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Experimental allergic encephalomyelitis in adult DA rats subjected to neonatal handling or gentling
BRAIN RESEARCH ELSEVIER
Brain Research 676 (1995) 133-140
Research report
Experimental allergic encephalomyelitis in adult DA rats subjected to neonatal handling or gentling Olgica Laban a, Miljana Dimitrijevid a,., Stephan von Hoersten b, Branislav M. Markovi6 a, Branislav D. Jankovi6 a a Immunology Research Center, Vojvode Stepe 458, 11221 Belgrade, Yugoslavia b Medizinische Hochschule, Konstanty-Gutsehow-Str. 8, D-30623 Hannover, Germany
Accepted 27 December 1994
Abstract
The present study investigated the effect of daily handling and gentling between postnatal days 1 and 28 on experimental allergic encephalomyelitis (E~ME) in 8-week old DA rats. Handling consisted of removing pups from the mother, and placing them in the novel cage for 15 min. The gentling procedure included handling accompanied by 3 min of dorsal tactile stimulation before returning the pups to 1Ehenest cage. Adult rats of both sexes handled in infancy showed increased susceptibility to EAE, as revealed by higher incidence of the disease, and more severe clinical signs. Anti-myelin basic protein (MBP) autoantibodies were increased in handled males, and decreased in handled females, compared to controls. Gentling induced aggravation of clinical signs and histopathological lesions of EAE in males, while in gentled females suppression was observed. These results indicated that both neonatal handling and gentling aggravated EAE induced in adult male rats. In female rats handling exacerbated, and gentling suppressed clinical EAE. The overall effect of neonatal manipulations was more pronounced in males. Furthermore, in mothers separated from their offspring due to handling and gentling, and immunized for EAE at day 28 postpartum, earlier appearance of clinical signs, and increased frequency of relapses compared to control dams was recorded. Keywords: Handling; Gentling; Neonatal period; Maternal separation; Stress; Experimental allergic encephalomyelitis; DA rat
1. Introduction
The early postnatal period is the time of maturation of various systems, including the nervous, endocrine, and immune system which are involved in maintaining the homeostasis [2], and are shown to have interdependent ontogenesis [20]. Stimuli provided by the mother are essential for normal development of these systems [19,441. Neonatal handling is a procedure that involves removing the pup from the mother for 3-15 rain daily, and placing it into a novel ,environment. Even so subtle disruption of the mother-infant interaction has been shown to produce a variety of physiological and behavioral changes in the offspring, that endure throughout the life. Rats handled in infancy exhibited a less pronounced adrenocortical response to stress [36], de-
* Corresponding author. Fax: (381) (11) 467 465. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0006-8993(95)00106-9
creased fearfulness and increased locomotor activity in a novel environment [29,49], fewer errors in a learning task [28], shortened immobility in a swim test and reduced voluntary alcohol consumption [18] when tested as adults. The reports dealing with the influence of neonatal handling on immune response measured later in life, i.e. antibody production to sheep red blood cells [49] and flaggellin [45], and proliferation of mitogen-stimulated lymphocytes [32], described potentiation of immune reactivity. However, a decreased number of plaque-forming cells in rodents subjected to handling was also demonstrated [42,49]. The only experiment dealing with neonatal handling and autoimmune disease revealed no effect on adjuvant-induced arthritis [3]. So far, the effects of gentling, i.e. handling accompanied by manual tactile stimulation, on physiological functioning have been poorly investigated [50]. A similar procedure, involving tactile stimulation with a brush, revealed prevention of decrease of growth hormone
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O. Laban et aL /Brain Research 676 (1995) 133-140
levels due to maternal deprivation [44] and differentially affected open-field behavior with respect to the duration of tactile stimulation [25]. According to our knowledge, no data are available on the effect of neonatal gentling on the immune response. In this study, we examined the influence of these two neonatal manipulations on EAE. EAE is an autoimmune disease of the central nervous system (CNS) which is induced by injection of brain tissue in complete Freund's adjuvant. Immunization induces activation of autoreactive T and B lymphocytes specific for MBP. It is well established that T lymphocytes specific for brain antigens cross the blood-brain barrier, and induce lesions of the nervous tissue [51], whereas the role of anti-brain antibodies is not yet resolved [41]. Clinically, the disease is characterized by gradual development of paralyses of tail, hind- and forelimbs and spontaneous recovery after several days. Stress [5,6,13,14,23,30,31] and stress-related hormones [33], transmitters [27] and neuropeptides [21] may alter the development of EAE. Bearing in mind that early postnatal manipulations may induce changes in neuroendocrine and immune functions, this study was designed to evaluate whether two stressful procedures, i.e. handling and gentling, applied during the neonatal period may affect the autoimmune disease of CNS in adulthood. In addition, lactating mothers deprived from their offspring due to handling and gentling were tested for susceptibility to EAE after weaning. 2. Materials and methods
2.1. Animals Inbred Dark August (DA) rats (MHC haplotype RT-1 avl) from breeding colony at Immunology Research Center were used in experiments. Animals were kept in standard plexiglas cages, with free access to food and water, and under natural lighting conditions. Temperature of the colony room was maintained at 20-23°C. At the age of 10-12 weeks, virgin females were mated with age-matched males. Pregnant females were daily checked for delivery and the day of offspring birth was designated as day 0. On postnatal day 28, pups were weaned from their mothers, and housed undisturbed until the day of immunization in the samesex, same-treatment groups (4-5 animals per cage). Animals used in this study were obtained from 18 litters which consisted of 6-8 pups. Entire litters were randomly assigned to experimental and control groups. Neonatal manipulations were done between 11.00 and 12.00 h, during the same year season, and at about 25°C ambient temperature. All experimental procedures were done in the same colony room with controlled traffic of personnel.
2.2. Neonatal handling Animals were handled daily from postnatal day 1 until postnatal day 28. Handling consisted of removing the pups from their cage, placing all the pups from a litter together into a novel cage with a clean bedding, and 15 rain later returning the animals back in the nest cage. The mother was removed before, and returned after its pups. Control animals were left undisturbed during the whole preweaning period, except for cage cleaning.
2.3. Neonatal gentling Gentling consisted of placing the pups into a novel cage for 15 min daily between postnatal day 1 and postnatal day 28, as already described for the handling procedure, with one exception. Before placing the pups in the novel cage, they were held for 3 min in the palm of the experimenter's hand and the animal's back was caressed with the other hand from the head toward the base of the tail, at the rate of approximately 50 strokes per minute. Control pups remained undisturbed in their nest cages throughout this time, except for regular change of bedding.
2.4. Separation of mothers Mothers of the animals subjected to neonatal manipulations were removed from their offspring and kept in separate cages for 15 min daily, as already described, between day 1 and day 28 postpartum (day of weaning). Control mothers spent the whole preweaning period with their offspring.
2.5. Induction and evaluation of EAE Rats subjected to neonatal handling and neonatal gentling and corresponding control animals were sensitized for EAE at the age of 8 weeks. The separated mother group and control mothers were immunized with encephalitogen on the day of weaning of the offspring, i.e. at the age of 17-20 weeks. All animals were immunized intradermally in the left hind foot pad with guinea pig spinal cord homogenate (20 rag/rat) emulsified in complete Freund's adjuvant (0.3 mg of Mycobacterium tuberculosis/rat). Each rat received an additional subcutaneous injection of Bordetella pertussis vaccine (9 × 109 microorganisms/rat) in the dorsum of the same foot. Clinical signs of EAE were recorded daily on a blind basis by one observer and graded from 0 to 4 depending on the disease intensity: 0, no clinical signs; 1, flaccid tail; 2, weakness of hind limbs; 3, paralysis of hind limbs, and 4, quadriplegia and moribund state. The clinical score represents maximal individual clini-
o. Laban et al. / Brain Research 676 (1995) 133-140 cal signs. The aggregate clinical score represents the sum of clinical signs for each day when the animal was paralyzed. The clinical score and aggregate clinical score include both diseased and non-diseased animals. In handling and gentling experiments, sacrifice was performed after full recow~ry of all animals (days 17-18 after immunization). The cerebrum, cerebellum and spinal cord were dissected out, fixed in 10% buffered formalin and processed for staining with hematoxylin and eosin. Serial sections of the brain and spinal cord were examined for histopathological lesions which were scored as follows: 0, no histopathological lesions; 1, 1 - 4 lesions per section; 2, 5 - 9 lesions per section; 3, 10-14 lesions per section; and 4, > 14 lesions per section. Histological changes characteristic of E A E were presented as a mean score, an arithmetic score reflecting an overall severity of lesions (0-4).
2.6. Determination of anti-MBP antibodies Neonatally manipulated rats and their controls were bled by cardiac puncture iln chloroform anesthesia on the day of sacrifice. Mothers were bled from retroorbital sinus on days 21, 28 and 35 postimmunization. Serum levels of antibodies against rat MBP were determined by E L I S A as previously described [12]. Briefly, plates were coated with 1 / x g / m l of rat MBP (kindly provided by Dr. George Hashim, St. Luke's-Roosevelt Hospital, New York) and saturated with 1% bovine serum albumin solution. Serum samples were tested in 1 / 5 0 dilution. Peroxidase-conjugated anti-rat I g G (Sigma, St. Louis) was used as the secondary antibody. Optical density (OD) was measured in a flow multiscan p h o t o m e t e r at 492 nm.
2. 7. Statistical analyses Data are presented as means + S.E.M. The statistical package S T A T V I E W II was used to analyze the data. Since experimental design in handling and gentling included variations in two factors, T r e a t m e n t and Sex, data were analyzed using the parametric test, two-factorial analysis of variance (ANOVA), except for
Table 1 EAE in handled rats Group Incidence (%) Males Handled Controls Females Handled Controls
135
aggregate clinical scores when the M a n n - W h i t n e y test was used. In mothers, the parametric Student t-test and non-parametric M a n n - W h i t n e y test (for scores) were used. Handling and gentling were run as independent experiments and separate statistical analyses were performed for each experiment.
3. Results 3.1. Survival of pups and body weights in rats subjected to neonatal manipulations Neonatal manipulations did not influence the survival of pups. However, both handled and gentled animals were 5 - 1 0 % heavier then controls (data not shown) one day before senzitation for E A E (Factor T r e a t m e n t F1,53 = 31.14, P < 0.001, and F1,7o = 4.92, P < 0.05, respectively).
3.2. E A E in adult rats subjected to neonatal handling The disease was aggravated in animals subjected to neonatal handling (Table 1) as revealed by a higher incidence and higher clinical score in comparison with controls (Factor T r e a t m e n t F1,52 = 9.26, P < 0.01). The aggregate clinical score (Fig. 1) was significantly increased in handled males ( P < 0.05) but not in handled females, thus indicating that the effect was more pronounced in male rats. Neonatal handling did not affect the onset and the peak of the disease (Table 1). However, the clinical signs appeared earlier (Factor Sex F1,36 = 4.99, P < 0.05) and earlier reached the maximal values (Factor Sex F1,36 ~-" 4.48, P < 0.05) in female rats. The severity of histopathological lesions did not differ among groups.
3.3. E A E in adult rats subjected to neonatal gentling The effect of neonatal gentling on clinical signs of E A E depended on the sex. Neonatally gentled males exhibited an increased incidence of the disease and a
Day of onset (~ 5: S.E.M.)
Day of maximum of the disease (~ + S.E.M.)
Duration in days (~ _+S.E.M.)
Clinical score (~ :1:S.E.M.)
Histological lesions (~ :t: S.E.M.)
13/15 (87) 6/12 (50)
7.2 _ 0.2 a 8.2 _ 0.7
8.1 + 0.2 a 8.3 _+0.9
2.2 + 0.4 2.5 + 0.6
1.9 + 0.2 b 0.8 + 0.3
1.2 _+0.4 0.7 _+0.2
12/14 (86) 9/15 (60)
8.6 + 0.5 8.9 + 0.5
9.3 + 0.5 9.2 + 0.5
3.0 + 0.5 2.8 + 1.4
1.7 + 0.3 1.2 + 0.3
0.7 + 0.4 1.0 + 0.3
Statistically significant differences (P < 0.05): a Factor Sex, and b Factor Treatment.
O. Laban et al. / Brain Research 676 (1995) 133-140
136
was also higher ( P < 0.02) in g e n t l e d males c o m p a r e d to controls, while t h e r e were n o differences b e t w e e n female groups (Fig. 2). T h e severity of histopathological lesions (Table 2) was increased in g e n t l e d males a n d decreased in g e n t l e d females c o m p a r e d to corres p o n d i n g controls ( I n t e r a c t i o n T r e a t m e n t × Sex F1,67 = 4.51, P < 0.05). As in the h a n d l i n g e x p e r i m e n t , the onset (Factor Sex F1,44 = 13.03, P < 0,001) a n d the p e a k ( F a c t o r Sex F1,44 = 13.03, P < 0.05) of the disease a p p e a r e d earlier in females, b u t t h e r e were n o differences due to gentling.
Males
8
"15
6
2
8
10
12
14
16
Females
3.4. E A E in mothers subjected to separation from the offspring
P
"0 c
6
8
10
12
14
16
Days after immunization
•
HANDLED +
CONTROLS
M o t h e r s showed a high susceptibility to E A E induction. I n c i d e n c e of the disease a n d severity of clinical signs were n o t affected by daily s e p a r a t i o n s from the offspring d u r i n g the p r e w e a n i n g period, b u t clinical signs a p p e a r e d earlier in s e p a r a t e d m o t h e r s ( T a b l e 3; Fig. 3.). A f t e r the recovery from E A E , relapses were m o r e f r e q u e n t in the e x p e r i m e n t a l g r o u p in comparison to controls. T h e relapses, which a p p e a r e d 3 - 1 0 days after recovery from acute disease, m a i n l y repres e n t e d 2 - 4 days of flaccid tail. However, 3 o u t of 7 s e p a r a t e d dams with relapses exhibited paresis or paralysis of h i n d limbs d u r i n g this second attack.
Fig. 1. Development of EAE in male and female rats subjected to neonatal handling. The aggregate clinical score was higher in handled males compared to control males (P < 0.05). * Statistically significant difference, handled males vs. control males (P < 0.05), in mean clinical score on days 7 and 8 postimmunization.
3.5. Anti-MBP antibodies
higher clinical score (Table 2) c o m p a r e d to control males a n d g e n t l e d females ( I n t e r a c t i o n T r e a t m e n t × Sex, F1,70 = 6.61, P < 0.02). T h e aggregate clinical score
Fig. 4 shows the s e r u m a n t i - M B P a n t i b o d y levels in a n i m a l s subjected to n e o n a t a l m a n i p u l a t i o n s . I n h a n dled males a n t i b o d y levels were increased, a n d in handled females decreased, c o m p a r e d to c o r r e s p o n d i n g
Table 2 EAE in gentled rats Group
Males Gentled Controls Females Gentled Controls
Incidence (%)
Day of onset (,~ + S.E.M.)
Day of m a x i m u m of the disease (~ __.S.E.M.)
Duration in days (~ + S,E.M.)
Clinical score (~ -t- S.E,M.)
Histological lesions (~ + S.E.M.)
15/19 (79) 10/19 (53)
6.5 + 0.1 a 6.7 + 0.2
7.4 + 0.3 a 7.2 ± 0.4
3.6 + 0.5 2.9 ± 0.4
2.1 _+0.3 b 1.1 + 0.3
1.9 J: 0.3 b 1.4 + 0.3
10/17 (59) 13/19 (68)
7.4 + 0.5 8.5 + 0.5
7.9 _+0.5 9.2 _+0.6
3.6 + 0.5 3.2 + 0.3
1.1 + 0.3 1.6 + 0.3
1.1 + 0.3 1.8 + 0.3
Statistically significant differences (P < 0.05): a Factor Sex, and b Interaction Treatment x Sex.
Table 3 EAE in adult female rats separated from their offspring Group
Incidence (%)
Separated from offspring Controls
12/12 (100) 11/12 (92)
a Statistically significant difference, P < 0.05.
Day of onset (~ _+S.E.M.) 7.7 J: 0.3 a 9.1 ± 0.4
Day of maximum of the disease (~ _+S.E.M.) 9.3 + 0.4 10.2 + 0.4
Duration in days (~ _+S.E.M.) 4.7 + 0.4 4.7 + 0.4
Mean clinical score (,~+ S.E.M.) 2.9 + 0.1 2.7 + 0.3
Frequency of relapses (%) 58 17
O. Laban et al. / Brain Research 676 (1995) 133-140 2 ¸
Males
600
*
137
(a)
500
I eq 400
/i
o 300 o
"0
x 200 t~ O
|
100
MALES
6
2
8
10
12
14
16
18
FEMALES
[ ] CONTROLS [ ] HANDLED
Females
600
(b)
500 <~ 400 o~
o 300
"0
o
x 200 O I
I
8
I
I
8
I -
I
I
10
I
GENTLED
12
~
W
100
I
14
16
18
MALES
Days after immunization ~ - -
'j
I
FEMALES
[ ] CONTROLS [ ] GENTLED
Fig. 4. Serum anti-MBP antibody level in handled and gentled rats.
CONTROLS
Fig. 2. Development of E A E in raale and female rats subjected to neonatal gentling. The aggregate clinical score was higher in gentled males compared to control males ( P < 0.02). * Statistically significant difference, gentled males vs. control males ( P < 0.05), in mean clinical score on days 6, 7 and 8 postimmunization.
controls (Interaction Treatment × Sex F1,51 = 6.13, P < 0.02). Gentling did not influence anti-MBP antibody production. There was no difference in anti-MBP anti-
body synthesis between separated and control mothers (Fig. 5).
4. Discussion
The present study showed that handling and gentling from birth until weaning aggravated clinical EAE in adulthood, and that this effect was more pro-
800 700 C
600
*
E ~ 500 "
"
o400 x 300
0
--~-
6
8
I0
12
14
16
18
20
22
24
Days after immunization ~---
SEPARATED
--o---
CONTROLS
Fig. 3. Development of E A E in mothers separated from their offspring during the preweaning period. There were no differences in the aggregate clinical score between separated and control mothers. • Statistically significant difference, separated mothers vs. control mothers ( P < 0.05), in mean clinical score on days 7 and 8 postimmunization.
0 200 100 i
21
q
t
i
28
i
3,5
after immunization •
SEPARATED ~
CONTROLS
Fig. 5. Serum anti-MBP antibody level in mothers separated from their offspring during the preweaning period.
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O. Laban et al. / Brain Research 676 (1995) 133-140
nounced in male rats. This finding is in agreement with previous reports of immunopotentiation due to early handling [32,45,49]. Histopathological lesions of EAE exhibited a similar pattern as clinical disease, but the differences between experimental and control animals were only statistically significant in the gentled groups. Another dissimilarity between handling and gentling was observed in the effect on anti-MBP antibody production. In handled males levels of these autoantibodies were increased. Although both procedures exerted a similar potentiation of clinical disease in males, it appears that handling and gentling affected mainly the humoral and cell-mediated autoimmune response, respectively. Increased anti-MBP antibody levels in handled males may be responsible for aggravation of EAE, since it was suggested that these antibodies are involved in the demyelinizating process in the CNS [48]. The pathogenic role of autoreactive CD4 ÷ T lymphocytes present in histopathological lesions of EAE is well documented [4,47]. Enhanced reactivity of T helper lymphocytes a n d / o r T cytotoxic lymphocytes specific for autoantigen could explain the potentiation of EAE in gentled animals. Thus, we may hypothesize that different mechanisms were responsible for the observed similar effect of handling and gentling on clinical disease. The effects of early handling and gentling on EAE in adults may be ascribed to alterations in the development of immune a n d / o r neuroendocrine systems. Since MBP appears late in ontogenesis and the maximum rate of myelinization in CNS is achieved during the neonatal period [40], it is possible that neonatal manipulations may have influenced the establishment of tolerance to MBP and thus affected EAE later in life. Environmental changes during the period of maturation of the adrenocortical system [43] may alter the development of this system. It was demonstrated that rats subjected to early handling exhibit a lower and shorter increase in glucocorticoids than non-handled animals after exposure to acute stress procedures in adulthood [35]. Lower circulating levels of corticosterone due to handling [36] may favor the escape of autoreactive T cells at critical times during development [8], thus resulting in increased susceptibility to autoimmune diseases later in life. Furthermore, a rise of glucocorticoids in EAE, due to production of cytokines and stress of paralyses, was reported to be important for the recovery from the disease [30,33]. Glucocorticoids are supposed to be involved in the recovery from EAE by suppression of the cellular immune response through a shift of T cell response from the Thl type to the Th2 type [52]. It may be hypothesized that handled rats did not mount an adequate corticosterone response during EAE, thus exhibiting a more severe disease when compared to non-
handled controls. There are no reports on the effect of gentling on the maturation of the adrenocortical system, but the discrepancy between the effects of handling and gentling on EAE support the view that corticosterone production is not the only mechanism responsible for the results obtained in our study. In accordance with this assumption is the recent report of restraint-induced suppression of clinical signs of EAE accompanied by lowered levels of corticosterone, reduced frequency of MBP-reactive lymph node cells and decreased interleukin-2 and interferon 3' production [13]. Besides glucocorticoids, growth hormones could also be involved. The acute decrease in serum levels of growth hormones in rats deprived from their mother for several hours may be rendered to normal by dorsal tactile stimulation of pups [44]. Unfortunately, there are no data on the effect of tactile stimulation alone on serum hormone levels. Furthermore, in rats deprived from their mother for 15 min no decrease, but some tendency toward enhancement of growth hormone release was noticed. Increased body weight in handled and gentled animals subjected to daily repeated, shortlasting separations from the mother during the entire neonatal period may be due to enhanced secretion of growth hormone. Since growth hormone is necessary for normal development of the immune system [9] and has an immuno-enhancing effect [22], the possibility of its involvement in potentiation of EAE should not be neglected. Our results pointed out to sex-differences in the effect of neonatal manipulations on the development of EAE. The effect of handling and gentling on clinical and histopathological characteristics of EAE were more pronounced in male rats. As for the production of anti-MBP antibodies, potentiation in males and suppression in females were observed. In addition, the disease appeared earlier in experimental and control male groups. Gender differences in the effect of handling on corticosterone levels [37] and humoral immune response [49] in adult rats were described. Furthermore, it was shown that neonatal handling may emphasize neuroanatomical sex-differences [10]. The differential effect of handling on susceptibility to EAE in males and females may be due to gender differences in baseline hormone production [15], immune reactivity [17] and response to stress [16]. Apart from the sex, another factor that influenced the effect of neonatal stress on EAE was the strain of rats. Namely, neonatal sound stress aggravated EAE in adult Lewis rats, and did not affect the disease in DA rats [11]. In the present study DA rats were used since our preliminary experiments, carried out in both DA and Lewis rats, revealed a more pronounced potentiating effect of the gentling procedure on EAE in DA rats. This was probably due to the fact that Lewis rats in the control group developed a high incidence of the
O. Laban et al. / Brain Research 676 (1995) 133-140
disease (92-100%) and severe clinical EAE (mean score 2.6-2.9), so further aggravation was hard to obtain. Susceptibility to EAE markedly varies among inbred rat strains and was suggested to be determined by both MHC and non-MHC genes, the latter being responsible for neuroendocrine-immune interactions. Both DA and Lewis rats after sensitization with encephalitogen develop EAE with high incidence and severity. High susceptibility to the disease in these two strains has been partly ascribed to a higher interferon-y-induced Ia expression on brain endothelium in comparison to EAE-resistant rats [34]. However, it seems that in DA rats susceptibility to EAE develops with age, since the incidence of EAE at the age of 5 to 8 weeks is 50-81%, compared to 90-.100% at the age of 12 weeks or more (unpublished observations). Although it is generally accepted that immune [39] and adrenocortical systems [43] fully develop by the age of 8 weeks, it may be speculated that in the DA strain immunological a n d / o r neuroendocrine alterations, which are most probably responsible for high susceptibility to EAE, progress with age. Both procedures used in this experiment included removal of the mother from its pups. During the experiment we noticed that the mothers were disturbed due to this short-lasting disruption of contact with their offspring, i.e. they spent time in the new cage looking for their pups. In monkeys, immune alterations in mothers due to separation from their offspring wele demonstrated [7,26], while for rodents there are no data available. We report here on earlier appearance of clinical EAE, and increased frequency of relapses in rat dams separated from their progeny. Previous investigations showed that EAE may be suppressed [6,14,23,30,31] and enhanced [5] by stress applied after immunization, but this is the first report on the modulatory effect of stress applied in adults before immunization with encephalitogen. The effect of stressful procedures applied during the early postnatal period depended on the type of stressor and immune function examined. More drastic procedures such as prolonged maternal deprivation [38,49], early weaning [1], and restraint [46] had a suppressive effect on humoral and cellular immune responses. Handling appears to be the procedure in which the potentiating effect on imnmne reactivity was observed [32,45,49]. However, the results obtained in our laboratory showed that neonatal sound stress in Lewis rats [11] and early weaning i~: DA rats aggravated EAE, while maternal deprivation reduced susceptibility to EAE in adult DA rats [24]. In the present study we further extend our findings on the effect of early experiences on autoimmunity induced in adults, showing that handling and gentling increased susceptibility to EAE in DA strain of rats. Thus, several factors, such
139
as treatment procedure, sex, and strain, may influence the effect of neonatal manipulations on EAE. Bearing in mind that EAE is an animal model for multiple sclerosis, it may be of interest to investigate whether early life events can predispose an individual to autoimmune diseases later in life.
Acknowledgements The present work was supported by the Ministry for Science and Technology, Belgrade.
References [1] Ackerman, S.H., Keller, S.E., Schleifer, S.J., Shindledecker, R.D., Camerino, M., Hofer, M.A., Weiner, H. and Stein, M., Premature maternal separation and lymphocyte function, Brain Behav. Immunol., 2 (1988) 161-165. [2] Ader, R., Developmental psychoneuroimmunology, Dev. Psychobiol., 16 (1983) 251-267. [3] Amkraut, A.A., Solomon, G.F. and Kraemer, H., Stress, early experience and adjuvant-induced arthritis in the rat, Psychosom. Med., 33 (1971) 203-214. [4] Ben-Nun, A., Wekerle, H. and Cohen, I.R., The rapid isolation of clonable antigen-specific T lymphocyte lines capable of mediating autoimmune encephalomyelitis, Eur. J. lmmunol., 11 (1981) 195-199. [5] Bernard, C.C.A., Grgacic, E. and Singer, G., The effect of stress, corticosterone and adrenalectomy on the development of experimental autoimmune encephalomyelitis, J. Neuroimmunol., 17 (1988) 254. [6] Bukilica, M., Djordjevi6, S., Mari6, I., Dimitrijevi6, M., Markovi6, B.M. and Jankovi6, B.D., Stress-induced suppression of experimental allergic encephalomyelitis in the rat, Int. J. Neurosci., 59 (1991) 167-175. [7] Coe, C.L., Rosenberg, L.T. and Levine, S., Prolonged effect of psychological disturbance on macrophage chemiluminiscence in the squirrel monkey, Brain. Behav. Irnmunol., 2 (1988) 151-160. [8] Craddock, C.G., Corticosteroid-induced lymphopenia, immunosuppression, and body defence, Ann. Intern. Med., 88 (1978) 564-566. [9] Denckla, W.D., Interactions between age and the neuroendocrine and immune system, FASEB J., 37 (1978) 1263-1267. [10] Denenberg, V.H., Fitch, R.H., Schrott, L.M., Cowell, P.E. and Waters, N.S., Corpus callosum: interactive effects of infantile handling and testosterone in rat, Behav. Neurosci., 105 (1991) 562-566. [11] Dimitrijevi6, M., Laban, O., von Hoersten, S., Markovi6, B.M. and Jankovi6, B.D., Neonatal sound stress and development of experimental allergic encephalomyelitis in Lewis and DA rats, Int. J. Neurosci., 78 (1994) 135-143. [12] Djordjevi~, S., Bukilica, M., Dimitrijevi~, M., Markovi6, B.M. and Jankovi6, B.D., Anti-myelin basic protein autoantibodies in rats stressed by overcrowding, Ann. N.Y. Acad. Sci., 650 (1992) 302-306. [13] Griffin, A.C., Lo, D.W., Wolny, A.C. and Whitacre, C.C., Suppression of experimental autoimmune encephalomyelitis by restraint stress: sex differences, J. Neuroimrnunol., 44 (1993) 103116. [14] Griffin, A.C. and Whitacre, C.C., Differential effects of stress on disease outcome in experimental autoimmune encephalomyelitis, FASEB J., 4 (1990) A2045.
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[15] Griffin, A. and Whitacre, C., Sex and strain differences in the circadian rhytm fluctuation of endocrine and immune functions in the rat: implications for rodent models of autoimmune disease, J. Neuroimmunol., 35 (1991) 53-64. [16] Griffin, A. and Whitacre, C., Sex differences in stress effects on disease course in experimental autoimmune encephalomyelitis (EAE), FASEB J., 5 (1991) 5870. [17] Grossman, C.J., Are there underlying immune-neuroendocrine interactions responsible for immunological sexual dimorphism?, Prog. NeuroEndocrinlmmunol., 3 (1990) 75-82. [18] Hilakivi-Clarke, L.A., Turkka, J., Lister, R.G. and Linnoila, M., Effects of early postnatal handling on brain /3-adrenoreceptors and behavior in tests related to stress, Brain Res., 542 (1991) 286-292. [19] Hofer, M.A., Relationships as regulators: a psychobiologic perspective on bereavement, Psychosom. Med., 46 (1984) 183-197. [20] Jankovi6, B.D., Isakovid, K., Mi6i~, M. and Kne~evi6, Z., The embryonic lympho-neuro-endocrine relationship, Clin. lmmunol. Immunopathol., 18 (1981) 108-120. [21] Jankovi6, B.D. and Mari6, D., Enkephalins and autoimmunity: differential effect of methionine-enkephalin on experimental allergic encephalomyelitis in Wistar and Lewis rats, J. Neurosci. Res., 18 (1987) 88-94. [22] Kelley, K.W., The role of growth hormone in modulation of the immune response, Ann. N.Y. Acad. Sci., 594 (1990) 95-103. [23] Kuroda, Y., Mori, T. and Hori, T., Restraint stress suppresses experimental allergic encephalomyelitis in Lewis rats, Brain. Res. Bull., 34 (1994) 15-17. [24] Laban, O., Markovid, B.M., Dimitrijevi6, M. and Jankovi6, B.D., Maternal deprivation and early weaning modulate experimental allergic encephalomyelitis in the rat, Brain, Behav. Immun., in press. [25] LaBarba, R.C., Fernandez, B., White, J.L. and Stewart, A., The effects of neonatal tactile stimulation on adult emotional reactivity in BALB/c mice, Dev. Psychobiol., 7 (1974) 393-398. [26] Laudenslager, M., Reite, M. and Harbeck, R., Immune status during mother-infant separation, Psychosom. Med., 44 (1982) 303. [27] Leonard, J.P., MacKenzie, F.J., Patel, H.A. and Cuzner, M.L., Hypothalamic noradrenergic pathways exert an influence on neuroendocrine and clinical status in experimental autoimmune encephalomyelitis, Brain Behav. Imrnun., 5 (1991) 328-338. [28] Levine, S., A further study of infantile handling and adult avoidance learning, J. Pers., 25 (1956) 70-80. [29] Levine, S., Haltmeyer, G.C., Karas, G. and Denenberg, V., Physiological and behavioral effects of infantile stimulation, Physiol. Behav., 2 (1967) 55-59. [30] Levine, S. and Saltzman A., Nonspecific stress prevents relapses of experimental allergic encephalomyelitis in rats, Brain. Behav. Immunol., 1 (1987) 336-341. [31] Levine, S., Strebel, R., Wenk, E.J. and Harman, P.J., Suppression of experimental allergic encephalomyelitis by stress, Proc. Soc. Exp. Biol. Med., 109 (1962) 294-298. [32] Lown, B.A and Dutka, M.E., Early handling enhances mitogen responses of splenic cells in adult C3H Mice, Brain Behav. Immunol., 1 (1987) 356-360. [33] MacPhee, I.A.M., Antoni, F.A. and Mason, D.W., Spontaneous recovery of rats from experimental allergic encephalomielitis is dependent on regulation of the immune system by endogenous adrenal corticoids, J. Exp. Med., 169 (1989) 431-445. [34] Male, D. and Pryce, G., Induction of Ia molecules on brain endothelium is related to susceptibility to experimental allergic encephalomyelitis, J. Neuroirnmunol., 21 (1989) 87-90.
[35] Meaney, M.J., Aitken, D.H., Bodnoff, S.R., Iny, L.J. and Sapolsky, R.M., The effects of postnatal handling on the development of the glucocorticoid receptor systems and stress recovery in the rat, Prog. Neuropsychopharmacol. Biol. Psychiat., 7 (1985) 731734. [36] Meaney, M.J., Aitken, D.H., Viau, V., Sharma, S. and Sarrieau, A., Neonatal handling alters adrenocortical negative feed-back sensitivity and hippocampal type II glucocorticoid receptor binding in the rat, Neuroendocrinology, 50 (1989) 597-604. [37] Meaney, M.J., Viau, V., Bhatangar, S., Betito, K., Iny, L.J., O'Donnell, D. and Mitchell, J.B., Cellular mechanisms underlying the development and expression of individual differences in the hypothalamic-pituitary-adrenal stress response, Steroid Biochem. Mol. Biol., 39 (1991) 265-274. [38] Michaut, R.J., Dechambre, R.P., Doumerc, S., Lesourd, B., Devillechabrolle, A. and Moulais, R., Influence of early maternal deprivation on adult humoral immune response in mice, Physiol. Behav., 26 (1981) 189-191. [39] Murgita, R.A. and Wigzell, H., Regulation of immune response functions in the fetus and newborn, Prog. Allergy, 29 (1981) 54-133. [40] Norton, W.T. and Poduslo, S.E., Myelination in rat brain: changes in myelin composition during brain maturation, J. Neurochem., 21 (1973) 759-773. [41] Paterson, P.Y. and Halberg, M.K., Antineural antibodies in experimental allergic encephalomyelitis. In E.J. Goetzl and N.H. Spector (Eds.), Neuroimmune Networks: Physiology and Diseases, Alan R. Liss, Inc., New York, 1989, pp. 251-257. [42] Raymond, L.N., Reyes, E., Tokuda, S. and Jones, B.C., Differential immune response in two handled inbred strains of mice, Physiol. Behav., 37 (1986) 295-297. [43] Sapolsky, R.M. and Meaney, M.J., Maturation of adrenocortical stress response: neuroendocrine control mechanisms and the stress hyporesponsive period, Brain Res. Rev., 11 (1986) 65-76. [44] Schanberg, S.M., Evoniuk, G. and Kuhn, C.M., Tactile and nutritional aspects of maternal care: specific regulators of neuroendocrine function and cellular development, Proc. Soc, Exp. Biol. Med., 175 (1984) 135-146. [45] Solomon, G.F., Levine, S. and Kraft, J.K., Early experience and immunity, Nature, 220 (1968) 821-822. [46] Taylor, C.E. and Ross, L.L., Alteration of antibody response to Pneumococcal polysaccharide Type III in rats by neonatal immobilization stress, Brain Behav. Immunol., 3 (1989) 160-165. [47] Traugott, U., McFarlin, D.E. and Raine, C.S., Immunopathology of the lesions in chronic relapsing experimental autoimmune encephalomyelitis in the mouse, Cell. Immunol., 99 (1986) 395410. [48] Varitek, V.A. and Day, E.D., Studies of rat antibodies specific for myelin basic protein (MBP). Antibody-dependent cell-mediated lysis of MBP-sensitized targets in vitro, J. Neuroimmunol., 1 (1981) 27-39. [49] von Hoersten, S., Dimitrijevi~, M., Markovi6, B.M. and Jankovi6, B.D., Effect of early experience on behavior and immune response in the rat, Physiol. Behav., 54 (1993) 931-940. [50] Weininger, O., Mortality of albino rats under stress as a function of early handling, Can. Jour. Psych., 7 (1953) 111-114. [51] Wekerle, H., Linington, C., Lassmann, H. and Meyermann, R., Cellular immune reactivity within the CNS, Trends Neurosci., 9 (1986) 271-277. [52] Wick, G., Hu, Y., Schwartz, S. and Kroemer, G., Immunoendocrine communication via the hypothalamo-pituitary-adrenal axis in autoimmune diseases, Endocr. Rev., 14 (1993) 539-563.
Brain Research 676 (1995) 133-140
Research report
Experimental allergic encephalomyelitis in adult DA rats subjected to neonatal handling or gentling Olgica Laban a, Miljana Dimitrijevid a,., Stephan von Hoersten b, Branislav M. Markovi6 a, Branislav D. Jankovi6 a a Immunology Research Center, Vojvode Stepe 458, 11221 Belgrade, Yugoslavia b Medizinische Hochschule, Konstanty-Gutsehow-Str. 8, D-30623 Hannover, Germany
Accepted 27 December 1994
Abstract
The present study investigated the effect of daily handling and gentling between postnatal days 1 and 28 on experimental allergic encephalomyelitis (E~ME) in 8-week old DA rats. Handling consisted of removing pups from the mother, and placing them in the novel cage for 15 min. The gentling procedure included handling accompanied by 3 min of dorsal tactile stimulation before returning the pups to 1Ehenest cage. Adult rats of both sexes handled in infancy showed increased susceptibility to EAE, as revealed by higher incidence of the disease, and more severe clinical signs. Anti-myelin basic protein (MBP) autoantibodies were increased in handled males, and decreased in handled females, compared to controls. Gentling induced aggravation of clinical signs and histopathological lesions of EAE in males, while in gentled females suppression was observed. These results indicated that both neonatal handling and gentling aggravated EAE induced in adult male rats. In female rats handling exacerbated, and gentling suppressed clinical EAE. The overall effect of neonatal manipulations was more pronounced in males. Furthermore, in mothers separated from their offspring due to handling and gentling, and immunized for EAE at day 28 postpartum, earlier appearance of clinical signs, and increased frequency of relapses compared to control dams was recorded. Keywords: Handling; Gentling; Neonatal period; Maternal separation; Stress; Experimental allergic encephalomyelitis; DA rat
1. Introduction
The early postnatal period is the time of maturation of various systems, including the nervous, endocrine, and immune system which are involved in maintaining the homeostasis [2], and are shown to have interdependent ontogenesis [20]. Stimuli provided by the mother are essential for normal development of these systems [19,441. Neonatal handling is a procedure that involves removing the pup from the mother for 3-15 rain daily, and placing it into a novel ,environment. Even so subtle disruption of the mother-infant interaction has been shown to produce a variety of physiological and behavioral changes in the offspring, that endure throughout the life. Rats handled in infancy exhibited a less pronounced adrenocortical response to stress [36], de-
* Corresponding author. Fax: (381) (11) 467 465. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0006-8993(95)00106-9
creased fearfulness and increased locomotor activity in a novel environment [29,49], fewer errors in a learning task [28], shortened immobility in a swim test and reduced voluntary alcohol consumption [18] when tested as adults. The reports dealing with the influence of neonatal handling on immune response measured later in life, i.e. antibody production to sheep red blood cells [49] and flaggellin [45], and proliferation of mitogen-stimulated lymphocytes [32], described potentiation of immune reactivity. However, a decreased number of plaque-forming cells in rodents subjected to handling was also demonstrated [42,49]. The only experiment dealing with neonatal handling and autoimmune disease revealed no effect on adjuvant-induced arthritis [3]. So far, the effects of gentling, i.e. handling accompanied by manual tactile stimulation, on physiological functioning have been poorly investigated [50]. A similar procedure, involving tactile stimulation with a brush, revealed prevention of decrease of growth hormone
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O. Laban et aL /Brain Research 676 (1995) 133-140
levels due to maternal deprivation [44] and differentially affected open-field behavior with respect to the duration of tactile stimulation [25]. According to our knowledge, no data are available on the effect of neonatal gentling on the immune response. In this study, we examined the influence of these two neonatal manipulations on EAE. EAE is an autoimmune disease of the central nervous system (CNS) which is induced by injection of brain tissue in complete Freund's adjuvant. Immunization induces activation of autoreactive T and B lymphocytes specific for MBP. It is well established that T lymphocytes specific for brain antigens cross the blood-brain barrier, and induce lesions of the nervous tissue [51], whereas the role of anti-brain antibodies is not yet resolved [41]. Clinically, the disease is characterized by gradual development of paralyses of tail, hind- and forelimbs and spontaneous recovery after several days. Stress [5,6,13,14,23,30,31] and stress-related hormones [33], transmitters [27] and neuropeptides [21] may alter the development of EAE. Bearing in mind that early postnatal manipulations may induce changes in neuroendocrine and immune functions, this study was designed to evaluate whether two stressful procedures, i.e. handling and gentling, applied during the neonatal period may affect the autoimmune disease of CNS in adulthood. In addition, lactating mothers deprived from their offspring due to handling and gentling were tested for susceptibility to EAE after weaning. 2. Materials and methods
2.1. Animals Inbred Dark August (DA) rats (MHC haplotype RT-1 avl) from breeding colony at Immunology Research Center were used in experiments. Animals were kept in standard plexiglas cages, with free access to food and water, and under natural lighting conditions. Temperature of the colony room was maintained at 20-23°C. At the age of 10-12 weeks, virgin females were mated with age-matched males. Pregnant females were daily checked for delivery and the day of offspring birth was designated as day 0. On postnatal day 28, pups were weaned from their mothers, and housed undisturbed until the day of immunization in the samesex, same-treatment groups (4-5 animals per cage). Animals used in this study were obtained from 18 litters which consisted of 6-8 pups. Entire litters were randomly assigned to experimental and control groups. Neonatal manipulations were done between 11.00 and 12.00 h, during the same year season, and at about 25°C ambient temperature. All experimental procedures were done in the same colony room with controlled traffic of personnel.
2.2. Neonatal handling Animals were handled daily from postnatal day 1 until postnatal day 28. Handling consisted of removing the pups from their cage, placing all the pups from a litter together into a novel cage with a clean bedding, and 15 rain later returning the animals back in the nest cage. The mother was removed before, and returned after its pups. Control animals were left undisturbed during the whole preweaning period, except for cage cleaning.
2.3. Neonatal gentling Gentling consisted of placing the pups into a novel cage for 15 min daily between postnatal day 1 and postnatal day 28, as already described for the handling procedure, with one exception. Before placing the pups in the novel cage, they were held for 3 min in the palm of the experimenter's hand and the animal's back was caressed with the other hand from the head toward the base of the tail, at the rate of approximately 50 strokes per minute. Control pups remained undisturbed in their nest cages throughout this time, except for regular change of bedding.
2.4. Separation of mothers Mothers of the animals subjected to neonatal manipulations were removed from their offspring and kept in separate cages for 15 min daily, as already described, between day 1 and day 28 postpartum (day of weaning). Control mothers spent the whole preweaning period with their offspring.
2.5. Induction and evaluation of EAE Rats subjected to neonatal handling and neonatal gentling and corresponding control animals were sensitized for EAE at the age of 8 weeks. The separated mother group and control mothers were immunized with encephalitogen on the day of weaning of the offspring, i.e. at the age of 17-20 weeks. All animals were immunized intradermally in the left hind foot pad with guinea pig spinal cord homogenate (20 rag/rat) emulsified in complete Freund's adjuvant (0.3 mg of Mycobacterium tuberculosis/rat). Each rat received an additional subcutaneous injection of Bordetella pertussis vaccine (9 × 109 microorganisms/rat) in the dorsum of the same foot. Clinical signs of EAE were recorded daily on a blind basis by one observer and graded from 0 to 4 depending on the disease intensity: 0, no clinical signs; 1, flaccid tail; 2, weakness of hind limbs; 3, paralysis of hind limbs, and 4, quadriplegia and moribund state. The clinical score represents maximal individual clini-
o. Laban et al. / Brain Research 676 (1995) 133-140 cal signs. The aggregate clinical score represents the sum of clinical signs for each day when the animal was paralyzed. The clinical score and aggregate clinical score include both diseased and non-diseased animals. In handling and gentling experiments, sacrifice was performed after full recow~ry of all animals (days 17-18 after immunization). The cerebrum, cerebellum and spinal cord were dissected out, fixed in 10% buffered formalin and processed for staining with hematoxylin and eosin. Serial sections of the brain and spinal cord were examined for histopathological lesions which were scored as follows: 0, no histopathological lesions; 1, 1 - 4 lesions per section; 2, 5 - 9 lesions per section; 3, 10-14 lesions per section; and 4, > 14 lesions per section. Histological changes characteristic of E A E were presented as a mean score, an arithmetic score reflecting an overall severity of lesions (0-4).
2.6. Determination of anti-MBP antibodies Neonatally manipulated rats and their controls were bled by cardiac puncture iln chloroform anesthesia on the day of sacrifice. Mothers were bled from retroorbital sinus on days 21, 28 and 35 postimmunization. Serum levels of antibodies against rat MBP were determined by E L I S A as previously described [12]. Briefly, plates were coated with 1 / x g / m l of rat MBP (kindly provided by Dr. George Hashim, St. Luke's-Roosevelt Hospital, New York) and saturated with 1% bovine serum albumin solution. Serum samples were tested in 1 / 5 0 dilution. Peroxidase-conjugated anti-rat I g G (Sigma, St. Louis) was used as the secondary antibody. Optical density (OD) was measured in a flow multiscan p h o t o m e t e r at 492 nm.
2. 7. Statistical analyses Data are presented as means + S.E.M. The statistical package S T A T V I E W II was used to analyze the data. Since experimental design in handling and gentling included variations in two factors, T r e a t m e n t and Sex, data were analyzed using the parametric test, two-factorial analysis of variance (ANOVA), except for
Table 1 EAE in handled rats Group Incidence (%) Males Handled Controls Females Handled Controls
135
aggregate clinical scores when the M a n n - W h i t n e y test was used. In mothers, the parametric Student t-test and non-parametric M a n n - W h i t n e y test (for scores) were used. Handling and gentling were run as independent experiments and separate statistical analyses were performed for each experiment.
3. Results 3.1. Survival of pups and body weights in rats subjected to neonatal manipulations Neonatal manipulations did not influence the survival of pups. However, both handled and gentled animals were 5 - 1 0 % heavier then controls (data not shown) one day before senzitation for E A E (Factor T r e a t m e n t F1,53 = 31.14, P < 0.001, and F1,7o = 4.92, P < 0.05, respectively).
3.2. E A E in adult rats subjected to neonatal handling The disease was aggravated in animals subjected to neonatal handling (Table 1) as revealed by a higher incidence and higher clinical score in comparison with controls (Factor T r e a t m e n t F1,52 = 9.26, P < 0.01). The aggregate clinical score (Fig. 1) was significantly increased in handled males ( P < 0.05) but not in handled females, thus indicating that the effect was more pronounced in male rats. Neonatal handling did not affect the onset and the peak of the disease (Table 1). However, the clinical signs appeared earlier (Factor Sex F1,36 = 4.99, P < 0.05) and earlier reached the maximal values (Factor Sex F1,36 ~-" 4.48, P < 0.05) in female rats. The severity of histopathological lesions did not differ among groups.
3.3. E A E in adult rats subjected to neonatal gentling The effect of neonatal gentling on clinical signs of E A E depended on the sex. Neonatally gentled males exhibited an increased incidence of the disease and a
Day of onset (~ 5: S.E.M.)
Day of maximum of the disease (~ + S.E.M.)
Duration in days (~ _+S.E.M.)
Clinical score (~ :1:S.E.M.)
Histological lesions (~ :t: S.E.M.)
13/15 (87) 6/12 (50)
7.2 _ 0.2 a 8.2 _ 0.7
8.1 + 0.2 a 8.3 _+0.9
2.2 + 0.4 2.5 + 0.6
1.9 + 0.2 b 0.8 + 0.3
1.2 _+0.4 0.7 _+0.2
12/14 (86) 9/15 (60)
8.6 + 0.5 8.9 + 0.5
9.3 + 0.5 9.2 + 0.5
3.0 + 0.5 2.8 + 1.4
1.7 + 0.3 1.2 + 0.3
0.7 + 0.4 1.0 + 0.3
Statistically significant differences (P < 0.05): a Factor Sex, and b Factor Treatment.
O. Laban et al. / Brain Research 676 (1995) 133-140
136
was also higher ( P < 0.02) in g e n t l e d males c o m p a r e d to controls, while t h e r e were n o differences b e t w e e n female groups (Fig. 2). T h e severity of histopathological lesions (Table 2) was increased in g e n t l e d males a n d decreased in g e n t l e d females c o m p a r e d to corres p o n d i n g controls ( I n t e r a c t i o n T r e a t m e n t × Sex F1,67 = 4.51, P < 0.05). As in the h a n d l i n g e x p e r i m e n t , the onset (Factor Sex F1,44 = 13.03, P < 0,001) a n d the p e a k ( F a c t o r Sex F1,44 = 13.03, P < 0.05) of the disease a p p e a r e d earlier in females, b u t t h e r e were n o differences due to gentling.
Males
8
"15
6
2
8
10
12
14
16
Females
3.4. E A E in mothers subjected to separation from the offspring
P
"0 c
6
8
10
12
14
16
Days after immunization
•
HANDLED +
CONTROLS
M o t h e r s showed a high susceptibility to E A E induction. I n c i d e n c e of the disease a n d severity of clinical signs were n o t affected by daily s e p a r a t i o n s from the offspring d u r i n g the p r e w e a n i n g period, b u t clinical signs a p p e a r e d earlier in s e p a r a t e d m o t h e r s ( T a b l e 3; Fig. 3.). A f t e r the recovery from E A E , relapses were m o r e f r e q u e n t in the e x p e r i m e n t a l g r o u p in comparison to controls. T h e relapses, which a p p e a r e d 3 - 1 0 days after recovery from acute disease, m a i n l y repres e n t e d 2 - 4 days of flaccid tail. However, 3 o u t of 7 s e p a r a t e d dams with relapses exhibited paresis or paralysis of h i n d limbs d u r i n g this second attack.
Fig. 1. Development of EAE in male and female rats subjected to neonatal handling. The aggregate clinical score was higher in handled males compared to control males (P < 0.05). * Statistically significant difference, handled males vs. control males (P < 0.05), in mean clinical score on days 7 and 8 postimmunization.
3.5. Anti-MBP antibodies
higher clinical score (Table 2) c o m p a r e d to control males a n d g e n t l e d females ( I n t e r a c t i o n T r e a t m e n t × Sex, F1,70 = 6.61, P < 0.02). T h e aggregate clinical score
Fig. 4 shows the s e r u m a n t i - M B P a n t i b o d y levels in a n i m a l s subjected to n e o n a t a l m a n i p u l a t i o n s . I n h a n dled males a n t i b o d y levels were increased, a n d in handled females decreased, c o m p a r e d to c o r r e s p o n d i n g
Table 2 EAE in gentled rats Group
Males Gentled Controls Females Gentled Controls
Incidence (%)
Day of onset (,~ + S.E.M.)
Day of m a x i m u m of the disease (~ __.S.E.M.)
Duration in days (~ + S,E.M.)
Clinical score (~ -t- S.E,M.)
Histological lesions (~ + S.E.M.)
15/19 (79) 10/19 (53)
6.5 + 0.1 a 6.7 + 0.2
7.4 + 0.3 a 7.2 ± 0.4
3.6 + 0.5 2.9 ± 0.4
2.1 _+0.3 b 1.1 + 0.3
1.9 J: 0.3 b 1.4 + 0.3
10/17 (59) 13/19 (68)
7.4 + 0.5 8.5 + 0.5
7.9 _+0.5 9.2 _+0.6
3.6 + 0.5 3.2 + 0.3
1.1 + 0.3 1.6 + 0.3
1.1 + 0.3 1.8 + 0.3
Statistically significant differences (P < 0.05): a Factor Sex, and b Interaction Treatment x Sex.
Table 3 EAE in adult female rats separated from their offspring Group
Incidence (%)
Separated from offspring Controls
12/12 (100) 11/12 (92)
a Statistically significant difference, P < 0.05.
Day of onset (~ _+S.E.M.) 7.7 J: 0.3 a 9.1 ± 0.4
Day of maximum of the disease (~ _+S.E.M.) 9.3 + 0.4 10.2 + 0.4
Duration in days (~ _+S.E.M.) 4.7 + 0.4 4.7 + 0.4
Mean clinical score (,~+ S.E.M.) 2.9 + 0.1 2.7 + 0.3
Frequency of relapses (%) 58 17
O. Laban et al. / Brain Research 676 (1995) 133-140 2 ¸
Males
600
*
137
(a)
500
I eq 400
/i
o 300 o
"0
x 200 t~ O
|
100
MALES
6
2
8
10
12
14
16
18
FEMALES
[ ] CONTROLS [ ] HANDLED
Females
600
(b)
500 <~ 400 o~
o 300
"0
o
x 200 O I
I
8
I
I
8
I -
I
I
10
I
GENTLED
12
~
W
100
I
14
16
18
MALES
Days after immunization ~ - -
'j
I
FEMALES
[ ] CONTROLS [ ] GENTLED
Fig. 4. Serum anti-MBP antibody level in handled and gentled rats.
CONTROLS
Fig. 2. Development of E A E in raale and female rats subjected to neonatal gentling. The aggregate clinical score was higher in gentled males compared to control males ( P < 0.02). * Statistically significant difference, gentled males vs. control males ( P < 0.05), in mean clinical score on days 6, 7 and 8 postimmunization.
controls (Interaction Treatment × Sex F1,51 = 6.13, P < 0.02). Gentling did not influence anti-MBP antibody production. There was no difference in anti-MBP anti-
body synthesis between separated and control mothers (Fig. 5).
4. Discussion
The present study showed that handling and gentling from birth until weaning aggravated clinical EAE in adulthood, and that this effect was more pro-
800 700 C
600
*
E ~ 500 "
"
o400 x 300
0
--~-
6
8
I0
12
14
16
18
20
22
24
Days after immunization ~---
SEPARATED
--o---
CONTROLS
Fig. 3. Development of E A E in mothers separated from their offspring during the preweaning period. There were no differences in the aggregate clinical score between separated and control mothers. • Statistically significant difference, separated mothers vs. control mothers ( P < 0.05), in mean clinical score on days 7 and 8 postimmunization.
0 200 100 i
21
q
t
i
28
i
3,5
after immunization •
SEPARATED ~
CONTROLS
Fig. 5. Serum anti-MBP antibody level in mothers separated from their offspring during the preweaning period.
138
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nounced in male rats. This finding is in agreement with previous reports of immunopotentiation due to early handling [32,45,49]. Histopathological lesions of EAE exhibited a similar pattern as clinical disease, but the differences between experimental and control animals were only statistically significant in the gentled groups. Another dissimilarity between handling and gentling was observed in the effect on anti-MBP antibody production. In handled males levels of these autoantibodies were increased. Although both procedures exerted a similar potentiation of clinical disease in males, it appears that handling and gentling affected mainly the humoral and cell-mediated autoimmune response, respectively. Increased anti-MBP antibody levels in handled males may be responsible for aggravation of EAE, since it was suggested that these antibodies are involved in the demyelinizating process in the CNS [48]. The pathogenic role of autoreactive CD4 ÷ T lymphocytes present in histopathological lesions of EAE is well documented [4,47]. Enhanced reactivity of T helper lymphocytes a n d / o r T cytotoxic lymphocytes specific for autoantigen could explain the potentiation of EAE in gentled animals. Thus, we may hypothesize that different mechanisms were responsible for the observed similar effect of handling and gentling on clinical disease. The effects of early handling and gentling on EAE in adults may be ascribed to alterations in the development of immune a n d / o r neuroendocrine systems. Since MBP appears late in ontogenesis and the maximum rate of myelinization in CNS is achieved during the neonatal period [40], it is possible that neonatal manipulations may have influenced the establishment of tolerance to MBP and thus affected EAE later in life. Environmental changes during the period of maturation of the adrenocortical system [43] may alter the development of this system. It was demonstrated that rats subjected to early handling exhibit a lower and shorter increase in glucocorticoids than non-handled animals after exposure to acute stress procedures in adulthood [35]. Lower circulating levels of corticosterone due to handling [36] may favor the escape of autoreactive T cells at critical times during development [8], thus resulting in increased susceptibility to autoimmune diseases later in life. Furthermore, a rise of glucocorticoids in EAE, due to production of cytokines and stress of paralyses, was reported to be important for the recovery from the disease [30,33]. Glucocorticoids are supposed to be involved in the recovery from EAE by suppression of the cellular immune response through a shift of T cell response from the Thl type to the Th2 type [52]. It may be hypothesized that handled rats did not mount an adequate corticosterone response during EAE, thus exhibiting a more severe disease when compared to non-
handled controls. There are no reports on the effect of gentling on the maturation of the adrenocortical system, but the discrepancy between the effects of handling and gentling on EAE support the view that corticosterone production is not the only mechanism responsible for the results obtained in our study. In accordance with this assumption is the recent report of restraint-induced suppression of clinical signs of EAE accompanied by lowered levels of corticosterone, reduced frequency of MBP-reactive lymph node cells and decreased interleukin-2 and interferon 3' production [13]. Besides glucocorticoids, growth hormones could also be involved. The acute decrease in serum levels of growth hormones in rats deprived from their mother for several hours may be rendered to normal by dorsal tactile stimulation of pups [44]. Unfortunately, there are no data on the effect of tactile stimulation alone on serum hormone levels. Furthermore, in rats deprived from their mother for 15 min no decrease, but some tendency toward enhancement of growth hormone release was noticed. Increased body weight in handled and gentled animals subjected to daily repeated, shortlasting separations from the mother during the entire neonatal period may be due to enhanced secretion of growth hormone. Since growth hormone is necessary for normal development of the immune system [9] and has an immuno-enhancing effect [22], the possibility of its involvement in potentiation of EAE should not be neglected. Our results pointed out to sex-differences in the effect of neonatal manipulations on the development of EAE. The effect of handling and gentling on clinical and histopathological characteristics of EAE were more pronounced in male rats. As for the production of anti-MBP antibodies, potentiation in males and suppression in females were observed. In addition, the disease appeared earlier in experimental and control male groups. Gender differences in the effect of handling on corticosterone levels [37] and humoral immune response [49] in adult rats were described. Furthermore, it was shown that neonatal handling may emphasize neuroanatomical sex-differences [10]. The differential effect of handling on susceptibility to EAE in males and females may be due to gender differences in baseline hormone production [15], immune reactivity [17] and response to stress [16]. Apart from the sex, another factor that influenced the effect of neonatal stress on EAE was the strain of rats. Namely, neonatal sound stress aggravated EAE in adult Lewis rats, and did not affect the disease in DA rats [11]. In the present study DA rats were used since our preliminary experiments, carried out in both DA and Lewis rats, revealed a more pronounced potentiating effect of the gentling procedure on EAE in DA rats. This was probably due to the fact that Lewis rats in the control group developed a high incidence of the
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disease (92-100%) and severe clinical EAE (mean score 2.6-2.9), so further aggravation was hard to obtain. Susceptibility to EAE markedly varies among inbred rat strains and was suggested to be determined by both MHC and non-MHC genes, the latter being responsible for neuroendocrine-immune interactions. Both DA and Lewis rats after sensitization with encephalitogen develop EAE with high incidence and severity. High susceptibility to the disease in these two strains has been partly ascribed to a higher interferon-y-induced Ia expression on brain endothelium in comparison to EAE-resistant rats [34]. However, it seems that in DA rats susceptibility to EAE develops with age, since the incidence of EAE at the age of 5 to 8 weeks is 50-81%, compared to 90-.100% at the age of 12 weeks or more (unpublished observations). Although it is generally accepted that immune [39] and adrenocortical systems [43] fully develop by the age of 8 weeks, it may be speculated that in the DA strain immunological a n d / o r neuroendocrine alterations, which are most probably responsible for high susceptibility to EAE, progress with age. Both procedures used in this experiment included removal of the mother from its pups. During the experiment we noticed that the mothers were disturbed due to this short-lasting disruption of contact with their offspring, i.e. they spent time in the new cage looking for their pups. In monkeys, immune alterations in mothers due to separation from their offspring wele demonstrated [7,26], while for rodents there are no data available. We report here on earlier appearance of clinical EAE, and increased frequency of relapses in rat dams separated from their progeny. Previous investigations showed that EAE may be suppressed [6,14,23,30,31] and enhanced [5] by stress applied after immunization, but this is the first report on the modulatory effect of stress applied in adults before immunization with encephalitogen. The effect of stressful procedures applied during the early postnatal period depended on the type of stressor and immune function examined. More drastic procedures such as prolonged maternal deprivation [38,49], early weaning [1], and restraint [46] had a suppressive effect on humoral and cellular immune responses. Handling appears to be the procedure in which the potentiating effect on imnmne reactivity was observed [32,45,49]. However, the results obtained in our laboratory showed that neonatal sound stress in Lewis rats [11] and early weaning i~: DA rats aggravated EAE, while maternal deprivation reduced susceptibility to EAE in adult DA rats [24]. In the present study we further extend our findings on the effect of early experiences on autoimmunity induced in adults, showing that handling and gentling increased susceptibility to EAE in DA strain of rats. Thus, several factors, such
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as treatment procedure, sex, and strain, may influence the effect of neonatal manipulations on EAE. Bearing in mind that EAE is an animal model for multiple sclerosis, it may be of interest to investigate whether early life events can predispose an individual to autoimmune diseases later in life.
Acknowledgements The present work was supported by the Ministry for Science and Technology, Belgrade.
References [1] Ackerman, S.H., Keller, S.E., Schleifer, S.J., Shindledecker, R.D., Camerino, M., Hofer, M.A., Weiner, H. and Stein, M., Premature maternal separation and lymphocyte function, Brain Behav. Immunol., 2 (1988) 161-165. [2] Ader, R., Developmental psychoneuroimmunology, Dev. Psychobiol., 16 (1983) 251-267. [3] Amkraut, A.A., Solomon, G.F. and Kraemer, H., Stress, early experience and adjuvant-induced arthritis in the rat, Psychosom. Med., 33 (1971) 203-214. [4] Ben-Nun, A., Wekerle, H. and Cohen, I.R., The rapid isolation of clonable antigen-specific T lymphocyte lines capable of mediating autoimmune encephalomyelitis, Eur. J. lmmunol., 11 (1981) 195-199. [5] Bernard, C.C.A., Grgacic, E. and Singer, G., The effect of stress, corticosterone and adrenalectomy on the development of experimental autoimmune encephalomyelitis, J. Neuroimmunol., 17 (1988) 254. [6] Bukilica, M., Djordjevi6, S., Mari6, I., Dimitrijevi6, M., Markovi6, B.M. and Jankovi6, B.D., Stress-induced suppression of experimental allergic encephalomyelitis in the rat, Int. J. Neurosci., 59 (1991) 167-175. [7] Coe, C.L., Rosenberg, L.T. and Levine, S., Prolonged effect of psychological disturbance on macrophage chemiluminiscence in the squirrel monkey, Brain. Behav. Irnmunol., 2 (1988) 151-160. [8] Craddock, C.G., Corticosteroid-induced lymphopenia, immunosuppression, and body defence, Ann. Intern. Med., 88 (1978) 564-566. [9] Denckla, W.D., Interactions between age and the neuroendocrine and immune system, FASEB J., 37 (1978) 1263-1267. [10] Denenberg, V.H., Fitch, R.H., Schrott, L.M., Cowell, P.E. and Waters, N.S., Corpus callosum: interactive effects of infantile handling and testosterone in rat, Behav. Neurosci., 105 (1991) 562-566. [11] Dimitrijevi6, M., Laban, O., von Hoersten, S., Markovi6, B.M. and Jankovi6, B.D., Neonatal sound stress and development of experimental allergic encephalomyelitis in Lewis and DA rats, Int. J. Neurosci., 78 (1994) 135-143. [12] Djordjevi~, S., Bukilica, M., Dimitrijevi~, M., Markovi6, B.M. and Jankovi6, B.D., Anti-myelin basic protein autoantibodies in rats stressed by overcrowding, Ann. N.Y. Acad. Sci., 650 (1992) 302-306. [13] Griffin, A.C., Lo, D.W., Wolny, A.C. and Whitacre, C.C., Suppression of experimental autoimmune encephalomyelitis by restraint stress: sex differences, J. Neuroimrnunol., 44 (1993) 103116. [14] Griffin, A.C. and Whitacre, C.C., Differential effects of stress on disease outcome in experimental autoimmune encephalomyelitis, FASEB J., 4 (1990) A2045.
140
O. Laban et al. /Brain Research 676 (1995) 133-140
[15] Griffin, A. and Whitacre, C., Sex and strain differences in the circadian rhytm fluctuation of endocrine and immune functions in the rat: implications for rodent models of autoimmune disease, J. Neuroimmunol., 35 (1991) 53-64. [16] Griffin, A. and Whitacre, C., Sex differences in stress effects on disease course in experimental autoimmune encephalomyelitis (EAE), FASEB J., 5 (1991) 5870. [17] Grossman, C.J., Are there underlying immune-neuroendocrine interactions responsible for immunological sexual dimorphism?, Prog. NeuroEndocrinlmmunol., 3 (1990) 75-82. [18] Hilakivi-Clarke, L.A., Turkka, J., Lister, R.G. and Linnoila, M., Effects of early postnatal handling on brain /3-adrenoreceptors and behavior in tests related to stress, Brain Res., 542 (1991) 286-292. [19] Hofer, M.A., Relationships as regulators: a psychobiologic perspective on bereavement, Psychosom. Med., 46 (1984) 183-197. [20] Jankovi6, B.D., Isakovid, K., Mi6i~, M. and Kne~evi6, Z., The embryonic lympho-neuro-endocrine relationship, Clin. lmmunol. Immunopathol., 18 (1981) 108-120. [21] Jankovi6, B.D. and Mari6, D., Enkephalins and autoimmunity: differential effect of methionine-enkephalin on experimental allergic encephalomyelitis in Wistar and Lewis rats, J. Neurosci. Res., 18 (1987) 88-94. [22] Kelley, K.W., The role of growth hormone in modulation of the immune response, Ann. N.Y. Acad. Sci., 594 (1990) 95-103. [23] Kuroda, Y., Mori, T. and Hori, T., Restraint stress suppresses experimental allergic encephalomyelitis in Lewis rats, Brain. Res. Bull., 34 (1994) 15-17. [24] Laban, O., Markovid, B.M., Dimitrijevi6, M. and Jankovi6, B.D., Maternal deprivation and early weaning modulate experimental allergic encephalomyelitis in the rat, Brain, Behav. Immun., in press. [25] LaBarba, R.C., Fernandez, B., White, J.L. and Stewart, A., The effects of neonatal tactile stimulation on adult emotional reactivity in BALB/c mice, Dev. Psychobiol., 7 (1974) 393-398. [26] Laudenslager, M., Reite, M. and Harbeck, R., Immune status during mother-infant separation, Psychosom. Med., 44 (1982) 303. [27] Leonard, J.P., MacKenzie, F.J., Patel, H.A. and Cuzner, M.L., Hypothalamic noradrenergic pathways exert an influence on neuroendocrine and clinical status in experimental autoimmune encephalomyelitis, Brain Behav. Imrnun., 5 (1991) 328-338. [28] Levine, S., A further study of infantile handling and adult avoidance learning, J. Pers., 25 (1956) 70-80. [29] Levine, S., Haltmeyer, G.C., Karas, G. and Denenberg, V., Physiological and behavioral effects of infantile stimulation, Physiol. Behav., 2 (1967) 55-59. [30] Levine, S. and Saltzman A., Nonspecific stress prevents relapses of experimental allergic encephalomyelitis in rats, Brain. Behav. Immunol., 1 (1987) 336-341. [31] Levine, S., Strebel, R., Wenk, E.J. and Harman, P.J., Suppression of experimental allergic encephalomyelitis by stress, Proc. Soc. Exp. Biol. Med., 109 (1962) 294-298. [32] Lown, B.A and Dutka, M.E., Early handling enhances mitogen responses of splenic cells in adult C3H Mice, Brain Behav. Immunol., 1 (1987) 356-360. [33] MacPhee, I.A.M., Antoni, F.A. and Mason, D.W., Spontaneous recovery of rats from experimental allergic encephalomielitis is dependent on regulation of the immune system by endogenous adrenal corticoids, J. Exp. Med., 169 (1989) 431-445. [34] Male, D. and Pryce, G., Induction of Ia molecules on brain endothelium is related to susceptibility to experimental allergic encephalomyelitis, J. Neuroirnmunol., 21 (1989) 87-90.
[35] Meaney, M.J., Aitken, D.H., Bodnoff, S.R., Iny, L.J. and Sapolsky, R.M., The effects of postnatal handling on the development of the glucocorticoid receptor systems and stress recovery in the rat, Prog. Neuropsychopharmacol. Biol. Psychiat., 7 (1985) 731734. [36] Meaney, M.J., Aitken, D.H., Viau, V., Sharma, S. and Sarrieau, A., Neonatal handling alters adrenocortical negative feed-back sensitivity and hippocampal type II glucocorticoid receptor binding in the rat, Neuroendocrinology, 50 (1989) 597-604. [37] Meaney, M.J., Viau, V., Bhatangar, S., Betito, K., Iny, L.J., O'Donnell, D. and Mitchell, J.B., Cellular mechanisms underlying the development and expression of individual differences in the hypothalamic-pituitary-adrenal stress response, Steroid Biochem. Mol. Biol., 39 (1991) 265-274. [38] Michaut, R.J., Dechambre, R.P., Doumerc, S., Lesourd, B., Devillechabrolle, A. and Moulais, R., Influence of early maternal deprivation on adult humoral immune response in mice, Physiol. Behav., 26 (1981) 189-191. [39] Murgita, R.A. and Wigzell, H., Regulation of immune response functions in the fetus and newborn, Prog. Allergy, 29 (1981) 54-133. [40] Norton, W.T. and Poduslo, S.E., Myelination in rat brain: changes in myelin composition during brain maturation, J. Neurochem., 21 (1973) 759-773. [41] Paterson, P.Y. and Halberg, M.K., Antineural antibodies in experimental allergic encephalomyelitis. In E.J. Goetzl and N.H. Spector (Eds.), Neuroimmune Networks: Physiology and Diseases, Alan R. Liss, Inc., New York, 1989, pp. 251-257. [42] Raymond, L.N., Reyes, E., Tokuda, S. and Jones, B.C., Differential immune response in two handled inbred strains of mice, Physiol. Behav., 37 (1986) 295-297. [43] Sapolsky, R.M. and Meaney, M.J., Maturation of adrenocortical stress response: neuroendocrine control mechanisms and the stress hyporesponsive period, Brain Res. Rev., 11 (1986) 65-76. [44] Schanberg, S.M., Evoniuk, G. and Kuhn, C.M., Tactile and nutritional aspects of maternal care: specific regulators of neuroendocrine function and cellular development, Proc. Soc, Exp. Biol. Med., 175 (1984) 135-146. [45] Solomon, G.F., Levine, S. and Kraft, J.K., Early experience and immunity, Nature, 220 (1968) 821-822. [46] Taylor, C.E. and Ross, L.L., Alteration of antibody response to Pneumococcal polysaccharide Type III in rats by neonatal immobilization stress, Brain Behav. Immunol., 3 (1989) 160-165. [47] Traugott, U., McFarlin, D.E. and Raine, C.S., Immunopathology of the lesions in chronic relapsing experimental autoimmune encephalomyelitis in the mouse, Cell. Immunol., 99 (1986) 395410. [48] Varitek, V.A. and Day, E.D., Studies of rat antibodies specific for myelin basic protein (MBP). Antibody-dependent cell-mediated lysis of MBP-sensitized targets in vitro, J. Neuroimmunol., 1 (1981) 27-39. [49] von Hoersten, S., Dimitrijevi~, M., Markovi6, B.M. and Jankovi6, B.D., Effect of early experience on behavior and immune response in the rat, Physiol. Behav., 54 (1993) 931-940. [50] Weininger, O., Mortality of albino rats under stress as a function of early handling, Can. Jour. Psych., 7 (1953) 111-114. [51] Wekerle, H., Linington, C., Lassmann, H. and Meyermann, R., Cellular immune reactivity within the CNS, Trends Neurosci., 9 (1986) 271-277. [52] Wick, G., Hu, Y., Schwartz, S. and Kroemer, G., Immunoendocrine communication via the hypothalamo-pituitary-adrenal axis in autoimmune diseases, Endocr. Rev., 14 (1993) 539-563.