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PAG stimulation does not affect primary antibody responses in rats
387
Pain, 29 (1987) 387-392 Hsevier PA1 01048
PAG stimulation does not affect primary antibody responses in rats Ryoko Noguchi
*, Chuya Hamada * * and Koki Shimoji * * Department of Anest~e~io~o~ and * * Department of Virology, Niigata University School of Medicine, Niigata 951 (Japan] (Received 17 July 1986, revised received 10 November 1986, accepted 18 November 1986)
Adult male rats, which had electrodes chronically implanted in the periaqueductal gray were immunized with sheep red blood cells (SRBCs). The number of direct and indirect plaque-forming cells (PFCs) in the group receiving PAG stimulation after immunization did not differ significantly from that in the unstimulated group. Thus, the results indicate that short-term PAG stimulation does not suppress antibody-producing activity in the rat.
S-
(PAG),
Key wordsz Periaqueductal gray; Stimulation-pr~u~d cells; Rat
analgesia; Antibody production; Plaque-fo~ng
Introduction Since the initial report by Reynolds [ll], several workers [9] have demonstrated that analgesia produced by electrical stimulation of the midbrain structures (stimulation produced analgesia, SAP) can eliminate the responses to noxious stimuli, while behavioral, cardiac and respiratory responsiveness are unaffected during and after the stimulation. PAG stimulation also produces significant analgesia in patients with chronic intractable pain [6]. On the other hand, however, Simon et al. [13] have demonstrated that PAG stimulation increases the metastasis of cancer in rats and that naloxone does not block this effect. Although the possibi~ty of immune-suppressive effects by PAG stimulation is suggested, the necessary measurements of various parameters reflecting immune reactions have not been carried out. Schanker et al. [12] found that midbrain electrical stimulation did not appear to have any acute or chronic effects on cellular immunity in humans. However, the effects of electrical stimulation on humoral immunity remained unclear. In the present‘study, we report the effects of acute PAG stimulation on primary antibody responses.
Correspondence to: Koki Shimoji, M.D., Department of Anesthesiology, Niigata University School of Medicine, l-757 Asahimachi, Niigata 951, Japan. 0304-3959/87/%03.50 @ 1987 Elsevier Science Publishers B.V. (Biomedical Division)
388
Methods Twenty-eight male Wistar rats, g-12 weeks old and weighing 260-320 g, were anesthetized with sodium pentobarbital (60 mg/kg, i.p.). Then, a monopolar stainless steel electrode (100 pm in diameter insulated except for 0.5 mm at the tip) was stereotaxically implanted in the right side of the PAG at RC 0.0, AP + 1 .O, L 1.5 mm, based on the atlas of Pellegrino et al. [lo] with a reference electrode in the neck muscle. The sites of the electrode tips, verified histologically after the experiment, were almost identical to those made by the stereotaxic apparatus (Fig. 1). The surgical procedures were essentially the same as those described by Skinner [15]. After a postoperative recovery period of 14 days, 22 of 25 rats were immunized with an injection of 1.0 ml of a 10% suspension of SRBC (2 X lo9 cells), which had been washed 3 times with balanced salt solution (BSS) at pH 7.2, into the tail veins while the animals were awake. These immunized rats were assigned randomly into PAG-stimulated and non-stimulated groups. The animals received electrical stimulation (100 Hz monophasic square-wave pulses, 0.2 msec duration, 20-50 PA) for 35 min, 1 h after the immunization. During PAG stimulation at each intensity level, the animal was tested for nociception by means of Haffner’s tail-pinch method [5]. -3
-
2
--6
2
1
0
Fig. 1. The locations
1
2
of stimulating
3 electrodes
4
5 mm
in the PAG, reconstructed
from histological
examinations.
389
During the pinch test, the animal was gently restrained on a wooden platform. The limbs and tail hung freely over the side of the platform, allowing easy access and a full range of withdrawal and escape responses. After a few minutes of adaptation, the animals exhibited no struggling on the platform. Pinches were applied with a pair of forceps to the tail at least twice immediately before and 20-30 set after PAG stimulation, and the behavioral response was rated by two observers according to a 3-point scale (complete inhibition = analgesia (2); attenuated response = hypalgesia (1); normal response = no~ogesia (0)). In addition to the escape response, other signs of distress, such as increased alertness, freezing, pilo-erection, respiratory acceleration, defecation, urination and vocalization, were also assessed. Analgesia was defined as a rating of more than 1 by both observers during PAG stimulation. In 7 of 13 animals stimulated, 6 of 12 unstimulated and in the 3 unimmunized rats, the numbers of antigen-specific IgM-PFC were investigated using direct techniques at day 4 following the injection of SRBC. In 6 stimulated and 6 unstimulated rats, numbers of both IgM- and IgG-PFC were counted by direct and indirect techniques at day 7. The procedures of plaque technique in microchambers were based on those reported by Cunningham and Szenberg 131.For plaque assay, all animals were anesthetized with ether and then killed by exsanguination of circulating blood by puncture of the heart to avoid conta~ation of autologous erythrocytes in the plaque assay. The spleens were removed and placed in Petri dishes containing ice-cold BSS. The spleens were teased with fine forceps. The suspension was centrifuged at 400 x g for 1 min at room temperature and then resuspended in BSS. Dilution was performed whenever necessary. The liquid assay mixture was prepared by adding 0.3 ml of 33% SRBC and 0.2 ml of guinea pig complement to 0.5 ml of a lymphoid cell suspension at various dilutions for the direct method. For the indirect method the assay mixture was prepared by adding 0.2 ml of 50% SRBC, 0.2 ml of guinea pig complement and 0.1 ml of IgG-fraction rabbit anti-rat IgG (Litton Bionetics, Kensington, MD) to the same volume of a lymphoid cell suspension. The microchamber was made of two microscope slides laid on two pieces of double-side tape. The volume of the ~cr~hamber was 150 f 5 ~1 (means f S.E.M.). The assay mixture of 0.15 ml containing 1 x lo4 spleen cells was pipetted into each chamber. After sealing the orifice of the slide chamber with cellophane tape, the preparation was incubated for 4 h at 37 o C. At respective dilutions, spleen samples were run in triplicate. The plaques were counted microscopically under 40-fold magnification within 4 h after the establishment of incubation. Statistical analyses were performed using Student’s 1 test for non-paired data. Differences between the mean values were considered to be significant when P values were less than 0.05.
ReSUltS
The results are summarized in Fig. 2. The numbers of IgM-PFC at day 4 after immunization in stimulated (n = 7) and unstimulated (n = 6) groups were 43.3 f 15.1
390
-” $
5
2
600
c m
Stimulated
0
Unslimulated
fl=6
500
“0 ;
400
a
’
300
$ J % LL
200
c” E
100
0
IgM Plaque
day
4
IgM Plaque IgG Plaque
day 7
Fig. 2. The effect of PAG stimulation on responses of rat spleen cell suspensions to antigen challenge. The number of PFC per lo4 spleen cells (mean I S.E.M.) was assayed at days 4 and 7 after immunization with SRBC. The number of facilitated plaques (IgG-PFC) was calculated as the total number of plaques developed with antiserum minus the direct plaques (IBM-PFC). There were no significant differences between the stimulated and unstimulated groups at all values.
(mean k S.E.M.) and 71.5 t- 23.8 per lo4 spleen cells, respectively. There was no significant difference in the mean numbers of IgM-PFC between the two groups. In the uni~u~z~ group (n = 3), the number of IgM-PFC at day 4 was 1.8 t_ 0.2 per lo4 spleen cells. At day 7, the numbers of IgG-PFC in the stimulated (n = 6) and unstimulated (n = 6) groups were 546.2 I 63.8 and 456.8 _t 25.5 respectively, per lo4 spleen cells, while the numbers of IgM-PFC were 300.9 rir 67.8 and 247.9 rf 84.2 respectively, per lo4 spleen cells. There was no significant difference in the mean numbers of IgM- as well as IgG-PFC at day 7 between the two groups. There was a sig~ficant difference in the numbers of IgM-PFC between day 4 and day 7 in both the stimulated and unstimulated groups (P < 0.05). On analgesia testing, 2 of 7 rats at day 4 of the experiment and 1 of 6 rats at day 7 of the experiment did not achieve SPA. However, none of the responses in the rats which demonstrated SPA differed significantly from those in the rats which did not achieve SPA.
Discussion
The present study demonstrated that short-term PAG stimulation did not affect the primary antibody responses in rats. In investigating antibody responses, it is necessary to estimate both the level of antibody and the number of antibody-for~ng cells. The Jerne-Nordin plaque assay [7] has become a standard technique for identifying antibody-producing cells. In this assay, each plaque represents one antibody-secreting cell. It is found that the direct
391
(IgM) and indirect (IgG) plaque-forming cells increase to a maximum at day 4 and at day 7 following immunization, respectively. These increases parallel the rise of serum IgM and IgG antibody titers [7]. The SRBC we used in the present study is one of the T-dependent (T-dep) antigens. According to several lines of evidence presented so far, the primary immune responses to T-dep antigens depend on both T and B cells which recognize the antigens. Due to the large amounts of SRBC used in the present study, the number of IgM-PFC at day 7 was thought to be larger than that at day 4 in both PAG-stimulate and unstimulated rats. In the present study, the primary antibody responses were augmented with prolongation of the response peak, probably resulting from injection of SRBC at a high dose for primary antigenic challenge. Although Simon et al. [13] demonstrated that a single short-term electrical stimulation of the midbrain increased the metastatic activity of cancer, they did not study cellular or humoral i~~ity. Further, Brechner et al. [2] found that the group which received PAG stimulation subsequent to prolactin-sensitive tumor implantation had a statistically higher incidence of enhanced tumor growth than a non-PAG stimulated control group. They suggested that enhanced tumor growth with PAG stimulation may be related to prolactin release in rats. Furthermore, Johnson et al. [8] found that &endorphin suppressed the in vitro antibody response using mouse spleen cells. Simon et al. [14] have subsequently shown that &endorphin administration into the nucleus of the raphe magnus facilitates metastatic tumor growth. Release of endogenous opiates has been shown to increase during mid-brain stimulation [1,6]. On the other hand, Fessler et al. [4] have recently reported that there is a possibility of elevated /%endorphin due to contrast medium infusion but not due to electrical brain stimulation. Nevertheless, these findings tend to suggest a certain relationship between electrical stimulation of the midbrain and enhanced tumor growth. However, concerning the mechanism of immunologic aspects of electrical brain stimulation, there has been no report except the recent investigation by Schanker et al. [12]. They have shown that acute midbrain SPA reduces the B cell percentage, but does not appear to have any other acute or chronic effects on i~une functions in humans. In the present experiment, we investigated the effect of short-term (35 ruin) PAG stimulation in the initial phase of antigen recognition in the primary antibody response after immunization. PAG stimulation for 35 min was found to be sufficient to elicit clinical analgesia in patients [1,6,12]. The effects of PAG simulation at various stages in the course of antibody production with long-term stimulation was not examined in the present study. Nevertheless, the present results have demonstrated that SPA applied for a short term does not affect cellular or humoral immune activity.
We thank Mr. Yukio Sato for excellent technical assistance and Dr. Satoshi Abe for preparing the histological specimens.
392
This study was supported by a grant-in-aid for special project research of selected intractable neurological disorders from the Ministry of Education, Science and Culture, Japan.
References 1 Akil, H., Richardson, D.E., Hughes, J. and Barchas, J.D., Enkephalin-like material elevated in ventricular cerebrospinal fluid of pain patients after analgesic focal stimulation, Science, 201 (1978) 463-465. 2 Brechner, T., Motyka, D. and Sherman, J., Growth enhancement of prolactin-sensitive mammary tumor by periaqueductal gray stimulation, Life Sci., 32 (1983) 525-530. 3 Cunningham, A.J. and Szenberg, A., Further improvements in the plaque technique for detecting single antibody-forming cells, Immunology, 14 (1968) 599-600. 4 Fessler, R.G., Brown, F.D., Rachlin, J.R. and Mullan, S., Elevated /3-endorphin in cerebrospinal fluid after electrical brain stimulation: artifact of contrast infusion?, Science, 224 (1984) 1017-1019. 5 Haffner, F.. Experimentelle Prlifung schmerzstillender Mittel, Dtsch. med. Wschr., 55 (1929) 731-733. 6 Hosobuchi, Y., Adams, J.E. and Linchitz, R., Pain relief by electrical stimulation of the central gray matter in humans and its reversal by naloxone, Science, 197 (1977) 183-186. 7 Jerne, N.K. and Nordin, A.A., Plaque formation in agar by single antibody-producing cells, Science. 140 (1963) 405. 8 Johnson, H.M., Smith, E.M., Torres, B.A. and Blalock, J.E.. Regulation of the in vitro response by neuroendocrine hormones, Proc. nat. Acad. Sci. (Wash.), 79 (1982) 4177-4174. 9 Mayer, D.J., Wholfle. T.L.. Akil. H., Carder, B. and Liebeskind, J.C.. Analgesia from electrical stimulation in the brainstem of the rat, Science, 174 (1971) 1351-1354. 10 Pellegrino, L.J., Pellegrino. A.S. and Cushman, A.J., A Stereotaxic Atlas of the Rat Brain, 2nd edn, Plenum Press, New York, 1979, pp. 61, 104-105. 11 Reynolds, D.V., Surgery in the rat during electrical analgesia induced by focal brain stimulation, Science, 164 (1969) 444445. 12 Schanker. H.M., Young, R., Cahan. L., Schroff, R. and Saxon. A.. Effect of midbrain stimulus-mduced analgesia on immune function in humans, J. Neuroimmunol., 5 (1983) 1855189. 13 Simon, R.H.. Lovett, III, E.J. and Joel Lundy, D.T., Electrical stimulation of the midbrain mediates metastatic tumor growth, Science, 209 (1980) 1132-1133. 14 Simon, R.H., Arbo. T.E. and Lundy, J.. Beta-endorphin injected into the nucleus of the raphe magnus facilitates metastatic tumor growth, Brain Res. Bull., 12 (1984) 487-941. 15 Skinner. J.E.. Neuroscience: a Laboratory Manual, Saunders. Toronto, 1971. pp. X7-144.
Pain, 29 (1987) 387-392 Hsevier PA1 01048
PAG stimulation does not affect primary antibody responses in rats Ryoko Noguchi
*, Chuya Hamada * * and Koki Shimoji * * Department of Anest~e~io~o~ and * * Department of Virology, Niigata University School of Medicine, Niigata 951 (Japan] (Received 17 July 1986, revised received 10 November 1986, accepted 18 November 1986)
Adult male rats, which had electrodes chronically implanted in the periaqueductal gray were immunized with sheep red blood cells (SRBCs). The number of direct and indirect plaque-forming cells (PFCs) in the group receiving PAG stimulation after immunization did not differ significantly from that in the unstimulated group. Thus, the results indicate that short-term PAG stimulation does not suppress antibody-producing activity in the rat.
S-
(PAG),
Key wordsz Periaqueductal gray; Stimulation-pr~u~d cells; Rat
analgesia; Antibody production; Plaque-fo~ng
Introduction Since the initial report by Reynolds [ll], several workers [9] have demonstrated that analgesia produced by electrical stimulation of the midbrain structures (stimulation produced analgesia, SAP) can eliminate the responses to noxious stimuli, while behavioral, cardiac and respiratory responsiveness are unaffected during and after the stimulation. PAG stimulation also produces significant analgesia in patients with chronic intractable pain [6]. On the other hand, however, Simon et al. [13] have demonstrated that PAG stimulation increases the metastasis of cancer in rats and that naloxone does not block this effect. Although the possibi~ty of immune-suppressive effects by PAG stimulation is suggested, the necessary measurements of various parameters reflecting immune reactions have not been carried out. Schanker et al. [12] found that midbrain electrical stimulation did not appear to have any acute or chronic effects on cellular immunity in humans. However, the effects of electrical stimulation on humoral immunity remained unclear. In the present‘study, we report the effects of acute PAG stimulation on primary antibody responses.
Correspondence to: Koki Shimoji, M.D., Department of Anesthesiology, Niigata University School of Medicine, l-757 Asahimachi, Niigata 951, Japan. 0304-3959/87/%03.50 @ 1987 Elsevier Science Publishers B.V. (Biomedical Division)
388
Methods Twenty-eight male Wistar rats, g-12 weeks old and weighing 260-320 g, were anesthetized with sodium pentobarbital (60 mg/kg, i.p.). Then, a monopolar stainless steel electrode (100 pm in diameter insulated except for 0.5 mm at the tip) was stereotaxically implanted in the right side of the PAG at RC 0.0, AP + 1 .O, L 1.5 mm, based on the atlas of Pellegrino et al. [lo] with a reference electrode in the neck muscle. The sites of the electrode tips, verified histologically after the experiment, were almost identical to those made by the stereotaxic apparatus (Fig. 1). The surgical procedures were essentially the same as those described by Skinner [15]. After a postoperative recovery period of 14 days, 22 of 25 rats were immunized with an injection of 1.0 ml of a 10% suspension of SRBC (2 X lo9 cells), which had been washed 3 times with balanced salt solution (BSS) at pH 7.2, into the tail veins while the animals were awake. These immunized rats were assigned randomly into PAG-stimulated and non-stimulated groups. The animals received electrical stimulation (100 Hz monophasic square-wave pulses, 0.2 msec duration, 20-50 PA) for 35 min, 1 h after the immunization. During PAG stimulation at each intensity level, the animal was tested for nociception by means of Haffner’s tail-pinch method [5]. -3
-
2
--6
2
1
0
Fig. 1. The locations
1
2
of stimulating
3 electrodes
4
5 mm
in the PAG, reconstructed
from histological
examinations.
389
During the pinch test, the animal was gently restrained on a wooden platform. The limbs and tail hung freely over the side of the platform, allowing easy access and a full range of withdrawal and escape responses. After a few minutes of adaptation, the animals exhibited no struggling on the platform. Pinches were applied with a pair of forceps to the tail at least twice immediately before and 20-30 set after PAG stimulation, and the behavioral response was rated by two observers according to a 3-point scale (complete inhibition = analgesia (2); attenuated response = hypalgesia (1); normal response = no~ogesia (0)). In addition to the escape response, other signs of distress, such as increased alertness, freezing, pilo-erection, respiratory acceleration, defecation, urination and vocalization, were also assessed. Analgesia was defined as a rating of more than 1 by both observers during PAG stimulation. In 7 of 13 animals stimulated, 6 of 12 unstimulated and in the 3 unimmunized rats, the numbers of antigen-specific IgM-PFC were investigated using direct techniques at day 4 following the injection of SRBC. In 6 stimulated and 6 unstimulated rats, numbers of both IgM- and IgG-PFC were counted by direct and indirect techniques at day 7. The procedures of plaque technique in microchambers were based on those reported by Cunningham and Szenberg 131.For plaque assay, all animals were anesthetized with ether and then killed by exsanguination of circulating blood by puncture of the heart to avoid conta~ation of autologous erythrocytes in the plaque assay. The spleens were removed and placed in Petri dishes containing ice-cold BSS. The spleens were teased with fine forceps. The suspension was centrifuged at 400 x g for 1 min at room temperature and then resuspended in BSS. Dilution was performed whenever necessary. The liquid assay mixture was prepared by adding 0.3 ml of 33% SRBC and 0.2 ml of guinea pig complement to 0.5 ml of a lymphoid cell suspension at various dilutions for the direct method. For the indirect method the assay mixture was prepared by adding 0.2 ml of 50% SRBC, 0.2 ml of guinea pig complement and 0.1 ml of IgG-fraction rabbit anti-rat IgG (Litton Bionetics, Kensington, MD) to the same volume of a lymphoid cell suspension. The microchamber was made of two microscope slides laid on two pieces of double-side tape. The volume of the ~cr~hamber was 150 f 5 ~1 (means f S.E.M.). The assay mixture of 0.15 ml containing 1 x lo4 spleen cells was pipetted into each chamber. After sealing the orifice of the slide chamber with cellophane tape, the preparation was incubated for 4 h at 37 o C. At respective dilutions, spleen samples were run in triplicate. The plaques were counted microscopically under 40-fold magnification within 4 h after the establishment of incubation. Statistical analyses were performed using Student’s 1 test for non-paired data. Differences between the mean values were considered to be significant when P values were less than 0.05.
ReSUltS
The results are summarized in Fig. 2. The numbers of IgM-PFC at day 4 after immunization in stimulated (n = 7) and unstimulated (n = 6) groups were 43.3 f 15.1
390
-” $
5
2
600
c m
Stimulated
0
Unslimulated
fl=6
500
“0 ;
400
a
’
300
$ J % LL
200
c” E
100
0
IgM Plaque
day
4
IgM Plaque IgG Plaque
day 7
Fig. 2. The effect of PAG stimulation on responses of rat spleen cell suspensions to antigen challenge. The number of PFC per lo4 spleen cells (mean I S.E.M.) was assayed at days 4 and 7 after immunization with SRBC. The number of facilitated plaques (IgG-PFC) was calculated as the total number of plaques developed with antiserum minus the direct plaques (IBM-PFC). There were no significant differences between the stimulated and unstimulated groups at all values.
(mean k S.E.M.) and 71.5 t- 23.8 per lo4 spleen cells, respectively. There was no significant difference in the mean numbers of IgM-PFC between the two groups. In the uni~u~z~ group (n = 3), the number of IgM-PFC at day 4 was 1.8 t_ 0.2 per lo4 spleen cells. At day 7, the numbers of IgG-PFC in the stimulated (n = 6) and unstimulated (n = 6) groups were 546.2 I 63.8 and 456.8 _t 25.5 respectively, per lo4 spleen cells, while the numbers of IgM-PFC were 300.9 rir 67.8 and 247.9 rf 84.2 respectively, per lo4 spleen cells. There was no significant difference in the mean numbers of IgM- as well as IgG-PFC at day 7 between the two groups. There was a sig~ficant difference in the numbers of IgM-PFC between day 4 and day 7 in both the stimulated and unstimulated groups (P < 0.05). On analgesia testing, 2 of 7 rats at day 4 of the experiment and 1 of 6 rats at day 7 of the experiment did not achieve SPA. However, none of the responses in the rats which demonstrated SPA differed significantly from those in the rats which did not achieve SPA.
Discussion
The present study demonstrated that short-term PAG stimulation did not affect the primary antibody responses in rats. In investigating antibody responses, it is necessary to estimate both the level of antibody and the number of antibody-for~ng cells. The Jerne-Nordin plaque assay [7] has become a standard technique for identifying antibody-producing cells. In this assay, each plaque represents one antibody-secreting cell. It is found that the direct
391
(IgM) and indirect (IgG) plaque-forming cells increase to a maximum at day 4 and at day 7 following immunization, respectively. These increases parallel the rise of serum IgM and IgG antibody titers [7]. The SRBC we used in the present study is one of the T-dependent (T-dep) antigens. According to several lines of evidence presented so far, the primary immune responses to T-dep antigens depend on both T and B cells which recognize the antigens. Due to the large amounts of SRBC used in the present study, the number of IgM-PFC at day 7 was thought to be larger than that at day 4 in both PAG-stimulate and unstimulated rats. In the present study, the primary antibody responses were augmented with prolongation of the response peak, probably resulting from injection of SRBC at a high dose for primary antigenic challenge. Although Simon et al. [13] demonstrated that a single short-term electrical stimulation of the midbrain increased the metastatic activity of cancer, they did not study cellular or humoral i~~ity. Further, Brechner et al. [2] found that the group which received PAG stimulation subsequent to prolactin-sensitive tumor implantation had a statistically higher incidence of enhanced tumor growth than a non-PAG stimulated control group. They suggested that enhanced tumor growth with PAG stimulation may be related to prolactin release in rats. Furthermore, Johnson et al. [8] found that &endorphin suppressed the in vitro antibody response using mouse spleen cells. Simon et al. [14] have subsequently shown that &endorphin administration into the nucleus of the raphe magnus facilitates metastatic tumor growth. Release of endogenous opiates has been shown to increase during mid-brain stimulation [1,6]. On the other hand, Fessler et al. [4] have recently reported that there is a possibility of elevated /%endorphin due to contrast medium infusion but not due to electrical brain stimulation. Nevertheless, these findings tend to suggest a certain relationship between electrical stimulation of the midbrain and enhanced tumor growth. However, concerning the mechanism of immunologic aspects of electrical brain stimulation, there has been no report except the recent investigation by Schanker et al. [12]. They have shown that acute midbrain SPA reduces the B cell percentage, but does not appear to have any other acute or chronic effects on i~une functions in humans. In the present experiment, we investigated the effect of short-term (35 ruin) PAG stimulation in the initial phase of antigen recognition in the primary antibody response after immunization. PAG stimulation for 35 min was found to be sufficient to elicit clinical analgesia in patients [1,6,12]. The effects of PAG simulation at various stages in the course of antibody production with long-term stimulation was not examined in the present study. Nevertheless, the present results have demonstrated that SPA applied for a short term does not affect cellular or humoral immune activity.
We thank Mr. Yukio Sato for excellent technical assistance and Dr. Satoshi Abe for preparing the histological specimens.
392
This study was supported by a grant-in-aid for special project research of selected intractable neurological disorders from the Ministry of Education, Science and Culture, Japan.
References 1 Akil, H., Richardson, D.E., Hughes, J. and Barchas, J.D., Enkephalin-like material elevated in ventricular cerebrospinal fluid of pain patients after analgesic focal stimulation, Science, 201 (1978) 463-465. 2 Brechner, T., Motyka, D. and Sherman, J., Growth enhancement of prolactin-sensitive mammary tumor by periaqueductal gray stimulation, Life Sci., 32 (1983) 525-530. 3 Cunningham, A.J. and Szenberg, A., Further improvements in the plaque technique for detecting single antibody-forming cells, Immunology, 14 (1968) 599-600. 4 Fessler, R.G., Brown, F.D., Rachlin, J.R. and Mullan, S., Elevated /3-endorphin in cerebrospinal fluid after electrical brain stimulation: artifact of contrast infusion?, Science, 224 (1984) 1017-1019. 5 Haffner, F.. Experimentelle Prlifung schmerzstillender Mittel, Dtsch. med. Wschr., 55 (1929) 731-733. 6 Hosobuchi, Y., Adams, J.E. and Linchitz, R., Pain relief by electrical stimulation of the central gray matter in humans and its reversal by naloxone, Science, 197 (1977) 183-186. 7 Jerne, N.K. and Nordin, A.A., Plaque formation in agar by single antibody-producing cells, Science. 140 (1963) 405. 8 Johnson, H.M., Smith, E.M., Torres, B.A. and Blalock, J.E.. Regulation of the in vitro response by neuroendocrine hormones, Proc. nat. Acad. Sci. (Wash.), 79 (1982) 4177-4174. 9 Mayer, D.J., Wholfle. T.L.. Akil. H., Carder, B. and Liebeskind, J.C.. Analgesia from electrical stimulation in the brainstem of the rat, Science, 174 (1971) 1351-1354. 10 Pellegrino, L.J., Pellegrino. A.S. and Cushman, A.J., A Stereotaxic Atlas of the Rat Brain, 2nd edn, Plenum Press, New York, 1979, pp. 61, 104-105. 11 Reynolds, D.V., Surgery in the rat during electrical analgesia induced by focal brain stimulation, Science, 164 (1969) 444445. 12 Schanker. H.M., Young, R., Cahan. L., Schroff, R. and Saxon. A.. Effect of midbrain stimulus-mduced analgesia on immune function in humans, J. Neuroimmunol., 5 (1983) 1855189. 13 Simon, R.H.. Lovett, III, E.J. and Joel Lundy, D.T., Electrical stimulation of the midbrain mediates metastatic tumor growth, Science, 209 (1980) 1132-1133. 14 Simon, R.H., Arbo. T.E. and Lundy, J.. Beta-endorphin injected into the nucleus of the raphe magnus facilitates metastatic tumor growth, Brain Res. Bull., 12 (1984) 487-941. 15 Skinner. J.E.. Neuroscience: a Laboratory Manual, Saunders. Toronto, 1971. pp. X7-144.