- Email: [email protected]
Pavlovian conditioning of lung anaphylactic response in rats
Life Sciences 68 (2000) 611–623
Pavlovian conditioning of lung anaphylactic response in rats J. Palermo-Neto*, R.K. Guimarães Applied Pharmacology and Toxicology Laboratory, Department of Pathology, School of Veterinary Medicine, University of São Paulo, São Paulo, Brazil Received 20 March 2000; accepted 7 June 2000
Abstract The present experiment was undertaken to verify if it is possible to impose Pavlovian conditioning on a lung anaphylactic response (LAR) in rats. Two experiments were done. In the 1st, egg albumin (OVA) aerosol inhalation, which induces signs and symptoms of LAR in OVA- sensitized rats, was paired with an audiovisual cue (conditional stimulus, CS). After reexposure to the CS, the signs and symptoms of LAR were quantitatively measured using a scoring system specially developed for this evaluation; the levels of stress response and anxiety were also quantified. Results showed that the rats reexposed to CS only, displayed LAR scores not significantly different from those reexposed to both CS and the antigen; animals of these groups showed significantly higher LAR scores than rats that received no OVA aerosol challenge. High levels of stress and anxiety were observed 30–40 min after the challenge with OVA aerosol. In the 2nd experiment, rats sensitized with OVA and submitted or not to Pavlovian conditioning were observed in the open-field and in the plus maze apparatus in the absence of OVA aerosol but in the presence of the CS; after behavioral observations the animals were sacrificed for serum corticosterone level determination. Both behavioral and biochemical data showed high levels of stress and anxiety in rats for which the antigen was previously paired with the CS; these changes were not observed in animals which received the antigen 24 h after the presentation of the CS (unpaired) or in those exposed to PBS aerosol (the OVA vehicle) only. The present data show not only that LAR can be submitted to Pavlovian conditioning, but also and importantly, that high levels of stress and anxiety are related to the course of LAR. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Anaphylaxis; Lung; Pavlovian conditioning; Stress; Anxiety; Neuroimmunomodulation
Introduction Several studies have focused on the existence of body-mind interactions both in health and in sickness. The long-held view that homeostatic mechanisms are integrated by nervous and * Corresponding author: Faculdade de Medicina Veterinária e Zootecnica da Universidade de São Paulo, Laboratório de Farmacologia Aplicada e Toxicologia, Av. Prof. Dr. Orlando Marques de Paiva, 87, CEP:05508-900, São Paulo - SP, Brazil. Tel.: 55 011 211.3074; fax: 55-011 210.2224. E-mail address: [email protected] (J. Palermo-Neto) 0024-3205/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 9 6 6 -8
612
J. Palermo-Neto, R.K. Guimarães / Life Sciences 68 (2000) 611–623
endocrine systems has been expanded by information that these systems interact with the immune system. Immune responses alter neural and endocrine functions, and in turn, neural and endocrine activity modifies immunologic function (1,2). Several studies have suggested that anaphylaxis can occur in the absence of any antigenic stimulus or physical irritant and may represent a conditioned response (3–5). In other words, neutral stimuli such as sensory events (conditional stimulus, CS) may become associated with antigens in such a way that subsequent exposure to these stimuli will elicit anaphylatic responses. In Pavlovian conditioning terms, the antigen is an unconditioned stimulus (US) that elicits an unconditioned response, i.e. anaphylaxis. In this respect, the observation that the immune system is influenced by Pavlovian conditioning (6) has provided strong evidence for neuro-immune interactions. The existence of a conditioned immunomodulation was described after treatment with both immunossupressive (6) and immunoenhancing agents (7); conditioned alterations of immune function in animals trained with antigens were also reported (8). The Central Nervous System (CNS) was also shown to play an important role in the conditioned secretion of histamine (8), acting as a functional effector of mast cell function in the allergic state (5). Taken together, these findings support the increasing evidence for CNS involvement in the regulation of both immune function and response in the allergic state. Symptoms of anaphylaxis, particularly those related to lung function, are known to be increased during stressful situations and/or high levels of anxiety. In addition, high levels of anxiety and stress are frequently reported in the emergence of an asthmatic crisis. However, experimental laboratory data supporting a relationship between lung anaphylactic response (LAR) and anxiety levels are lacking. Thus, the present experiment was designed to determine if it is possible to impose Pavlovian conditioning on LAR and also to analyze some possible relationships between behavioral and biochemical data indicative of levels of stress/ anxiety and LAR. Material and methods Animals One hundred pathogen free and genetically similar male Wistar rats from our colony, weighing 230–250 g and about 75–80 days of age, were used. The animals were housed in temperature-controlled (21–238C) and artificially lighted rooms on a 12-h light/12-h dark cycle (lights on at 7:00 a.m.) with free access to food and water. The experiments were performed in a different room, at the same temperature as the animal colony, to which the animals were transferred and maintained in their home cages 2 h before the experiments. Animals were housed and used in accordance with the guidelines of the Committee on Care and Use of Laboratory Animal Resources of the School of Veterinary Medicine, University of São Paulo; these guidelines are similar to those of the National Research Council, USA. Sensitization and conditioning protocol Aluminum-precipitated egg albumin, OVA (Sigma Chemical Company, USA) prepared in phosphate buffered saline (PBS), was intraperitonially (ip) administered to the rats at a dose of 4.0 mg/kg (1.0 ml/kg solution); this was considered to be 0 day of sensitization. The alu-
J. Palermo-Neto, R.K. Guimarães / Life Sciences 68 (2000) 611–623
613
minum precipitate acts as an adjuvant to promote IgE production (9). After this treatment, animals were randomly and equally divided into different groups (see ahead, under experimental design) being repeatedly given an antigen (OVA) aerosol inhalation challenge after sensitization; on these days, we imposed or not the typical Pavlovian conditioning procedure, summarized in Table 1. Aerosol exposure was accomplished by placing the rats individually in a 40 3 50 3 20 cm glass chamber connected to an inhalation nebulizer (Inalamax, model S3; NS Indústria de Apar eˆlhos Médicos, Brasil) that generated an aerosol mist pumped into the exposure chamber by the airflow supplied by a small animal ventilator set at 60 strokes/min with a pumping volume of 10 ml. Some rats were exposed to an aerosol of 1% OVA solution in PBS (w/v) for 15 min. The Pavlovian conditioning imposed was based on the method described elsewhere (5). Briefly, a CS consisted of an audiovisual cue (the sound of the motor of the inhalation apparatus plus the flashing of a 10 Watt green lamp placed 10 cm above the chamber) was paired with the US (the 1% OVA challenge) during the three sessions of aerosol exposure. Some animals (negative controls) were placed for the same period of time (15 min), and also for three times, in identical chambers and in the presence of the audiovisual cue (CS), but received no OVA, i.e., during the 15 min period of inhalation they were exposed to PBS buffer only (see Table 1). Behavioral studies The animals were observed between 8:00 and 12:00 a.m. for signs and symptoms of LAR, exploratory behavior in an open-field and free explorations of a plus-maze apparatus. To minimize the influence of possible circadian changes in the behavioral parameters measured, animals of the different groups were alternated and always observed at the same time of day.
Table 1 Sensitization and conditioning protocol used to impose a Pavlovian Conditioning of a lung anaphylactic response in rats Treatments (days) Experiment
Groups PC1-positive control P1-paired NC1-negative control PC2-positive control U2-umpaired NC2-negative control N-naive
1
2
n 20 20 20 10 10 10 10
0 (1) OVA OVA OVA OVA OVA OVA PBS
34,41 48
35,42 49
— — — — CS(4) — —
OVA 1 CS OVA 1 CS PBS 1 CS(3) OVA 1 CS OVA(4) PBS 1 CS —
56 (2)
5 number of animals sensitization day 5 4.0 mg/kg OVA, i.p. (2) 1% OVA solution aerosol challenge for 15 min, in the presence of an audiovisual cue (CS) (3) PBS buffer aerosol inhalation for 15 min in the presence CS (4) OVA and CS were given alone (5) CS was delivered during the open-field and plus maze observations n
(1)
OVA 1 CS PBS 1 CS PBS 1 CS CS(5) CS CS —
614
J. Palermo-Neto, R.K. Guimarães / Life Sciences 68 (2000) 611–623
The experimental devices used were washed with an alcohol-water solution (5%) before placing the animals in the chamber, to obviate possible biasing effects due to odor clues left by previous rats. The signs and symptoms of LAR were quantified in the inhalation chambers during the 15 min period of OVA or PBS buffer inhalation and CS presentation. Briefly, scores from 0 to 5 were attributed to each animal’s behavior by an observer who was not aware of the treatments. The grading system was as follows: 0, asleep or stationary; 1, active, with few bursts of head scratching; 2, predominantly active, with bursts of head scratching and grooming; 3, constant activity characterized by rearing, head scratching, sniffing, grooming, and presence of a few sneezes; 4, constant rearing, head scratching, sniffing, grooming, presence of a large number of sneezes and a few mouth openings (forced inspirations); 5, constant mouth openings, and presence of postural alterations (animals tend to keep their trunk in an upright position). This criterion was not subjective as shown by the excellent scoring agreement found, in previously conducted pilot studies, by two different and independent observers that analyzed the behavior of rats similarly sensitized and challenged with OVA (Pearson’s correlation, r 5 0.91).The open-field used was constructed as described elsewhere (10). Briefly, it comprises a round wooden arena (97 cm in diameter and 32 cm high walls), painted white and divided by black lines into 25 similar parts; during the experiment the device was lit by two 40 Watt white bulb. For the observations, each rat was individually placed in the center of the apparatus and behavioral parameters were measured for 6 min. Each animal was observed remotely using a video camera; hand-operated counters were employed to score locomotion frequency (number of floor units entered with both front feet) and rearing frequency (number of times the animals stood on their hind legs). For behavioral analyses in the plus maze the rats were placed in the central square of the apparatus (10 3 10 cm), and allowed 5 min of free exploration. The plus-maze device used was made of wood and had two open arms (50 3 10 cm) and two enclosed arms of the same size with 40 cm high walls, and was elevated 50 cm above the ground. Each rat was observed remotely using a video camera for a period of 5 min for the number of entries into each type of arm (all four paws defining an entry) and the time spent in open and closed arms. The measures that reflect anxiety in this test are the percentage of entries into open arms and the percentage of time spent in open arms (11, 12). Serum corticosterone determination Corticosterone is the most abundant circulating steroid secreted by rats, being considered a good indicator of adrenocortical function in this animal species (13). This hormone was determined in serum using commercial kits (Coat-A-Count, USA); this procedure is based on a solid-phase radioimmunoassay in which 125I-labeled corticosterone competes for a fixed time with corticosterone in the rat sample for antibody sites. Serum samples were assayed directly without extraction or purification. In order to decrease data variability and to avoid possible effects of handling stress on serum corticosterone levels, the rats were handled daily for habituation to the experimental conditions of blood collection and were kept far from the room where animals were sacrificed. In addition, animals were sacrificed at the same time of day (between 8:00 and 9:00 a.m.) in order to minimize the reported circadian effects on serum glucocorticoid levels (14).
J. Palermo-Neto, R.K. Guimarães / Life Sciences 68 (2000) 611–623
615
Experimental design Two experiments were done according to standardized GLP guideliness. In the first, 60 rats were randomly divided into three equal groups PC1 (positive control), P1 (paired) and NC1 (negative control) and submitted to the sensitization, challenge and conditioning protocols described above and summarized in Table 1. The rats of groups PC1 and P1 were repeatedly given a 1% OVA aerosol challenge on days 35, 42 and 49 of sensitization in the presence of CS, when Pavlovian conditioning was imposed. On those days, the rats of group NC1 received PBS buffer in the inhalation chamber for the same period of time and in the presence of the audiovisual cue. These animals were then tested 56 days after sensitization; for this purpose, animals of group PC1 were again challenged for 15 min. with a 1% OVA solution, whereas those of groups P1 and NC1 received PBS buffer in the inhalation chamber for the same period of time. During the 4 sessions of inhalation, animals were observed for LAR, that was quantified using the scoring system presented above. Thirty min after the end of the inhalation test (56th day), each rat was individually placed in the center of an open-field apparatus for the determination of locomotion, and rearing frequencies; 4 min later (40 min after the end of the inhalation session), rats were individually transferred to the plus maze apparatus for behavioral evaluations. In the 2nd experiment, 40 rats were divided at random into 4 groups of 10 animals each: PC2 (positive control), U2 (unpaired), NC2 (negative control), and N (naive) as summarized in Table 1. The rats of groups PC2, U2 and NC2 were submitted to the sensitization, challenge and conditioning protocols described above; rats of group N were not sensitized, i.e., they were i.p treated with 0.1 ml/kg of PBS on day 0. The same audiovisual cue (CS) as used in the 1st experiment (the sound of the inhalation chamber plus the flashing of the 10 Watt green bulb) was now employed. The animals of groups PC2 (positive control) were challenged with a 1% OVA solution aerosol for 15 min and in the presence of CS on days 35, 42 and 49 of sensitization. On those days, the rats of group NC2 received PBS buffer in the inhalation chamber for the same period of time and in the presence of the audiovisual cue. Animals of group U2 (unpaired) were placed in the inhalation chamber for 15 min and in the presence of the CS, on days 34, 41 and 48 of sensitization, but received the 1% OVA solution aerosol challenge on the following days, in the absence of the audiovisual cue. On days 35, 42 and 49, the rats of group N were placed for 15 min. in the inhalation chambers free of OVA and PBS aerosols. Fifty six days after sensitization, animals of groups PC2, U2 and NC2 were observed in the open-field and immediately after in the plus-maze apparatus for behavioral alterations in the presence of the audiovisual cue used (CS); rats of group N were observed in both apparatuses in the absence of sound and light. During these behavioral observations, the animals were not exposed to OVA solution or PBS buffer aerosols. Immediately after the end of the plus maze test, all rats were sacrificed by decapitation and blood was collected for the determination of serum corticosterone levels. Statistical analysis Bartlett’s test (15) showed that the data concerning locomotion and rearing frequencies and serum corticosterone levels as well as those obtained in the plus maze were parametric (P,0.05). Thus, one-way analysis of variance followed by the Tukey test for comparison of
616
J. Palermo-Neto, R.K. Guimarães / Life Sciences 68 (2000) 611–623
Fig. 1. Effects of a Pavlovian conditioning on scores of a lung anaphylactic responses (LAR) in rats. Aluminumprecipitated OVA (4.0 mg/kg) was i.p. administered to rats of groups PC1, P1 and NC1 on experimental day 0. On days 35, 42 and 49 of sensitization, rats of groups PC1 and P1 were given 1% OVA aerosol challenge in the presence of CS (an audiovisual cue); on those days, the rats of group NC1 were exposed to aerosol of PBS buffer. On day 56, animals of groups PC1 were challenged with 1% OVA and those of groups P1 and NC1 with PBS buffer in the presence of CS. An asterisk on top of columns mean a statistically significant difference at P,0,05 (KruskalWallis and Dunnet tests).
cell means were used to analyze these data. The LAR scores were analyzed by Kruskal-Wallis (KW) analysis of variance for nonparametric data, followed by the Dunnett test for multiple comparisons. The StatPac Statistic Analysis Package was used throughout with the level of significance set at P,0.05 for all comparisons. Results Figure 1 shows that rats sensitized with OVA displayed signs and symptoms of anaphylaxis when challenged by the respiratory route with a 1% OVA solution; indeed, the LAR scores observed in animals of groups PC1 (positive control) and P1 (paired) during the 15 min inhalation period were significantly higher (KW2,57 5 15.73, 18.24, 10.76 and 12.01, p,0.05, for post-sensitization days 35, 42, 49 and 56, respectively) than those observed in rats of the control group (group NC1: negative control). This Figure also shows that the Pavlovian conditioning imposed was successful. Thus, on post-sensitization day 56, the CS presentation per se was able to induce high levels of LAR in the animals of group P1 (paired). In this day, no significant differences were found between data for groups PC1 (positive control) and P1 (paired). Table 2 presents the frequencies of locomotion and rearing and the plus-maze data for rats of groups PC1, P1 and NC1, obtained 30 min after the end of the inhalation challenge on postimmunization day 56. As can be seen, no differences were found between the rearing (F2,57 5
J. Palermo-Neto, R.K. Guimarães / Life Sciences 68 (2000) 611–623
617
Table 2 Effects of Pavlovian conditioning of a lung analphylactic response on open-field and plus maze data of OVA-sensitized rats observed 30–40 min after the challenge test performed on day 56 of sensitization Plus maze Open-field (1)
Groups PC1 P1 NC1
Locomotion
Rearing
% Open arm entries
Time spent in open arms
82.4 6 16.0*(2) 108.2 6 23.8 131.3 6 16.5
30.7 6 0.7 24.2 6 1.5 21.8 6 1.7
10.0 6 5.0* 23.4 6 8.3 29.5 6 11.2
106.6 6 38.1* 190.0 6 60.0 193.2 6 27.4
PC1,P1 and NC1 5 positive control, paired and negative groups, respectively (n5 20 rats/group). Data are mean 6 SEM. * p,0.05 compared to data for group C (ANOVA and Tuckey tests).
(1) (2)
1.28 P.0.05) frequencies of rats of groups PC1 (positive control), P1 (paired) and NC1 (negative control). Nevertheless, the locomotion frequency of rats of group PC1 (positive control) was lower than that of animals of group NC1 (negative control) (F2,57 5 9.62, p,0.05). No differences were found between data for groups P1 (paired) and NC1. Rats of group PC1 (sensitized with OVA and exposed to an OVA inhalation challenge) presented a decreased percentage of entries into the plus maze open arms (F2,575 10.3 P,0.05) and a decrease in time spent in open arm exploration (F2,57 5 8.4 P,0.05) compared to rats of group NC1. Further analysis showed no differences between the plus maze data for rats of groups P1 and NC1; thus, exposure to the CS in the inhalation chamber was unable to change per se the behavior of the animals observed in the apparatus 40 min after the end of the inhalation section. The behavioral data obtained in the 2nd experiment are presented in Table 3. No differences were found among the rearing data for the animals of the different groups (F2,27 5 2,01, p,0.05). Nevertheless, the locomotion frequency of animals of group PC2 (positive control) was significantly lower (F2,275 12.8 p,0.05) than those of rats of groups U2 (unpaired), NC2 Table 3 Effects of Pavlovian conditioning of a lung analphylactic response on open-field and plus maze data of OVA-sensitized rats observed in both apparatuses in the absence of antigen (US). Rats of groups PC2, U2 and NC2 were observed in the presence of the audiovisual cue (CS) Plus maze Open-field Groups(1) PC2 U2 NC2 N
Locomotion
Rearing
% of open arm entries
Time spent in open arms
54.1 6 18.9(2)* 128.7 6 10.2 111.2 6 9.3** 187.0 6 8.8
18.4 6 6.4 16.5 6 4.4 24.1 6 5.8 32.7 6 10.2
13.1 6 6.1* 25.5 6 7.0 31.0 6 12.0 43.1 6 9.4
56.3 6 19.1* 93.9 6 27.0 118.4 6 10.5** 182.2 6 21.6
PC2, U2, NC2 and N 5 positive control, unpaired, negative control and naive, respectively (n5 10 rats/ group). (2) Data are mean 6 SEM. * p,0.05 compared to data for groups U2, NC2 and N. * p,0.05 compared to data for group N. (1)
618
J. Palermo-Neto, R.K. Guimarães / Life Sciences 68 (2000) 611–623
(negative control) and N (naive). Thus, CS presentation per se induced a significant decrease in the open-field parameters of rats, since animals of group PC2 were observed in the apparatus in the presence of the audiovisual cue and in the absence of the US (1% OVA challenge) paired on inhalation days 35, 42 and 49 post-sensitization. In contrast, data for groups U2 did not differ from those for group NC2. Furthermore, the open-field data for rats of group NC2 were smaller than those of group N (P,0.05), suggesting that the audiovisual stimuli decreased per se (P,0.05) the animals’ locomotion and rearing frequencies in the open-field. The obtained plus-maze data point in the same direction. Thus, the percentage of open arm exploration and the total time spent in open arm exploration by rats of group PC2 (observed in the apparatus in the presence of CS, and in the absence of US) were smaller (F2,27 5 7.2 and 9.8, p,0.05 respectively) than those measured in rats of groups U2, NC2 and N. No differences were found between data of groups U2 and NC2 in the plus maze. Finally, the total time spent in open arm exploration by rats of group NC2 was smaller than that spent by animals of group N. Biochemical analysis of serum corticosterone levels showed that CS presentation per se in the animals submitted to Pavlovian conditioning (group PC2) increased (F2,275 18.1, p,0.05) the serum levels of this hormone compared to those measured in animals of the control groups U2 (unpaired) NC2 (negative control) and N (naive) : NC25 183.14 6 22.31; N 5 107.12 6 16.40; PC2 5 321.66 6 23.41 and U2 5 202.12 6 16.7. Significant differences were also observed between data of groups U2 and NC2 in relation to those of group N. Discussion The present findings demonstrate that in certain conditioning situations the CNS can induce anaphylaxis in previously sensitized animals. This conclusion is based in the appearance of signs and symptoms of LAR in animals submitted to the Pavlovian conditioning and challenged 56 days after OVA sensitization in the absence of the antigen and in the presence of the audiovisual cue. This fact is relevant and suggests that the conditioned response now imposed is physiopathologically meaningful. Indeed, the present investigation agrees with other studies suggesting that conditioned responses are relevant for the appearance, intensity and maintenance of an anaphylactic response (4–6). In the context of this discussion, stress is defined as a complex process by which an organism responds to either external environmental or psychological events that pose a challenge or danger to the organism (16); thus, the stressful stimuli will be referred to as “stressor” and the response as the “stress response”. Also, “anxiety level” will be operationally inferred as suggested elsewhere (11,12), i.e., as the response to a situation in which behavior is influenced by two opposing motivational forces (e.g. a natural curiosity to explore unexplored or novel areas versus an aversion to open areas). In this respect, it is known that behavioral evaluations performed in the open-field and in the plus maze apparatus as well as biochemical analysis of serum corticosterone levels are good indexes of both stress response and anxiety levels (17,18). Thus, the present results also showed high levels of stress response and/or anxiety (SRAL) after the course of LAR (1st experiment) and, importantly, they showed that these conditions are also susceptible to Pavlovian conditioning (2nd experiment). Taken together, the present data support the increasing evidence for CNS involvement in the regulation of immune function (5,6).
J. Palermo-Neto, R.K. Guimarães / Life Sciences 68 (2000) 611–623
619
In an experiment similar to the present one, OVA injections, which stimulate mucosal mast cells to secrete mediators, were paired with an audiovisual cue; after reexposure to the audiovisual cue, a mediator (rat mast cell protease II - RMCPII) was measured. It was observed that animals reexposed to only the audiovisual cue released a quantity of protease not significantly different from that released by animals reexposed to both cue and antigen (5). Since RMCPII is present not only in the intestine but also in the mucosal lamina of the lung (19, 20) it seems feasible to think that this protease might be also involved in the present data. Indeed, RMCPII plays an important role in the secretion of histamine (8) and it is common knowledge that histamine increases vascular permeability and produces bronchoconstriction (21). Thus CNS might be regulating the secretion of a mediator known to occur in mucosal mast cells. Indeed, there is considerable in vitro and in vivo evidence supporting both morphological and functional nerve-mast cell interactions (22–24). OVA exposure of sensitized animals has been previously associated with the presence of activated T cells in the airway mucosa (25). According to Holgate (26), in response to allergens, T-cells produce a restricted array of cytokines encoded in a small cluster on the long arm of chromosome 5 at bands 31–33, which has been shown to be the IL-4 gene cluster. In particular, proinflammatory cytokines like IL-4 were reported to be produced by a subtype of T helper cells known as TH2. Nevertheless, TH2 cells are not the only source of proallergic cytokines. Mast cells, basophils, eosinophyls, Cd81 T cells, other cytokines and, under certain circumstances, bronchial epithelial cells, fibroblasts, dendritic cells and smooth muscle can all produce cytokines encoded by the IL-4 gene cluster (27). Thus, it is not simple to define the targeting point of the conditioning discussed here. It was reported that stressors inhibit exploratory behavior when the test animal is placed in an open-field (18, 28–30). This effect was also reported after other behavioral tests which rely on exploratory behavior (31). In contrast, anxiolytic compounds, in particular benzodiazepines (12,32) and 5-HT2 antagonists such as ritanserin (33), have been reported to cause increases in exploratory behavior in different situations. Accordingly, decrements in open arm exploration in the plus maze apparatus have been reported to indicate high levels of anxiety (11,12,17). Thus, the open-field and plus maze data of the 1st experiment show that stress and anxiety were observed 30–40 min after LAR. Indeed, in this experiment, the locomotion frequency of rats of group PC1 (positive control) observed 30 min after the OVA aerosol challenge was lower than that measured in rats of group NC1 (negative control). Furthermore, the percentage of entries into the plus maze open arms and the time spent in open arm exploration were smaller in animals of group PC1 compared to group NC1. In contrast, the behavioral data of rats of groups P1 (paired) and NC1 were not significantly different. Thus, if the Pavlovian conditioning was indeed imposed, as evidenced by LAR scores, how is it possible to explain the absence of differences now detected in experiment 1 between the open-field and plus maze data of rats of groups PC1 (positive control) and P1 (paired)? These differences are probably due to two possible underlying variables: the absence of the audiovisual cue (CS) during behavioral observations in both apparatuses, and/or the time interval elapsed between the inhalation tests and the open-field and plus maze experiments. Indeed, when the rats were tested in both apparatus in the presence of the imposed CS (2nd experiment) differences were found between the behavioral data of the animals of the four groups. Thus, the SRAL were higher in rats of group PC2 (positive control) in relation to
620
J. Palermo-Neto, R.K. Guimarães / Life Sciences 68 (2000) 611–623
those measured in animals of groups U2 (unpaired), NC2 (negative control) and N (naive). Biochemical data related to serum corticosterone levels obtained for these rats immediately after the behavioral observations supported this assumption since they pointed in the same direction. Indeed, a positive correlation was found between the behavioral and biochemical data indicative of stress and anxiety in the present experiment (Pearson’s correlation r 5 0.95 and 0.93 for corticosterone 3 open-field and corticosterone 3 plus maze, respectively). Thus, the data of the 2nd experiment also show that stress and/or anxiety are related to LAR and that these behavioral states are susceptible of Pavlovian conditioning. In a review, Dunn (34) provided several lines of evidence indicating that immune challenge can activate the hypothalamus-pituitary-adrenocortical axis (HPA). Briefly, interleukin-1 (IL-1) produced by lymphocytes during the immune response activates noradrenergic projections from the brainstem to the hypothalamic paraventricular nucleus; this input activates the HPA, stimulating the release of corticotropin-releasing factor (CRF), which in turn stimulates the secretion of both pituitary ACTH and adrenal glucocorticoid hormone (34–36). As already stated, inflammatory changes and cytokines have been associated with airway hyper-responsiveness and inflammatory events observed in asthma (37,38) or in OVAexposed rats (24). Thus, it is not at all impossible to suggest that IL-1 took part in the events that led to the behavioral changes now observed in the 2nd experiment. Indeed, according to Connor et al. (39), IL-1b induced “anxiogenic-like” effects in rats analyzed in the elevated plus maze. Furthermore, according to Dunn et al (36), brain CRF is involved in the reduction of exploratory behavior induced by ip injection of IL-1. In this context, the production and release of this cytokine might also explain the behavioral differences found in the 1st experiment between rats of group PC1 (positive control) and P1 (paired). Indeed, animals of group PC1 were challenged with antigen in the presence of the audiovisual cue, whereas those of group P1 received no antigen in the presence of CS. Under such conditions, the inflammatory component of the airway hyper-responsiveness and the production and release of interleukins might be thought to be qualitatively and/or quantitatively different. Furthermore, these animals were observed in the behavioral apparatus at least 40 min after the inhalation session and in the absence of CS. These facts might be also relevant to the understanding of the present data, since changes in HPA activation induced by immune stimulation were already reported to be time dependent (40–41). Finally, it seems important to point out that the audiovisual cue per se was stressful and anxiogenic, a fact already reported in different experimental contexts (42, 43). Indeed, some of the behavioral and biochemical data now obtained showed that the audiovisual presentation increased the animals’ SRAL compared to rats observed in the absence of environmental stimuli (group N). Nevertheless, particularly relevant for the present discussion are the significant differences now found between the behavioral and biochemical data of rats of groups PC2 (positive control) and U2 (unpaired) and the lack of differences between those of groups U2 (unpaired) and NC2 (negative control). Thus, it seems indisputable that the Pavlovian conditioning presently imposed clearly displayed a relationship between SRAL and LAR. If interleukins are indeed involved in the generation of airway hyper-responsiveness it seems feasible to think that the CNS can regulate their production and/or release, as suggested before by MacQueen et al. (5) for RMCPII secretion that, as discussed previously, might be also involved with the present data. In this respect, a pituitary-independent stress-
J. Palermo-Neto, R.K. Guimarães / Life Sciences 68 (2000) 611–623
621
induced suppression of peripheral blood lymphocyte proliferation has been related to central and peripheral catecholaminergic systems, which have been shown to regulate the immune processes (44). Thus, it might be that the final neural link between SRAL and airway hyperresponsiveness after OVA solution aerosol challenge in OVA-sensitized rats involves also the neural fibers that have been shown to terminate in close association not only with T lymphocytes and macrophages (45) but also with mast cells (20, 21, 23). Indeed, it is common knowledge that lymphocytes, macrophages and mast cells are closely linked both to lung anaphylactic responses (24, 25, 46) and to interleukin production (26).
Acknowledgments This research was supported by FAPESP (No 99/04228-7) and CNPq (No 520050/975Nv) to whom the authors wish to express sincere thanks.
References 1. Hopkins SJ, Rotwell NJ. Cytokines and the nervous system I: Expression and recognition. Trends in Neuroscicence 1995;18:83–87. 2. Dunn AJ. Interactions between the Nervous System and the Immune System. In: Bloom FE, Kupfer DJ, editors. Psychopharmacology: The Fourth Generation of Progress. New York: Raven Press, Ltd., 1995, pp. 719–731. 3. Khan AU, Bonk C, Gordon Y. Non-allergic asthma and conditioning process. Annals of Allergy 1974; 32: 245. 4. Khan AU. Effectiveness of biofeedback and counter-conditioning in the treatment of bronchial asthma. Journal of Psychosomatic Research 1977; 21(2):97–104. 5. MacQueen G, Marshall J, Perdue M, Siegel S, Bienenstock J. Science 1989;243:83–85. 6. Ader R, Cohen N. Psychoneuroimmunology: Conditioning and stress. Annals Review of Psychology 1993; 44:53–85. 7. Dyck DG, Greenberg AH, Osachuck TA. Tolerance to drug-induced (poly I:C) natural killer cell activation: congruence with a Pavlovian conditioning model. Journal of Experimental Psychology and Animal Behavioral Processes 1986;12:25–31. 8. Russell M, Dark KA, Cummins RW, Ellman G, Callaway E, Peeke HV. Learned histamine release. Science 1984;225:733–734. 9. King SJ, Miller HR. Anaphylactic release of mucosal mast cell protease and its relationship to gut permeability in Nippostrongylus-primed rats. Immunology 1984;51:653–60. 10. Bernardi MM, De Souza H, Palermo-Neto J. Effects of single and long-term haloperidol administration on open field behavior of rats. Psychopharmacology 1981;73:171–175. 11. Pelow S, Chopin P, File SE, Briley M. Validation of open: closed are entries in an elevated plus-maze as a measure of anxiety in the rat. Journal of Neuroscience Methods 1985b;14:149–167. 12. File SE. Interactions of anxiolytic and antidepressant drugs with hormones of the hypothalamic-pituitaryadrenal axis. Psychopharmacology of Anxiolytics and Antiderpessants. Pergamon Press, New York, 1991. pp. 29–55. 13. Sutano W, De Kloet ER. Species-spedificity of corticosteroid receptors in hamster and rat brains. Endocrinology 1987;121:1405–1511. 14. Albers HE, Yogev L, Todd RB, Goldman BD. Adrenal corticoids in hamsters: role of circadian timing. American Journal of Physiology 1985;248:434–438. 15. Johnson N, Leone F. Statistics and Experimental Design. In: Johnson N, Leone F, editors. Engineering and Physical Sciences, New York: John Wiley, 1977. pp. 241–244.
622
J. Palermo-Neto, R.K. Guimarães / Life Sciences 68 (2000) 611–623
16. Gatchell RJ, Baum A. An Introduction to Health Psychology Reading. Maryland: Addison-Wesley Publishing Company, 1983. 17. Comissaris RL. Conflict behaviors as animal models for the study of anxiety. In: F. van Haaren, editor. Methods in behavioral pharmacology. New York: Elsevier Science Publishers, 1993. pp. 443–474. 18. Paré WP, Glavin GB. Animal models of stress in pharmacology. In: F. van Haaren, editor. Methods in behavioral pharmacology. New York: Elsevier Science Publishers, 1993. pp. 413–441. 19. Lee TD, Sweitzer M, Bienenstock J,. Befus AD. Heterogeneity in mast cell populations. Clinical Immunology Review 1985;4:143. 20. Schwartz LB. Mediators of human mast cells and human mast cell subsets. Annals of Allergy 1987;58: 262–35. 21. Barnes PJ. Therapeutic strategies for allergic diseases. Nature 1999; 402: B31–B38. 22. Olsson Y. Mast cells in human peripheral nerve. Acta Neurologica Scandinavica 1971;(47):357–368. 23. Harari Y, Russel DA, Castro GA. Anaphylaxis-mediated epithelial C1-secretion and parasite rejection in rat intestine. Journal of Immunology 1987;138(4):1250–5. 24. Hagermark O, Hockfelt T, Pernow B. Flare and itch induced by substance P in human skin. Journal of Investigation in Dermatology 1978;71:233–237. 25. Haczku A, Moqbel R, Jacobson M, Kay AB, Barnes PJ, CHUNG KF. T-cells subsets and activation in bronchial mucosa of sensitized Brown-Noway rats after single allergen exposure. Immunology 1995;85:591– 597. 26. Holgate ST. The epidemic of allergy and asthma. Nature 1999;402:2–4. 27. Chung KF, Barnes PJ. Cytokines in asthma. Thorax 1999;54:825–857. 28. Weiss JM, Baley WH, Pohorecky LA, Korzeniowski D, Grillone G. Stress-induced depression of motor activity correlates with regional changes in brain norepinephrine but not in dopamine. Neurochemical Research 1980;5:9–22. 29. Katz RJ. Animals models of human depressive disorders. Neuroscience and Biobehavioral Review 1981;5: 231–246. 30. Kennett GA, Chaouloff F, Marcou M, Curzon G. Female rats are more vulnerable than males in an animal model of depression: the possible role of serotonim. Brain Research 1986;382:416–421. 31. Crawley JN. Exploratory behavior models of anxiety in mice. Neuroscience and Biobehavioral Review 1985; 9:37–44. 32. File SE. How good is social interaction as a test of anxiety? In: Simon P, Soubrie P, Wildlocher D, editors. Psychopharmacology of Anxiolytics and Antidepressants. New York: Pergamon Press, 1991. pp. 2955. 33. Meert TF, Melis W, Aserts N, Clincke G. Antagonism of meta-chlorophenylpiperazine-induced inhibition of exploratory activity in an emergence procedure, the open field test, in rats. Behavioral Pharmacology 1997; 8(4):353–63. 34. Dunn AJ. Interleukin-1 as a stimulator of hormone secretion. Progress in Neurology Endocrinology and Immunology 1990;3:26–34. 35. Besedovsky H, Del Rey A, Sorkin E, Dinarello CA. Immunoregulatory feedback between interleukin-1 and glucocorticoid hormones. Science 1986;233:652–654. 36. Dunn AJ, Antoon M, Chapman Y. Reduction of exploratory behavior by intraperitoneal injection of interleukin-1 involves brain corticotropin-releasing factor. Brain Research Bulletin 1991;26:539–542. 37. Hamid Q, Azzawi M, Ying S. Expression of mRNA for interleukins in mucosal bronchial biopsies from asthma. Journal of Clinical Investigation 1991;87:1541. 38. Robinson D, Hamid Q, Bentley A, Sun Y, Kay AB, Durham SR. Activation of CD41 T cells, increased TH2type cytokine mRNA expression, and eosinophil recruitment in bronchoalveolar lavage after allergen inhalation challenge in patients with atopic asthma. Journal of Allergy and Clinical Immunology 1993;92: 313–324. 39. Connor TJ, Song C, Leonard BE, Merali Z, Anisman H. An assesment of the effects of central interleukin1beta, -2, -6, and tumor necrosis factor administration on some behavioral, neurochemical, endocrine and immune parameters of rat. Neuroscience 1998;84(3):923–33. 40. Dunn AJ. Endotoxin-induced activation of cerebral catecholamine and serotonin metabolism: comparison with interleukin-1. Journal Pharmacology and Experimental Therapeutics 1992;261:964–969.
J. Palermo-Neto, R.K. Guimarães / Life Sciences 68 (2000) 611–623
623
41. Stenzel-Poore M, Vale WW, Rivier C. Relationship between antigen-induced immune stimulation and activation of the hypothalamic-pituitary-adrenal axis in the rat. Endocrinology 1993;132:1313–1318. 42. Walsh RN, Cummins RA. The open-field test: a critical review. Psychology Bulletin 1976;86:482–504. 43. Porsolt RD, McArthur RA, Lenègre A. Psychotropic screening procedures. In: F. van Haaren, editor. Methods in behavioral pharmacology. New York: Elsevier Science Publishers, 1993. pp. 23–51. 44. Keller SE, Schleifer SJ, Liotta AS, Bond RN, Farhoody N, Stein M. Stress-induced alterations of immunity in hypophysectomized rats. Proceedings of the National Academy of Sciences 1988;85:9297–301. 45. Felten D, Felten SY, Carlson SL, Olschowka JA, Liunat S. Noradrenergic and peptidergic innervation of lymphoid tissue. Journal of Immunology 1985;135:755s–765s. 46. Holt PG, Macaubas C, Stumbles PA, Sly PD. The role of allergy in the development of asthma. Nature 1999; 402:12–17.
Pavlovian conditioning of lung anaphylactic response in rats J. Palermo-Neto*, R.K. Guimarães Applied Pharmacology and Toxicology Laboratory, Department of Pathology, School of Veterinary Medicine, University of São Paulo, São Paulo, Brazil Received 20 March 2000; accepted 7 June 2000
Abstract The present experiment was undertaken to verify if it is possible to impose Pavlovian conditioning on a lung anaphylactic response (LAR) in rats. Two experiments were done. In the 1st, egg albumin (OVA) aerosol inhalation, which induces signs and symptoms of LAR in OVA- sensitized rats, was paired with an audiovisual cue (conditional stimulus, CS). After reexposure to the CS, the signs and symptoms of LAR were quantitatively measured using a scoring system specially developed for this evaluation; the levels of stress response and anxiety were also quantified. Results showed that the rats reexposed to CS only, displayed LAR scores not significantly different from those reexposed to both CS and the antigen; animals of these groups showed significantly higher LAR scores than rats that received no OVA aerosol challenge. High levels of stress and anxiety were observed 30–40 min after the challenge with OVA aerosol. In the 2nd experiment, rats sensitized with OVA and submitted or not to Pavlovian conditioning were observed in the open-field and in the plus maze apparatus in the absence of OVA aerosol but in the presence of the CS; after behavioral observations the animals were sacrificed for serum corticosterone level determination. Both behavioral and biochemical data showed high levels of stress and anxiety in rats for which the antigen was previously paired with the CS; these changes were not observed in animals which received the antigen 24 h after the presentation of the CS (unpaired) or in those exposed to PBS aerosol (the OVA vehicle) only. The present data show not only that LAR can be submitted to Pavlovian conditioning, but also and importantly, that high levels of stress and anxiety are related to the course of LAR. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Anaphylaxis; Lung; Pavlovian conditioning; Stress; Anxiety; Neuroimmunomodulation
Introduction Several studies have focused on the existence of body-mind interactions both in health and in sickness. The long-held view that homeostatic mechanisms are integrated by nervous and * Corresponding author: Faculdade de Medicina Veterinária e Zootecnica da Universidade de São Paulo, Laboratório de Farmacologia Aplicada e Toxicologia, Av. Prof. Dr. Orlando Marques de Paiva, 87, CEP:05508-900, São Paulo - SP, Brazil. Tel.: 55 011 211.3074; fax: 55-011 210.2224. E-mail address: [email protected] (J. Palermo-Neto) 0024-3205/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 9 6 6 -8
612
J. Palermo-Neto, R.K. Guimarães / Life Sciences 68 (2000) 611–623
endocrine systems has been expanded by information that these systems interact with the immune system. Immune responses alter neural and endocrine functions, and in turn, neural and endocrine activity modifies immunologic function (1,2). Several studies have suggested that anaphylaxis can occur in the absence of any antigenic stimulus or physical irritant and may represent a conditioned response (3–5). In other words, neutral stimuli such as sensory events (conditional stimulus, CS) may become associated with antigens in such a way that subsequent exposure to these stimuli will elicit anaphylatic responses. In Pavlovian conditioning terms, the antigen is an unconditioned stimulus (US) that elicits an unconditioned response, i.e. anaphylaxis. In this respect, the observation that the immune system is influenced by Pavlovian conditioning (6) has provided strong evidence for neuro-immune interactions. The existence of a conditioned immunomodulation was described after treatment with both immunossupressive (6) and immunoenhancing agents (7); conditioned alterations of immune function in animals trained with antigens were also reported (8). The Central Nervous System (CNS) was also shown to play an important role in the conditioned secretion of histamine (8), acting as a functional effector of mast cell function in the allergic state (5). Taken together, these findings support the increasing evidence for CNS involvement in the regulation of both immune function and response in the allergic state. Symptoms of anaphylaxis, particularly those related to lung function, are known to be increased during stressful situations and/or high levels of anxiety. In addition, high levels of anxiety and stress are frequently reported in the emergence of an asthmatic crisis. However, experimental laboratory data supporting a relationship between lung anaphylactic response (LAR) and anxiety levels are lacking. Thus, the present experiment was designed to determine if it is possible to impose Pavlovian conditioning on LAR and also to analyze some possible relationships between behavioral and biochemical data indicative of levels of stress/ anxiety and LAR. Material and methods Animals One hundred pathogen free and genetically similar male Wistar rats from our colony, weighing 230–250 g and about 75–80 days of age, were used. The animals were housed in temperature-controlled (21–238C) and artificially lighted rooms on a 12-h light/12-h dark cycle (lights on at 7:00 a.m.) with free access to food and water. The experiments were performed in a different room, at the same temperature as the animal colony, to which the animals were transferred and maintained in their home cages 2 h before the experiments. Animals were housed and used in accordance with the guidelines of the Committee on Care and Use of Laboratory Animal Resources of the School of Veterinary Medicine, University of São Paulo; these guidelines are similar to those of the National Research Council, USA. Sensitization and conditioning protocol Aluminum-precipitated egg albumin, OVA (Sigma Chemical Company, USA) prepared in phosphate buffered saline (PBS), was intraperitonially (ip) administered to the rats at a dose of 4.0 mg/kg (1.0 ml/kg solution); this was considered to be 0 day of sensitization. The alu-
J. Palermo-Neto, R.K. Guimarães / Life Sciences 68 (2000) 611–623
613
minum precipitate acts as an adjuvant to promote IgE production (9). After this treatment, animals were randomly and equally divided into different groups (see ahead, under experimental design) being repeatedly given an antigen (OVA) aerosol inhalation challenge after sensitization; on these days, we imposed or not the typical Pavlovian conditioning procedure, summarized in Table 1. Aerosol exposure was accomplished by placing the rats individually in a 40 3 50 3 20 cm glass chamber connected to an inhalation nebulizer (Inalamax, model S3; NS Indústria de Apar eˆlhos Médicos, Brasil) that generated an aerosol mist pumped into the exposure chamber by the airflow supplied by a small animal ventilator set at 60 strokes/min with a pumping volume of 10 ml. Some rats were exposed to an aerosol of 1% OVA solution in PBS (w/v) for 15 min. The Pavlovian conditioning imposed was based on the method described elsewhere (5). Briefly, a CS consisted of an audiovisual cue (the sound of the motor of the inhalation apparatus plus the flashing of a 10 Watt green lamp placed 10 cm above the chamber) was paired with the US (the 1% OVA challenge) during the three sessions of aerosol exposure. Some animals (negative controls) were placed for the same period of time (15 min), and also for three times, in identical chambers and in the presence of the audiovisual cue (CS), but received no OVA, i.e., during the 15 min period of inhalation they were exposed to PBS buffer only (see Table 1). Behavioral studies The animals were observed between 8:00 and 12:00 a.m. for signs and symptoms of LAR, exploratory behavior in an open-field and free explorations of a plus-maze apparatus. To minimize the influence of possible circadian changes in the behavioral parameters measured, animals of the different groups were alternated and always observed at the same time of day.
Table 1 Sensitization and conditioning protocol used to impose a Pavlovian Conditioning of a lung anaphylactic response in rats Treatments (days) Experiment
Groups PC1-positive control P1-paired NC1-negative control PC2-positive control U2-umpaired NC2-negative control N-naive
1
2
n 20 20 20 10 10 10 10
0 (1) OVA OVA OVA OVA OVA OVA PBS
34,41 48
35,42 49
— — — — CS(4) — —
OVA 1 CS OVA 1 CS PBS 1 CS(3) OVA 1 CS OVA(4) PBS 1 CS —
56 (2)
5 number of animals sensitization day 5 4.0 mg/kg OVA, i.p. (2) 1% OVA solution aerosol challenge for 15 min, in the presence of an audiovisual cue (CS) (3) PBS buffer aerosol inhalation for 15 min in the presence CS (4) OVA and CS were given alone (5) CS was delivered during the open-field and plus maze observations n
(1)
OVA 1 CS PBS 1 CS PBS 1 CS CS(5) CS CS —
614
J. Palermo-Neto, R.K. Guimarães / Life Sciences 68 (2000) 611–623
The experimental devices used were washed with an alcohol-water solution (5%) before placing the animals in the chamber, to obviate possible biasing effects due to odor clues left by previous rats. The signs and symptoms of LAR were quantified in the inhalation chambers during the 15 min period of OVA or PBS buffer inhalation and CS presentation. Briefly, scores from 0 to 5 were attributed to each animal’s behavior by an observer who was not aware of the treatments. The grading system was as follows: 0, asleep or stationary; 1, active, with few bursts of head scratching; 2, predominantly active, with bursts of head scratching and grooming; 3, constant activity characterized by rearing, head scratching, sniffing, grooming, and presence of a few sneezes; 4, constant rearing, head scratching, sniffing, grooming, presence of a large number of sneezes and a few mouth openings (forced inspirations); 5, constant mouth openings, and presence of postural alterations (animals tend to keep their trunk in an upright position). This criterion was not subjective as shown by the excellent scoring agreement found, in previously conducted pilot studies, by two different and independent observers that analyzed the behavior of rats similarly sensitized and challenged with OVA (Pearson’s correlation, r 5 0.91).The open-field used was constructed as described elsewhere (10). Briefly, it comprises a round wooden arena (97 cm in diameter and 32 cm high walls), painted white and divided by black lines into 25 similar parts; during the experiment the device was lit by two 40 Watt white bulb. For the observations, each rat was individually placed in the center of the apparatus and behavioral parameters were measured for 6 min. Each animal was observed remotely using a video camera; hand-operated counters were employed to score locomotion frequency (number of floor units entered with both front feet) and rearing frequency (number of times the animals stood on their hind legs). For behavioral analyses in the plus maze the rats were placed in the central square of the apparatus (10 3 10 cm), and allowed 5 min of free exploration. The plus-maze device used was made of wood and had two open arms (50 3 10 cm) and two enclosed arms of the same size with 40 cm high walls, and was elevated 50 cm above the ground. Each rat was observed remotely using a video camera for a period of 5 min for the number of entries into each type of arm (all four paws defining an entry) and the time spent in open and closed arms. The measures that reflect anxiety in this test are the percentage of entries into open arms and the percentage of time spent in open arms (11, 12). Serum corticosterone determination Corticosterone is the most abundant circulating steroid secreted by rats, being considered a good indicator of adrenocortical function in this animal species (13). This hormone was determined in serum using commercial kits (Coat-A-Count, USA); this procedure is based on a solid-phase radioimmunoassay in which 125I-labeled corticosterone competes for a fixed time with corticosterone in the rat sample for antibody sites. Serum samples were assayed directly without extraction or purification. In order to decrease data variability and to avoid possible effects of handling stress on serum corticosterone levels, the rats were handled daily for habituation to the experimental conditions of blood collection and were kept far from the room where animals were sacrificed. In addition, animals were sacrificed at the same time of day (between 8:00 and 9:00 a.m.) in order to minimize the reported circadian effects on serum glucocorticoid levels (14).
J. Palermo-Neto, R.K. Guimarães / Life Sciences 68 (2000) 611–623
615
Experimental design Two experiments were done according to standardized GLP guideliness. In the first, 60 rats were randomly divided into three equal groups PC1 (positive control), P1 (paired) and NC1 (negative control) and submitted to the sensitization, challenge and conditioning protocols described above and summarized in Table 1. The rats of groups PC1 and P1 were repeatedly given a 1% OVA aerosol challenge on days 35, 42 and 49 of sensitization in the presence of CS, when Pavlovian conditioning was imposed. On those days, the rats of group NC1 received PBS buffer in the inhalation chamber for the same period of time and in the presence of the audiovisual cue. These animals were then tested 56 days after sensitization; for this purpose, animals of group PC1 were again challenged for 15 min. with a 1% OVA solution, whereas those of groups P1 and NC1 received PBS buffer in the inhalation chamber for the same period of time. During the 4 sessions of inhalation, animals were observed for LAR, that was quantified using the scoring system presented above. Thirty min after the end of the inhalation test (56th day), each rat was individually placed in the center of an open-field apparatus for the determination of locomotion, and rearing frequencies; 4 min later (40 min after the end of the inhalation session), rats were individually transferred to the plus maze apparatus for behavioral evaluations. In the 2nd experiment, 40 rats were divided at random into 4 groups of 10 animals each: PC2 (positive control), U2 (unpaired), NC2 (negative control), and N (naive) as summarized in Table 1. The rats of groups PC2, U2 and NC2 were submitted to the sensitization, challenge and conditioning protocols described above; rats of group N were not sensitized, i.e., they were i.p treated with 0.1 ml/kg of PBS on day 0. The same audiovisual cue (CS) as used in the 1st experiment (the sound of the inhalation chamber plus the flashing of the 10 Watt green bulb) was now employed. The animals of groups PC2 (positive control) were challenged with a 1% OVA solution aerosol for 15 min and in the presence of CS on days 35, 42 and 49 of sensitization. On those days, the rats of group NC2 received PBS buffer in the inhalation chamber for the same period of time and in the presence of the audiovisual cue. Animals of group U2 (unpaired) were placed in the inhalation chamber for 15 min and in the presence of the CS, on days 34, 41 and 48 of sensitization, but received the 1% OVA solution aerosol challenge on the following days, in the absence of the audiovisual cue. On days 35, 42 and 49, the rats of group N were placed for 15 min. in the inhalation chambers free of OVA and PBS aerosols. Fifty six days after sensitization, animals of groups PC2, U2 and NC2 were observed in the open-field and immediately after in the plus-maze apparatus for behavioral alterations in the presence of the audiovisual cue used (CS); rats of group N were observed in both apparatuses in the absence of sound and light. During these behavioral observations, the animals were not exposed to OVA solution or PBS buffer aerosols. Immediately after the end of the plus maze test, all rats were sacrificed by decapitation and blood was collected for the determination of serum corticosterone levels. Statistical analysis Bartlett’s test (15) showed that the data concerning locomotion and rearing frequencies and serum corticosterone levels as well as those obtained in the plus maze were parametric (P,0.05). Thus, one-way analysis of variance followed by the Tukey test for comparison of
616
J. Palermo-Neto, R.K. Guimarães / Life Sciences 68 (2000) 611–623
Fig. 1. Effects of a Pavlovian conditioning on scores of a lung anaphylactic responses (LAR) in rats. Aluminumprecipitated OVA (4.0 mg/kg) was i.p. administered to rats of groups PC1, P1 and NC1 on experimental day 0. On days 35, 42 and 49 of sensitization, rats of groups PC1 and P1 were given 1% OVA aerosol challenge in the presence of CS (an audiovisual cue); on those days, the rats of group NC1 were exposed to aerosol of PBS buffer. On day 56, animals of groups PC1 were challenged with 1% OVA and those of groups P1 and NC1 with PBS buffer in the presence of CS. An asterisk on top of columns mean a statistically significant difference at P,0,05 (KruskalWallis and Dunnet tests).
cell means were used to analyze these data. The LAR scores were analyzed by Kruskal-Wallis (KW) analysis of variance for nonparametric data, followed by the Dunnett test for multiple comparisons. The StatPac Statistic Analysis Package was used throughout with the level of significance set at P,0.05 for all comparisons. Results Figure 1 shows that rats sensitized with OVA displayed signs and symptoms of anaphylaxis when challenged by the respiratory route with a 1% OVA solution; indeed, the LAR scores observed in animals of groups PC1 (positive control) and P1 (paired) during the 15 min inhalation period were significantly higher (KW2,57 5 15.73, 18.24, 10.76 and 12.01, p,0.05, for post-sensitization days 35, 42, 49 and 56, respectively) than those observed in rats of the control group (group NC1: negative control). This Figure also shows that the Pavlovian conditioning imposed was successful. Thus, on post-sensitization day 56, the CS presentation per se was able to induce high levels of LAR in the animals of group P1 (paired). In this day, no significant differences were found between data for groups PC1 (positive control) and P1 (paired). Table 2 presents the frequencies of locomotion and rearing and the plus-maze data for rats of groups PC1, P1 and NC1, obtained 30 min after the end of the inhalation challenge on postimmunization day 56. As can be seen, no differences were found between the rearing (F2,57 5
J. Palermo-Neto, R.K. Guimarães / Life Sciences 68 (2000) 611–623
617
Table 2 Effects of Pavlovian conditioning of a lung analphylactic response on open-field and plus maze data of OVA-sensitized rats observed 30–40 min after the challenge test performed on day 56 of sensitization Plus maze Open-field (1)
Groups PC1 P1 NC1
Locomotion
Rearing
% Open arm entries
Time spent in open arms
82.4 6 16.0*(2) 108.2 6 23.8 131.3 6 16.5
30.7 6 0.7 24.2 6 1.5 21.8 6 1.7
10.0 6 5.0* 23.4 6 8.3 29.5 6 11.2
106.6 6 38.1* 190.0 6 60.0 193.2 6 27.4
PC1,P1 and NC1 5 positive control, paired and negative groups, respectively (n5 20 rats/group). Data are mean 6 SEM. * p,0.05 compared to data for group C (ANOVA and Tuckey tests).
(1) (2)
1.28 P.0.05) frequencies of rats of groups PC1 (positive control), P1 (paired) and NC1 (negative control). Nevertheless, the locomotion frequency of rats of group PC1 (positive control) was lower than that of animals of group NC1 (negative control) (F2,57 5 9.62, p,0.05). No differences were found between data for groups P1 (paired) and NC1. Rats of group PC1 (sensitized with OVA and exposed to an OVA inhalation challenge) presented a decreased percentage of entries into the plus maze open arms (F2,575 10.3 P,0.05) and a decrease in time spent in open arm exploration (F2,57 5 8.4 P,0.05) compared to rats of group NC1. Further analysis showed no differences between the plus maze data for rats of groups P1 and NC1; thus, exposure to the CS in the inhalation chamber was unable to change per se the behavior of the animals observed in the apparatus 40 min after the end of the inhalation section. The behavioral data obtained in the 2nd experiment are presented in Table 3. No differences were found among the rearing data for the animals of the different groups (F2,27 5 2,01, p,0.05). Nevertheless, the locomotion frequency of animals of group PC2 (positive control) was significantly lower (F2,275 12.8 p,0.05) than those of rats of groups U2 (unpaired), NC2 Table 3 Effects of Pavlovian conditioning of a lung analphylactic response on open-field and plus maze data of OVA-sensitized rats observed in both apparatuses in the absence of antigen (US). Rats of groups PC2, U2 and NC2 were observed in the presence of the audiovisual cue (CS) Plus maze Open-field Groups(1) PC2 U2 NC2 N
Locomotion
Rearing
% of open arm entries
Time spent in open arms
54.1 6 18.9(2)* 128.7 6 10.2 111.2 6 9.3** 187.0 6 8.8
18.4 6 6.4 16.5 6 4.4 24.1 6 5.8 32.7 6 10.2
13.1 6 6.1* 25.5 6 7.0 31.0 6 12.0 43.1 6 9.4
56.3 6 19.1* 93.9 6 27.0 118.4 6 10.5** 182.2 6 21.6
PC2, U2, NC2 and N 5 positive control, unpaired, negative control and naive, respectively (n5 10 rats/ group). (2) Data are mean 6 SEM. * p,0.05 compared to data for groups U2, NC2 and N. * p,0.05 compared to data for group N. (1)
618
J. Palermo-Neto, R.K. Guimarães / Life Sciences 68 (2000) 611–623
(negative control) and N (naive). Thus, CS presentation per se induced a significant decrease in the open-field parameters of rats, since animals of group PC2 were observed in the apparatus in the presence of the audiovisual cue and in the absence of the US (1% OVA challenge) paired on inhalation days 35, 42 and 49 post-sensitization. In contrast, data for groups U2 did not differ from those for group NC2. Furthermore, the open-field data for rats of group NC2 were smaller than those of group N (P,0.05), suggesting that the audiovisual stimuli decreased per se (P,0.05) the animals’ locomotion and rearing frequencies in the open-field. The obtained plus-maze data point in the same direction. Thus, the percentage of open arm exploration and the total time spent in open arm exploration by rats of group PC2 (observed in the apparatus in the presence of CS, and in the absence of US) were smaller (F2,27 5 7.2 and 9.8, p,0.05 respectively) than those measured in rats of groups U2, NC2 and N. No differences were found between data of groups U2 and NC2 in the plus maze. Finally, the total time spent in open arm exploration by rats of group NC2 was smaller than that spent by animals of group N. Biochemical analysis of serum corticosterone levels showed that CS presentation per se in the animals submitted to Pavlovian conditioning (group PC2) increased (F2,275 18.1, p,0.05) the serum levels of this hormone compared to those measured in animals of the control groups U2 (unpaired) NC2 (negative control) and N (naive) : NC25 183.14 6 22.31; N 5 107.12 6 16.40; PC2 5 321.66 6 23.41 and U2 5 202.12 6 16.7. Significant differences were also observed between data of groups U2 and NC2 in relation to those of group N. Discussion The present findings demonstrate that in certain conditioning situations the CNS can induce anaphylaxis in previously sensitized animals. This conclusion is based in the appearance of signs and symptoms of LAR in animals submitted to the Pavlovian conditioning and challenged 56 days after OVA sensitization in the absence of the antigen and in the presence of the audiovisual cue. This fact is relevant and suggests that the conditioned response now imposed is physiopathologically meaningful. Indeed, the present investigation agrees with other studies suggesting that conditioned responses are relevant for the appearance, intensity and maintenance of an anaphylactic response (4–6). In the context of this discussion, stress is defined as a complex process by which an organism responds to either external environmental or psychological events that pose a challenge or danger to the organism (16); thus, the stressful stimuli will be referred to as “stressor” and the response as the “stress response”. Also, “anxiety level” will be operationally inferred as suggested elsewhere (11,12), i.e., as the response to a situation in which behavior is influenced by two opposing motivational forces (e.g. a natural curiosity to explore unexplored or novel areas versus an aversion to open areas). In this respect, it is known that behavioral evaluations performed in the open-field and in the plus maze apparatus as well as biochemical analysis of serum corticosterone levels are good indexes of both stress response and anxiety levels (17,18). Thus, the present results also showed high levels of stress response and/or anxiety (SRAL) after the course of LAR (1st experiment) and, importantly, they showed that these conditions are also susceptible to Pavlovian conditioning (2nd experiment). Taken together, the present data support the increasing evidence for CNS involvement in the regulation of immune function (5,6).
J. Palermo-Neto, R.K. Guimarães / Life Sciences 68 (2000) 611–623
619
In an experiment similar to the present one, OVA injections, which stimulate mucosal mast cells to secrete mediators, were paired with an audiovisual cue; after reexposure to the audiovisual cue, a mediator (rat mast cell protease II - RMCPII) was measured. It was observed that animals reexposed to only the audiovisual cue released a quantity of protease not significantly different from that released by animals reexposed to both cue and antigen (5). Since RMCPII is present not only in the intestine but also in the mucosal lamina of the lung (19, 20) it seems feasible to think that this protease might be also involved in the present data. Indeed, RMCPII plays an important role in the secretion of histamine (8) and it is common knowledge that histamine increases vascular permeability and produces bronchoconstriction (21). Thus CNS might be regulating the secretion of a mediator known to occur in mucosal mast cells. Indeed, there is considerable in vitro and in vivo evidence supporting both morphological and functional nerve-mast cell interactions (22–24). OVA exposure of sensitized animals has been previously associated with the presence of activated T cells in the airway mucosa (25). According to Holgate (26), in response to allergens, T-cells produce a restricted array of cytokines encoded in a small cluster on the long arm of chromosome 5 at bands 31–33, which has been shown to be the IL-4 gene cluster. In particular, proinflammatory cytokines like IL-4 were reported to be produced by a subtype of T helper cells known as TH2. Nevertheless, TH2 cells are not the only source of proallergic cytokines. Mast cells, basophils, eosinophyls, Cd81 T cells, other cytokines and, under certain circumstances, bronchial epithelial cells, fibroblasts, dendritic cells and smooth muscle can all produce cytokines encoded by the IL-4 gene cluster (27). Thus, it is not simple to define the targeting point of the conditioning discussed here. It was reported that stressors inhibit exploratory behavior when the test animal is placed in an open-field (18, 28–30). This effect was also reported after other behavioral tests which rely on exploratory behavior (31). In contrast, anxiolytic compounds, in particular benzodiazepines (12,32) and 5-HT2 antagonists such as ritanserin (33), have been reported to cause increases in exploratory behavior in different situations. Accordingly, decrements in open arm exploration in the plus maze apparatus have been reported to indicate high levels of anxiety (11,12,17). Thus, the open-field and plus maze data of the 1st experiment show that stress and anxiety were observed 30–40 min after LAR. Indeed, in this experiment, the locomotion frequency of rats of group PC1 (positive control) observed 30 min after the OVA aerosol challenge was lower than that measured in rats of group NC1 (negative control). Furthermore, the percentage of entries into the plus maze open arms and the time spent in open arm exploration were smaller in animals of group PC1 compared to group NC1. In contrast, the behavioral data of rats of groups P1 (paired) and NC1 were not significantly different. Thus, if the Pavlovian conditioning was indeed imposed, as evidenced by LAR scores, how is it possible to explain the absence of differences now detected in experiment 1 between the open-field and plus maze data of rats of groups PC1 (positive control) and P1 (paired)? These differences are probably due to two possible underlying variables: the absence of the audiovisual cue (CS) during behavioral observations in both apparatuses, and/or the time interval elapsed between the inhalation tests and the open-field and plus maze experiments. Indeed, when the rats were tested in both apparatus in the presence of the imposed CS (2nd experiment) differences were found between the behavioral data of the animals of the four groups. Thus, the SRAL were higher in rats of group PC2 (positive control) in relation to
620
J. Palermo-Neto, R.K. Guimarães / Life Sciences 68 (2000) 611–623
those measured in animals of groups U2 (unpaired), NC2 (negative control) and N (naive). Biochemical data related to serum corticosterone levels obtained for these rats immediately after the behavioral observations supported this assumption since they pointed in the same direction. Indeed, a positive correlation was found between the behavioral and biochemical data indicative of stress and anxiety in the present experiment (Pearson’s correlation r 5 0.95 and 0.93 for corticosterone 3 open-field and corticosterone 3 plus maze, respectively). Thus, the data of the 2nd experiment also show that stress and/or anxiety are related to LAR and that these behavioral states are susceptible of Pavlovian conditioning. In a review, Dunn (34) provided several lines of evidence indicating that immune challenge can activate the hypothalamus-pituitary-adrenocortical axis (HPA). Briefly, interleukin-1 (IL-1) produced by lymphocytes during the immune response activates noradrenergic projections from the brainstem to the hypothalamic paraventricular nucleus; this input activates the HPA, stimulating the release of corticotropin-releasing factor (CRF), which in turn stimulates the secretion of both pituitary ACTH and adrenal glucocorticoid hormone (34–36). As already stated, inflammatory changes and cytokines have been associated with airway hyper-responsiveness and inflammatory events observed in asthma (37,38) or in OVAexposed rats (24). Thus, it is not at all impossible to suggest that IL-1 took part in the events that led to the behavioral changes now observed in the 2nd experiment. Indeed, according to Connor et al. (39), IL-1b induced “anxiogenic-like” effects in rats analyzed in the elevated plus maze. Furthermore, according to Dunn et al (36), brain CRF is involved in the reduction of exploratory behavior induced by ip injection of IL-1. In this context, the production and release of this cytokine might also explain the behavioral differences found in the 1st experiment between rats of group PC1 (positive control) and P1 (paired). Indeed, animals of group PC1 were challenged with antigen in the presence of the audiovisual cue, whereas those of group P1 received no antigen in the presence of CS. Under such conditions, the inflammatory component of the airway hyper-responsiveness and the production and release of interleukins might be thought to be qualitatively and/or quantitatively different. Furthermore, these animals were observed in the behavioral apparatus at least 40 min after the inhalation session and in the absence of CS. These facts might be also relevant to the understanding of the present data, since changes in HPA activation induced by immune stimulation were already reported to be time dependent (40–41). Finally, it seems important to point out that the audiovisual cue per se was stressful and anxiogenic, a fact already reported in different experimental contexts (42, 43). Indeed, some of the behavioral and biochemical data now obtained showed that the audiovisual presentation increased the animals’ SRAL compared to rats observed in the absence of environmental stimuli (group N). Nevertheless, particularly relevant for the present discussion are the significant differences now found between the behavioral and biochemical data of rats of groups PC2 (positive control) and U2 (unpaired) and the lack of differences between those of groups U2 (unpaired) and NC2 (negative control). Thus, it seems indisputable that the Pavlovian conditioning presently imposed clearly displayed a relationship between SRAL and LAR. If interleukins are indeed involved in the generation of airway hyper-responsiveness it seems feasible to think that the CNS can regulate their production and/or release, as suggested before by MacQueen et al. (5) for RMCPII secretion that, as discussed previously, might be also involved with the present data. In this respect, a pituitary-independent stress-
J. Palermo-Neto, R.K. Guimarães / Life Sciences 68 (2000) 611–623
621
induced suppression of peripheral blood lymphocyte proliferation has been related to central and peripheral catecholaminergic systems, which have been shown to regulate the immune processes (44). Thus, it might be that the final neural link between SRAL and airway hyperresponsiveness after OVA solution aerosol challenge in OVA-sensitized rats involves also the neural fibers that have been shown to terminate in close association not only with T lymphocytes and macrophages (45) but also with mast cells (20, 21, 23). Indeed, it is common knowledge that lymphocytes, macrophages and mast cells are closely linked both to lung anaphylactic responses (24, 25, 46) and to interleukin production (26).
Acknowledgments This research was supported by FAPESP (No 99/04228-7) and CNPq (No 520050/975Nv) to whom the authors wish to express sincere thanks.
References 1. Hopkins SJ, Rotwell NJ. Cytokines and the nervous system I: Expression and recognition. Trends in Neuroscicence 1995;18:83–87. 2. Dunn AJ. Interactions between the Nervous System and the Immune System. In: Bloom FE, Kupfer DJ, editors. Psychopharmacology: The Fourth Generation of Progress. New York: Raven Press, Ltd., 1995, pp. 719–731. 3. Khan AU, Bonk C, Gordon Y. Non-allergic asthma and conditioning process. Annals of Allergy 1974; 32: 245. 4. Khan AU. Effectiveness of biofeedback and counter-conditioning in the treatment of bronchial asthma. Journal of Psychosomatic Research 1977; 21(2):97–104. 5. MacQueen G, Marshall J, Perdue M, Siegel S, Bienenstock J. Science 1989;243:83–85. 6. Ader R, Cohen N. Psychoneuroimmunology: Conditioning and stress. Annals Review of Psychology 1993; 44:53–85. 7. Dyck DG, Greenberg AH, Osachuck TA. Tolerance to drug-induced (poly I:C) natural killer cell activation: congruence with a Pavlovian conditioning model. Journal of Experimental Psychology and Animal Behavioral Processes 1986;12:25–31. 8. Russell M, Dark KA, Cummins RW, Ellman G, Callaway E, Peeke HV. Learned histamine release. Science 1984;225:733–734. 9. King SJ, Miller HR. Anaphylactic release of mucosal mast cell protease and its relationship to gut permeability in Nippostrongylus-primed rats. Immunology 1984;51:653–60. 10. Bernardi MM, De Souza H, Palermo-Neto J. Effects of single and long-term haloperidol administration on open field behavior of rats. Psychopharmacology 1981;73:171–175. 11. Pelow S, Chopin P, File SE, Briley M. Validation of open: closed are entries in an elevated plus-maze as a measure of anxiety in the rat. Journal of Neuroscience Methods 1985b;14:149–167. 12. File SE. Interactions of anxiolytic and antidepressant drugs with hormones of the hypothalamic-pituitaryadrenal axis. Psychopharmacology of Anxiolytics and Antiderpessants. Pergamon Press, New York, 1991. pp. 29–55. 13. Sutano W, De Kloet ER. Species-spedificity of corticosteroid receptors in hamster and rat brains. Endocrinology 1987;121:1405–1511. 14. Albers HE, Yogev L, Todd RB, Goldman BD. Adrenal corticoids in hamsters: role of circadian timing. American Journal of Physiology 1985;248:434–438. 15. Johnson N, Leone F. Statistics and Experimental Design. In: Johnson N, Leone F, editors. Engineering and Physical Sciences, New York: John Wiley, 1977. pp. 241–244.
622
J. Palermo-Neto, R.K. Guimarães / Life Sciences 68 (2000) 611–623
16. Gatchell RJ, Baum A. An Introduction to Health Psychology Reading. Maryland: Addison-Wesley Publishing Company, 1983. 17. Comissaris RL. Conflict behaviors as animal models for the study of anxiety. In: F. van Haaren, editor. Methods in behavioral pharmacology. New York: Elsevier Science Publishers, 1993. pp. 443–474. 18. Paré WP, Glavin GB. Animal models of stress in pharmacology. In: F. van Haaren, editor. Methods in behavioral pharmacology. New York: Elsevier Science Publishers, 1993. pp. 413–441. 19. Lee TD, Sweitzer M, Bienenstock J,. Befus AD. Heterogeneity in mast cell populations. Clinical Immunology Review 1985;4:143. 20. Schwartz LB. Mediators of human mast cells and human mast cell subsets. Annals of Allergy 1987;58: 262–35. 21. Barnes PJ. Therapeutic strategies for allergic diseases. Nature 1999; 402: B31–B38. 22. Olsson Y. Mast cells in human peripheral nerve. Acta Neurologica Scandinavica 1971;(47):357–368. 23. Harari Y, Russel DA, Castro GA. Anaphylaxis-mediated epithelial C1-secretion and parasite rejection in rat intestine. Journal of Immunology 1987;138(4):1250–5. 24. Hagermark O, Hockfelt T, Pernow B. Flare and itch induced by substance P in human skin. Journal of Investigation in Dermatology 1978;71:233–237. 25. Haczku A, Moqbel R, Jacobson M, Kay AB, Barnes PJ, CHUNG KF. T-cells subsets and activation in bronchial mucosa of sensitized Brown-Noway rats after single allergen exposure. Immunology 1995;85:591– 597. 26. Holgate ST. The epidemic of allergy and asthma. Nature 1999;402:2–4. 27. Chung KF, Barnes PJ. Cytokines in asthma. Thorax 1999;54:825–857. 28. Weiss JM, Baley WH, Pohorecky LA, Korzeniowski D, Grillone G. Stress-induced depression of motor activity correlates with regional changes in brain norepinephrine but not in dopamine. Neurochemical Research 1980;5:9–22. 29. Katz RJ. Animals models of human depressive disorders. Neuroscience and Biobehavioral Review 1981;5: 231–246. 30. Kennett GA, Chaouloff F, Marcou M, Curzon G. Female rats are more vulnerable than males in an animal model of depression: the possible role of serotonim. Brain Research 1986;382:416–421. 31. Crawley JN. Exploratory behavior models of anxiety in mice. Neuroscience and Biobehavioral Review 1985; 9:37–44. 32. File SE. How good is social interaction as a test of anxiety? In: Simon P, Soubrie P, Wildlocher D, editors. Psychopharmacology of Anxiolytics and Antidepressants. New York: Pergamon Press, 1991. pp. 2955. 33. Meert TF, Melis W, Aserts N, Clincke G. Antagonism of meta-chlorophenylpiperazine-induced inhibition of exploratory activity in an emergence procedure, the open field test, in rats. Behavioral Pharmacology 1997; 8(4):353–63. 34. Dunn AJ. Interleukin-1 as a stimulator of hormone secretion. Progress in Neurology Endocrinology and Immunology 1990;3:26–34. 35. Besedovsky H, Del Rey A, Sorkin E, Dinarello CA. Immunoregulatory feedback between interleukin-1 and glucocorticoid hormones. Science 1986;233:652–654. 36. Dunn AJ, Antoon M, Chapman Y. Reduction of exploratory behavior by intraperitoneal injection of interleukin-1 involves brain corticotropin-releasing factor. Brain Research Bulletin 1991;26:539–542. 37. Hamid Q, Azzawi M, Ying S. Expression of mRNA for interleukins in mucosal bronchial biopsies from asthma. Journal of Clinical Investigation 1991;87:1541. 38. Robinson D, Hamid Q, Bentley A, Sun Y, Kay AB, Durham SR. Activation of CD41 T cells, increased TH2type cytokine mRNA expression, and eosinophil recruitment in bronchoalveolar lavage after allergen inhalation challenge in patients with atopic asthma. Journal of Allergy and Clinical Immunology 1993;92: 313–324. 39. Connor TJ, Song C, Leonard BE, Merali Z, Anisman H. An assesment of the effects of central interleukin1beta, -2, -6, and tumor necrosis factor administration on some behavioral, neurochemical, endocrine and immune parameters of rat. Neuroscience 1998;84(3):923–33. 40. Dunn AJ. Endotoxin-induced activation of cerebral catecholamine and serotonin metabolism: comparison with interleukin-1. Journal Pharmacology and Experimental Therapeutics 1992;261:964–969.
J. Palermo-Neto, R.K. Guimarães / Life Sciences 68 (2000) 611–623
623
41. Stenzel-Poore M, Vale WW, Rivier C. Relationship between antigen-induced immune stimulation and activation of the hypothalamic-pituitary-adrenal axis in the rat. Endocrinology 1993;132:1313–1318. 42. Walsh RN, Cummins RA. The open-field test: a critical review. Psychology Bulletin 1976;86:482–504. 43. Porsolt RD, McArthur RA, Lenègre A. Psychotropic screening procedures. In: F. van Haaren, editor. Methods in behavioral pharmacology. New York: Elsevier Science Publishers, 1993. pp. 23–51. 44. Keller SE, Schleifer SJ, Liotta AS, Bond RN, Farhoody N, Stein M. Stress-induced alterations of immunity in hypophysectomized rats. Proceedings of the National Academy of Sciences 1988;85:9297–301. 45. Felten D, Felten SY, Carlson SL, Olschowka JA, Liunat S. Noradrenergic and peptidergic innervation of lymphoid tissue. Journal of Immunology 1985;135:755s–765s. 46. Holt PG, Macaubas C, Stumbles PA, Sly PD. The role of allergy in the development of asthma. Nature 1999; 402:12–17.