time.
The
suppressed by 24 hours, whereas the All responses were normalized by at
least 4 days postinduction. These results are in accord with most of the studies performed in human patients following general anesthesia for major surgery (Park et a l . , 1971; Jubert et a l . , 1973; Berenbaum et a l . , 1973; Cullen and Belle, 1975; Slade et a l . , 1975). The only human study to show no effect was that of Kanto et al. (1974); however, they only evaluated t h e i r patients 30 to 50 minutes postinduction of anesthesia.
In those studies examining the kinetics of the blas-
togenic response (Jubert et a l . , 1973; Berenbaum et a l . , 1973; Slade et a l . , 1975), similar findings were observed as those reported in this study.
Maxi-
mal suppression was observed between 2 to 6 hours postinduction with responses returning to normal between 4 to 7 days.
The d i f f i c u l t y with these human
studies is that i t is d i f f i c u l t to determine whether the suppression was an effect of the anesthesia or an effect of the surgical trauma. Cullen and
68 Belle (1975) and Slade et al. (1975) suggested that at least part of the suppression observed in t h e i r studies
could be attributed to the release of
corticosteroids induced by the surgical trauma. M a j o r surgical procedures cause a rise in plasma cortisol levels within 2 hours following surgery and return to normal by 24 hours, whereas, minor surgical procedures have l i t t l e effect on plasma cortisol
levels (Plumpton et
al.,
1968; Carter et
al.,
1970). The hemograms in these studies revealed a leukocytosis with a marked neutrophilia and lymphopenia similar to that observed following the administration of corticosteroids.
Although cortisol
levels were not measured in
this present study, the role of corticosteroids in the suppression of the blastogenic response was probably minimal since the animals experienced minimal surgical trauma and the leukocytosis
observed in these animals was not
typical of a steroid-induced leukocytosis
(Fauci et a l . , 1976; Nara et a l . ,
1979).
In addition, the anesthetics used in this study have been reported to
lower plasma cortisol levels (Oyama, 1973). Kelly (1980) reported a marked suppression in the PHA response in 4 dogs 4 hours postinduction of general anesthesia for laparotomies. was s t i l l
The response
suppressed at 24 hours and back to normal by 7 days.
The only
other study in the dog examined the blastogenic response to PHA during the first
24 hours following general anesthesia in 12 dogs undergoing routine
hysterectomies (Medleau et a l . , 1983). They reported a suppression in 4 of the 12 dogs at some time interval within that 24-hour period (all dogs were not tested at the same time intervals).
One of the potential problems with
this study was that almost half of the dogs had very poor responses to PHA before the induction of anesthesia. An interesting finding in this study was that the response to PHA appeared to be the most sensitive and the response to PWM the least sensitive to suppression following general anesthesia. man (Berenbaum et a l . ,
Similar results have been observed in
1973; Jubert et a l . ,
stimulate both B and T cells
1973).
PWM has been shown to
in man (Mellstedt, 1975; Weksler and Kuntz,
1976) and the dog (Wulff et a l . , 1982).
On the other hand, PHA is thought to
stimulate primarily T cells since human (Weksler and Kuntz, 1976) and canine (Wulff et a l . , 1982) B cells respond poorly to stimulation with PHA. Therefore, these findings suggest that the T cell population may be more susceptible to the suppressive effects of general anesthesia than the B cells.
The
response to Con A supports this suggestion since i t has been recently shown to activate both canine B and T cells (Wulff et a l . , 1982). In summary, i t
appears that general anesthesia does have a transient
immunosuppressive effect on lymphocyte function at least as evaluated in a
69 lymphocyte transformation test.
The results of this study suggest that one
should wait at least 4 days following general anesthesia before evaluating lymphocyte function in a patient using the lymphocyte transformation test. ACKNOWLEDGEMENTS This study was supported in part by the B i l l i e McFadden--American Boxer Club Research Fund. REFERENCES Berenbaum, M.C., Fluck, P.A., and Hurst, N.P., 1973. Depression of lymphocyte responses after surgical trauma. Br. J. Exp. Path. 54:597-607. Carter, M.E., and James, V.H.T., 1970. Pituitary-adrenal response to surgical stress in patients receiving corticotrophin treatment. Lancet 1:328-331. Cullen, B.F., and Belle, G.V., 1975. Lymphocyte transformation and changes in leukocyte count: effects of anesthesia and operation. Anesthesiology 43:563-569. Fauci, A.S., Dale, D.C., and Balow, J.E., 1976. Glucocorticoid therapy: mechanisms of action and c l i n i c a l considerations. Ann. Int. Med. 84:304315. Felsburg, P.J., Reilley, M.T., and Sinnigen, J.C., 1980. A new micro whole blood lymphocyte transformation test for evaluating the in v i t r o lymphocyte responsiveness of canine lymphocytes. Vet. Immun--o~.~unopath. 1:251-261. Felsburg, P.J., Serra, D.A., Mandato, V.N., and Jezyk, P.F., 1983. Potentiation of the canine lymphocyte blastogenic response by indomethacin. Vet. Immunol. Immunopath. 4:533-543. Jubert, A.V., Lee, E.T., Hersh, E.M., and McBride, C.M., 1973. Effects of surgery, anesthesia and intraoperative blood loss on immunocompetence. J. Surg. Res. 15:399-403. Kanto, J., Vapaavuori, M., and Viljanen, M.K., 1974. Mitogen-induced lymphocyte transformation after general anesthesia. B r i t . J. Anes. 46:733-735. Kelly, G.E., 1980. The effect of surgery in dogs on the response to concomitant distemper vaccination. Aust. Vet. J. 56:556-557. Kristensen, F., Kristensen, B., and Lazary, S., 1982. The lymphocyte stimulation test in veterinary immlmology. Vet. Immunol. Immunopath. 3:203-277. Medleau, L., Crowe, D.T., and Dawe, D.L., 1983. Effect of surgery on the in v i t r o response of canine peripheral blood lymphocytes to phytohemag~u~ . Am. J. Vet. Res. 44:859-860. Mellstedt, H., 1975. In v i t r o activation of human T and B lymphocytes by pokeweed mitogen. ~ln.~-6~'-E'xp. Immunol. 19:75-82. Nara, P.L., Krakowka, S., and Powers, T.E., 1979. Effects of prednisolone on the development of immune responses to canine distemper virus in Beagle pups. Am. J. Vet. Res. 40:1742-1747. Osborne, C.A., Stevens, J.B., and Perman, V., 1974. Kidney biopsy. Vet Clinics N.A. 4:351-365. Oyama, T., 1973. Endocrine responses to anesthetic agents. B r i t . J. Anesth. 45:276-281. Park, S.W., Brody, J . I . , Wallace, H.A., and Blakemore, W.S., 1971. Immunosuppressive effect of surgery. Lancet 1:53-55. Plumpton, F.S., Besser, G.M., and Cole, P.V., 1968. Corticosteroid treatment and surgery. I. An investigation of the indications for steroid cover. Anesthesia 24:3-9. Slade, M.S., Simmons, R.L., Yunis, E., and Greenberg, L.J., 1975. Immunodepression after major surgery in normal patients. Surgery 78:363-372.
70 Weksler, M.E., and Kuntz, M.M., 1976. Synergy between human T and B lymphocytes in their response to phytohemagglutinin and pokeweed mitogen. Immunology 31:273-281. Wulff, J.C., Tsoi, M.S., Aprile, J., Deeg, H.J., Durkopp, N., and Storb, R., 1982. Stimulation of canine lymphocyte subpopulations separated non. lytically by monoclonal anti-T and polyclonal anti-B cell antibodies. Blut 45:309-316.