Journal of Neuroimmunology 104 (2000) 31–36 www.elsevier.com / locate / jneuroin
Behavioral aspects of experimental autoimmune encephalomyelitis Yehuda Pollak a , Haim Ovadia b , Inbal Goshen a , Ronnie Gurevich a , Keren Monsa a , Ronit Avitsur a , a, Raz Yirmiya * a
Department of Psychology, The Hebrew University of Jerusalem, Mount Scopus, Jerusalem 91905, Israel b Department of Neurology, Hadassah-Hebrew University Hospital, Jerusalem 91120, Israel Received 14 July 1999; received in revised form 28 September 1999; accepted 12 October 1999
Abstract Acute inflammation is known to induce a depressive-like sickness behavior syndrome in humans and in experimental animals. In the present study, we sought to determine whether a chronic neuroautoimmune inflammation is also associated with a similar behavioral syndrome. Experimental autoimmune encephalomyelitis (EAE) was induced in SJL / J female mice by adoptive transfer of lymph node cells, and sickness behavior symptoms, including anorexia, loss of hody weight, reduced social exploration, and decreased preference for sucrose solution were measured. We report that these components of sickness hehavior were induced during the acute phase of the disease, and recovered in later phases. Moreover, the onset and recovery of the behavioral symptoms preceded the onset and recovery of the neurological signs, respectively. Since EAE is considered a model for multiple sclerosis (MS), it is suggested that EAF-induced behavioral changes may serve as a model for the depressive symptomatology that characterizes most MS patients. 2000 Elsevier Science B.V. All rights reserved. Keywords: EAE; Sickness behavior; Multiple sclerosis; Depression; Mice
1. Introduction Sickness is associated with many typical behavioral phenomena, such as anorexia and loss of body weight, hypersomnia, reduced interest in social activity, decreased motor activity, and anhedonia (inability to experience pleasure from stimuli and activities used to induce pleasure) (Hart, 1988; Kruger and Majde, 1994; Sonti et al., 1996; Anisman and Merali, 1999; Dantzer et al., 1999; Yirmiya et al., 1999). Extensive neuroimmunological study revealed that immune activation and the secretion of cytokines in the periphery and the brain mediate these behavioral responses to disease (sickness behavior). Thus, exogenous administration of cytokines, or stimulation of endogenous cytokines production, elicit sickness behavior, whereas cytokines antagonists or blockers of their synthesis abolish the behavioral effects of immune challenges (Connor and Leonard, 1998; Maier and Watkins, 1998; Dantzer et al., 1999; Yirmiya et al., 1999). Most of the research on sickness behavior syndrome has been limited to the behavioral consequences of acute inflammation. Much less research has focused on the *Corresponding author. Tel.: 1972-2-5883695; fax: 1972-2-5881159. E-mail address:
[email protected] (R. Yirmiya)
behavioral correlates of more chronic disease models. One of the few exceptions is the reports on sickness behavior and disturbed emotionality in genetic models of systemic lupus erythematosus (SLE) in mice (Sakic et al., 1994, 1996; Schrott and Crnic, 1996). The aim of the present research was to substantiate and extend these reports by examining the behavioral alterations in a central nervous system (CNS) chronic inflammatory disease, using the experimental autoimmune encephalomyelitis (EAE) model. EAE is a T-cell-mediated autoimmune disease of the CNS in which CD4, TH1 cells are activated by a self myelin antigen and insult the myelin sheaths within the CNS. The tissue damage is mediated mainly by proinflammatory cytokines (Olsson, 1995; Eng et al., 1996), some of which (i.e., interleukin-1 (IL-1), interleukin-6 (IL-6) and tumor necrosis factor a (TNF-a)) are known to induce sickness behavior (Anisman and Merali, 1999; Yirmiya et al., 1999). Therefore, we hypothesized that EAE will be associated with marked sickness behavior symptoms. To detect and characterize sickness behavior in EAE mice, we measured four variables: food intake, body weight, social exploration, and sucrose solution preference during the different phases of the disease. These variables
0165-5728 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0165-5728( 99 )00257-X
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were previously found to be decreased following an acute immune challenge (Yirmiya, 1996, 1997). To elucidate the temporal relationship between the onset of the behavioral and the neurological symptoms, we analyzed the behavioral alterations during the days preceding and following the onset of the acute phase and the recovery phase.
2. Materials and methods
2.1. Subjects Female SJL / J mice (n528), aged 8-9 weeks (Harlan– Sprague–Dawley, USA), were housed individually in constant room temperature (23618C), and were provided with food, water and 2% sucrose solution ad lib. Manipulations and measurements were conducted during the dark phase of a reversed 12 h light?dark cycle (lights off at 0830 h).
Fig. 1. Clinical course of passive EAE. On day 0, mice were injected with either saline (SAL) or lymph node cells activated in vivo and in vitro against PLP (EAE). Clinical condition was scored on a 0–6 scale. The course of the disease was divided into three phases (marked by arrows).
2.2. Passive induction and clinical evaluation of EAE EAE was induced in mice by adoptive transfer of lymph node cells (Wekerle et al., 1994). Female SJL / J mice were sensitized with 150 mg mouse proteolipid protein, PLP 139–151, emulsified in complete Freund’s adjuvant (CFA) (1:1). Ten days later, draining lymph nodes (popliteal, inguinal, and paraaortic) were excised and homogenized to a single cell suspension. Cells were incubated with PLP (10 mg / 4310 6 cells / ml) in a CO 2 incubator at 378 for 4 days. Naive female SJL / J mice received 15–20310 6 activated lymph nodes cells intraperitoneally. The severity of the disease was scored on a scale of 0 to 6 as follows: 05no neurological signs; 15tail weakness; 25tail paralysis, 35loss of righting reflex (when mice have difficulty to turn over after being laid down on their back, but no locomotive difficulties are observed); 45hind limb paresis / paralysis (hind limbs are dragged); 55 quadriplegia (immobilityl) and 65death of the animal. The time course of the disease of each mouse was divided into three phases. The acute phase beings with the onset of neurological symptoms, and ends in the day preceding the first clinical improvement. The recovery phase begins with the first clinical improvement and ends when the animal?s clinical symptoms cease to improve. From that day on, an animal is considered to be in the chronic phase. In order to compare the EAE and control groups through the different phases, control mice observations were divided into three phases according to the average length of each phase in the EAE mice (Fig. 1).
2.3. Procedure During the week preceding EAE induction, mice were familiarized with the experimental conditions. Each mouse was provided ad lib with food pellets and two tubes
containing water and 2% sucrose solution. The food pellets were put on the floor of the cage and the drinking tubes were low enough to enable sick mice to eat and drink effortlessly. A conspecific juvenile was placed daily in each mouse’s home cage for 3 mm. In the 3 days preceding the inoculation, body weight, the intake of food, water and sucrose solution, and the time spent by the mouse in social exploration of a conspecific juvenile were measured, averaged, and considered as baseline level. Mice were divided into two groups (n512 and 16, for the saline and EAE groups, respectively) matched for all four measures. For 7 weeks following the inoculation, body weight, food, sucrose and water consumption, and social exploration were measured regularly. The experiment was conducted in two replications. In the first experiment (with n54 and n58 for the saline and EAE groups, respectively), the body weight and the intake of sucrose and water were measured twice a week. In the second experiment (with n58 for each of the two groups), the body weight, intake of food, sucrose and water, and social exploration, were measured daily (in order to analyze the temporal relationship between the neurological and the behavioral symptoms). Since there were no differences in body weight and the consumption of sucrose and water between these replications, data were combined for the final statistical analysis of the effect of EAE in the different phases.
2.4. Statistical analysis Measures in each phase were averaged for each animal. Variables were calculated for each mouse as percent of baseline, by dividing the averaged measure of the phase by the baseline, and multiplying it by 100. Results were analyzed by two-way ANOVAs with EAE / saline as a
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between-subject factor, and the phase of disease as a repeated measures, within-subject factor. Results were further analyzed by post-hoc corrected Student’s t-tests. To assess the temporal relationship between the neurological and the behavioral syndromes, the behavioral measures of each mouse in the days preceding and following the onset of the neurological signs were calculated as percent of baseline as described above. Results were analyzed by two-way ANOVAs with EAF / saline as a between-subject factor, and the day as a repeated measures, within-subject factor. Results were further analyzed using post-hoc corrected Student’s t-tests. The same statistical procedure was used for analyzing the behavioral measures in the days preceding and following the onset of the recovery phase.
3. Results
3.1. Effects of EAE on clinical score and sickness behavior The time course of EAE progression is presented in Fig. 1. Mean onset of neurological symptoms was 7 days following the injection. Thirteen days, in average, after the disease onset, EAE mice entered their recovery phase. Nine days later the chronic phase began, and was ended by the termination of the experiment, 53 days following the injection. EAE was accompanied by an overall decrease in body weight (F(1,26)559.52, p,0.001) with a significant difference between the groups at each phase (Fig. 2A). Within the EAE group, body weight during the chronic, but not the recovery phase, was significantly greater than during the acute phase, reflected by a significant interaction between EAE / saline injection and the disease phase (F(2,52)55.64, p,0.01). Food intake was decreased during the acute phase of the disease, and it returned to baseline level during the recovery and the chronic phases, reflected by a significant interaction between EAF / saline injection and the disease phase (F(2,36)521.92, p,0.001) (Fig. 2B). Post-hoc tests revealed a significant difference between the groups only in the acute phase. Within the EAE group, significant differences were found between the acute and both the recovery and the chronic phases. EAE was associated with a dramatic reduction of sucrose consumption during the acute phase, followed by an elevation above control level during the recovery and the chronic phases, reflected by a significant interaction between EAE / saline injection and the disease phase (F(2,52)55.48, p,0.01) (Fig. 2C). Post-hoc tests revealed a significant difference between the groups only in the acute phase. A trend toward increased sucrose consumption during the recovery and the chronic phases was observed in both groups. This increase was particularly
Fig. 2. Effect of EAE on body weight (A), food intake (B), sucrose and water intake (C?D), and social exploration (E) during the different phases. Data represent the mean (1SEM) of 8?16 mice / group. * p,0.05 vs. saline group. 1p,0.05 vs. EAE group in the acute phase.
evident in the EAE group; however, there was no significant difference between the EAE and the saline groups at these later phases. Within the EAE group, sucrose consumption during the recovery and chronic phases was significantly greater than during the acute phase. EAE was not accompanied by changes in water consumption at any phase, as reflected by the absence of neither significant main effect of EAE nor significant interaction between EAE and the disease phase ( p.0.1) (Fig. 2D). EAB was associated with a reduction in social exploration during the acute phase, but not the recovery and the chronic phases of the disease, reflected by a significant interaction between EAE / saline injection and the disease phase (F(2,36)512.45 p,0.05) (Fig. 2E). Post-hoc tests revealed a significant difference between the groups only in the acute phase. Within the EAE group, social exploration during the recovery and chronic phases was significantly greater than during the acute phase. To determine the relationship between the behavioral disturbances that were observed during the acute phase and the clinical score, we computed Pearson correlation coefficients between these variables. Correlations between clinical score and food consumption, sucrose intake and
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social exploration were 0.29, 20.26, and 20.24, respectively. None of these correlations was statistically significant.
3.2. Temporal relationships between the onsets of the neurological and the behavioral syndromes During the days preceding and following the onset of the acute phase, significant alterations in food intake, body weight, sucrose consumption, and social exploration were detected, as reflected by significant interactions between the groups and the days (F(4,56)58.58, 15.82, 7.56 and 7.18, p,0.001, respectively) (Fig. 3A–D). Significant alterations were also detected during the days preceding and following the onset of the recovery phase, as reflected by significant interactions between the groups and the days (F(6,84)57.61, 3.53 and 4.44, p,0.005, for food intake, body weight, and social exploration, respectively), and a tendency toward significance with respect to sucrose intake (F(6,84)52.1, p,0.1) (Fig. 4A–D). Alterations in food and sucrose intake and in social exploration began 1 day before the onset of the acute phase, and ended 3–4 days before the onset of the recovery phase. Post-hoc comparisons within the EAE group revealed significant reductions in food and sucrose intake from the day preceding the onset of the acute phase and went on, until 3 days before the onset of the recovery phase. One day preceding the onset of the recovery phase, food and sucrose intake were already significantly elevated. A somewhat different temporal pattern characterized the changes in body weight, i.e., significant differences between the groups began only with the onset of the acute phase, and continued until 2 days following the onset of
Fig. 3. Effect of EAE on body weight (A), food intake (B), sucrose intake (C), and social exploration (D) during the days preceding and following the onset of the acute phase. Data represent the mean (6SEM) of 8–16 mice / group. * p,0.05 vs. saline group. 1p,0.05 vs. EAE group 2 days preceding the acute phase onset.
Fig. 4. Effect of EAE on body weight (A), food intake (B), sucrose intake (C), and social exploration (D) during the days preceding and following the onset of the recovery phase. Data represent the mean (6SEM) of 8–16 mice / group. * p,0.05 vs. saline group. 1p,0.05 vs. EAE group 4 days preceding the recovery phase onset.
the recovery phase. Within the EAE group, body weight was significantly reduced from the onset day of the acute phase and onwards. Elevation in body weight began 1 day preceding the onset of the recovery phase, but achieved statistical significance only 2 days following onset.
4. Discussion The results indicate that EAE is associated with reduced body weight, food and sucrose intake, and social exploration. These findings confirm and extend previous studies from other laboratories, reporting that chronic inflammation and autoimmune conditions in animals are associated with substantial behavioral alterations (Sakic et al., 1994, 1996; Schrott and Crnic, 1996). The present study enabled us to draw conclusions regarding the time course of the behavioral syndrome associated with EAE. Food and sucrose consumption started to drop on the day preceding the onset of the neurological symptoms, began to recover several days before the onset of the recovery phase, and recovered completely when the mice began to show neurological improvement. A significant decrease in body weight was first detected together with the appearance of neurological symptoms. Body weight began to recover only during the chronic phase of the disease. It should be noted that during the recovery phase, body weight remained low in spite of the elevation in food consumption, suggesting alterations in metabolic processes. Reduced sucrose intake is considered to reflect alterations in hedonic behavior (Willner, 1997). The lack of changes in water intake in the EAE mice is consistent with this hypothesis, although the
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overall low quantities of water consumed make these data difficult for interpretation. Social exploration was also reduced in the days preceding the onset of the acute phase, showing the first improvement several days before the onset of the recovery phase, but fully recovering to baseline level only during the recovery phase. The distinct time course for each behavioral parameter suggests variations in the mechanisms underlying the different components of the behavioral syndrome. The mechanisms underlying the association between EAE and sickness behavior remain to be elucidated. However, several findings suggest that the behavioral consequences of EAE are not a mere result of motor impairment. (1) We excluded from the data analysis mice that could not move (i.e., with paralysis of both hind and fore limbs). (2) The behavioral syndrome preceded the onset of the neurological symptoms. (3) Reduction in liquid consumption is specific to sucrose, since no differences between the EAE and control mice were observed with respect to water consumption. (4) Within the acute phase, clinical score was not correlated with any of the behavioral measures. (5) The disease phase, rather than clinical score, is sometimes more important for determining the behavioral alterations of EAE mice. For example, food and sucrose intake exceeded normal levels in the recovery phase, although motor impairment was similar to that observed during the acute phase. Data obtained in our laboratory, as well as those in others, indicate that cylokines are critically involved in mediating sickness behavior symptoms. Pro-inflammatory cytokines, such as IL-1 and TNF-a, induce sickness behavior when administered either in the periphery or the brain, and are involved in mediating sickness behavior induced by several immune challenges (Anisman and Merali, 1999; Dantzer et al., 1999; Yirmiya et al., 1999). EAE is associated with intensive immune activation, including a dramatic increase in the production and secretion of cytokines. Our data support the hypothesis that this activation underlies the behavioral effects of the disease in two ways. First, high levels of pro-inflammatory cytokines, within immune organs and the CNS, precede the onset of the clinical symptoms (Kennedy et al., 1992; Renno et al., 1994; Okuda et al., 1995; Okuda et al., 1998). Similarly, in the present study the behavioral symptoms were found to precede the neurological signs. Second, the production of pro-inflammatory cytokines (e.g., IL-1, TNFa, IFN-g) by activated immune cells is markedly increased during the acute phase, and is attenuated by several antiinflammatory cytokines such as IL-10 and TGF-b during and even preceding the recovery phase (Kennedy et al., 1992; Renno et al., 1994; Okuda et al., 1995; Okuda et al., 1998). Thus, the present findings that food and sucrose consumption, as well as social exploration, were markedly decreased during the acute phase, but elevated before and during the recovery phase, suggest that alterations in cytokine production underlie the alterations in behavior.
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The symptoms of sickness behavior resemble the characteristic clinical symptoms of depression. Both of the two core symptoms of depression, depressed mood and anhedonia (DSM-IV, 1994), were shown to be produced by immune challenges and exogenous administration of cytokines in humans and in animal models (Meyers and Valentine, 1995; Yirmiya, 1996; Avitsur et al., 1997; Morag et al., 1998; Anisman and Merali, 1999). Other common symptoms of depression are changes in body weight, diminished concentration ability, fatigue, and sleep problems (DSM-IV, 1994), all of which were found to be associated with a variety of medical conditions which are accompanied by immune activation (Yirmiya, 1997). Furthermore, in a previous study, we found that pretreatment with antidepressants attenuated the behavioral alterations induced by an acute immune challenge, suggesting that sickness behavior and depression share common mechanisms (Yirmiya, 1996; Yirmiya et al., 1999). The behavioral syndrome observed in the EAE mice, and especially the specific reduction in sucrose intake which may reflect an anhedonic state (Willner, 1997), raises the possibility that EAE induces a depressive-like episode, since sucrose intake may reflect alteration in hedonic value, one of the two essential symptoms of a depressive episode (Willner, 1997). Multiple sclerosis (MS) is an autoimmune disease, characterized by an inflammation of the white matter of the nervous system, resulting in motor and sensory impairment. MS is associated with an intensive immune activation, including high levels of pro- and anti-inflammatory cytokine production and secretion within the nervous system (Cavallo et al., 1993; Olsson, 1995). At the psychological level, MS is associated with high prevalence of neuropsychological and neuropsychiatric phenomena, especially with high prevalence of depression (Whitlock and Siskind, 1980; Schiffer and Babigian, 1984; Minden and Schiffer, 1990). Several lines of evidence suggest that the depression observed in MS patients cannot be solely attributed to the psychological reaction to the functional impairment induced by the disease: (1) The prevalence of depression among MS patients is higher than in other neurological diseases, although those diseases cause no less functional impairment (Whitlock and Siskind, 1980; Schiffer and Babigian, 1984); (2) In many cases, the depressive symptoms precede the neurological signs (Schiffer and Babigian, 1984; Sullivan et al., 1995); (3) The quality of the depressive symptoms in MS patients was found to be less emotional or cognitive, and more vegetative and diurnal, suggesting ‘organic’ rather than ‘reactive’ depression (Whitlock and Siskind, 1980); (4) Immune dysregulation was found to precede depressive episodes in MS patients (Foley et al., 1992), and affective symptoms in patients with MS were correlated with cerebrospinal fluid white blood cell counts but not to degree of disability (Fassbender et al., 1998). Our data in EAE, the animal model of MS, resemble
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some of the findings related to the association between MS and depression. Depressive episodes in MS patients were found to accompany exacerbation and to dissolve on remission of the neurological symptoms (Minden and Schiffer, 1990). Our EAE mice also showed depressivelike symptoms mainly during the acute phase. In addition, in many cases depressive symptoms appear before the onset of MS (Schiffer and Babigian, 1984; Sullivan et al., 1995). Similarly, we found that the depressive symptoms preceded the neurological signs. In view of these similarities, the behavioral changes associated with EAE may serve as a model of ‘‘depression due to a general medical condition’’ (DSM-IV, 1994) in general, and MS-associated depression in particular.
Acknowledgements The authors thanks Boaz Horev, Rinat Koren, Shin Lavy, Einav Orion, YacI Perets and Gili Wolf for their excellent help in running the experiments. The research was supported by Grant 94-204 from the United States– Israel Binational Science Foundation (BSF), Jerusalem Israel.
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