Full text of DOI:10.1080/09629350124118

Short Communication
Mediators of Inflammation, 10, 229–232 (2001)
T
HIS study was designated to evaluate the influence of
Evidence that behavioral depression
does not influence airway cell influx
in allergic rats
behavioral depression on the airway leukocyte
recruitment in allergic animals. To achieve this, total
and differential cell counts in bronchoalveolar (BAL)
fluid of ovalbumin (OVA)-sensitized and depressed
rats was evaluated. Inescapable electric footshock,
applied on day 0, 7 and 13 after OVA sensitization, was
used as a model to induce depression. In both non-
depressed and depressed groups, the number of total
and differential cells (eosinophils and mononuclear
cells) in BAL fluid was significantly larger in sensitized
compared with non-sensitized animals. However, no
statistical differences were found between these
groups with respect to the number of total and
differentialleukocytes, irrespective of the day inescap-
able shock was applied. Thus, behavioral depression
does not influence the pattern of cell infiltration into
the airways of allergen-induced airway inflammation.
Marcos A. Varriano, Ana A. Varriano, Fernanda Datti,
Marcelo Datti, Edson Antunes and
CA
Nancy A. Teixeira
Department of Pharmacology, Faculty of Medical
Sciences, UNICAMP, P.O. Box 6111, 13081–970
Campinas (SP), Brazil
CA
Corresponding author
Tel: +55 19 3788 7185
Key words: Acute stress, Eosinophils, Allergic asthma,
Inflammation, Lungs
Fax: +55 19 3289 2968
E-mail: airoldi@obelix.unicamp.br
Introduction
studies have focused on the effects of behavioral
depression on the airway leukocyte recruitment in
allergic animals, we designed experiments to evaluate
the total and differential (neutrophils, eosinophils and
mononuclear cells) leukocyte counts in BAL fluid of
OVA-sensitized and depressed rats.
Asthma is one of the most common chronic pulmo-
nary diseases. It is characterized by bronchial hyper-
responsiveness and airway inflammation, both of
which result in obstruction of the breathing tubes.
Besides edema of the mucous membrane, enhanced
production of mucus, damage of mucociliary move-
ment and epithelium, a marked accumulation of
leukocytes, particularly eosinophils and lymphocytes,
is observed in the airways, where they are believed to
Materials and methods
Animals
1
play a key role in the pathogenesis of asthma.
Correlations between the eosinophil number and the
severity of this disease have also been observed.
Adult female Wistar rats (3–5 months old) provided
by the Biology Institute Laboratory Animal Center
(CEMIB) of the State University of Campinas (UNI-
CAMP) were used. They were grouped in propylene
cages with metal rung tops, with free access to water
and food.The light cycle was automatically controlled
(on 6:00 a.m./off 6:00 p.m.), and the room tem-
perature was thermostatically regulated to maintain
26 ± 2°C. Rats were housed in individual cages 3 days
before the experiment. All experiments were carried
out in accordance with the guidelines of UNICAMP.
2
Asthma-like airway inflammation is achieved in rats
by subcutaneous injection of ovalbumin (OVA),
which leads to an increased production of IgE
antibodies for this allergen. Thus, in OVA-sensitized
animals, challenge with this allergen evokes an initial
recruitment of neutrophils followed by a delayed
eosinophil influx, as detected in both the bronchoal-
veolar lavage (BAL) fluid and lung tissues. Using this
model, we recently showed that chronic mild unpre-
dictable prenatal stress produces a marked increase in
the allergen-induced airway inflammation in the rat
offspring, as evaluated by the large recruitment of
3
,4
Behavioral depression model
In this study, the inescapable electric footshock was
used asa modeltoinduce behavioraldepressioninrats,
5
eosinophils into their airways. Studies carried out in
1
2
humans and experimental animals have also sug-
gested that emotional acute stress can contribute to
the onset or severity of allergic asthma, particularly in
as previously described. Briefly, the shock box
3
consisted of a plexiglass box (25 ´ 19.5 ´ 19.5 cm )
equipped with a grid floor having stainless steel bars,
where inescapable 1.0 mA footshocks were delivered.
6
–11
association with behavioral depression.
Since no
ISSN 0962-9351 print/ISSN 1466-1861 online/01/040229-04 © 2001 Taylor & Francis Ltd
DOI: 10.1080/09629350120080438
229

M. A. Varriano et al.
The footshocks were generated by a shock generator
carried out on air-dried smears stained with May-
Grunwald-Giemsa. A minimum of 300 cells was
counted in optic microscope and classified as neu-
trophils, eosinophils and mononuclear cells based on
normal morphological criteria.
(
Albarsch, PortoAlegre, Brazil) connected to the shock
box and toa microcomputer(XT 2002; Microtec,Porto
Alegre, Brazil) executing a software program described
1
3
by Hermini et al. This program controlled the shock
on/offperiodsandwas basedontheescapelatenciesof
1
99 animals determined during a series of 60 foot-
Experimental procedure
shocks delivered over 30 sec in a shuttle box. The
duration of the shocks varied from 0.58 to 18.18 sec,
and each shock was separated by an interval of
The afore-described protocols resulted in four experi-
mental groups (seven to eight rats each): (1) non-
depressed, non-sensitized; (2) non-depressed, sensi-
tized; (3) depressed, non-sensitized; and (4)
depressed, sensitized. The inescapable electric foot-
shock was applied in three different time intervals
after OVA sensitization: day 0, day 7 or day 13,
considering day 1 as the day of sensitization.
5
–25 sec. One rat per session was placed in the shock
box andafter 1 min of adaptationthey were exposedto
6
1
0 inescapable scrambled electric footshocks of
.0 mA each. Non-depressedrats were only maintained
in the shock box and used as controls.This procedure
leads to an escape deficit, observed exclusively in
inescapably shocked animals when exposed to a
sessionof escapable shocks,24 h later. Such behavioral
deficit, extensively described in literature, is con-
Statistical analysis
All data are expressed as mean and SEM. Data were
analyzed with the use of analysis of variance followed
by theTurkey-Kramer test for multiple comparisons. p
< 0.05 was considered to indicate significance.
1
4
sidered an animal model of depression.
Sensitization procedure and antigen challenge
Rats were sensitized against OVA chicken egg (Grade
III; Sigma, St. Louis, MO, USA). Active sensitization
was performed by subcutaneous injection of 0.15 ml
of solution containing 200 mg of OVA and 8mg of
Results
Total and differential leukocyte counts in
bronchoalveolar lavage (BAL) fluid
Al(OH) , prepared in saline according to a previous
3
3
study, with slight modifications. Non-sensitized rats
In the non-depressed group, the content of total
leukocytes in BAL fluid in response to intratracheal
injection of OVA was significantly larger (p < 0.05) in
OVA-sensitized (48 h) compared with non-sensitized
animals, as expected (Table 1).This was associatedwith
marked influxes of eosinophils (ranging from 35 to
50%) and mononuclear cells (ranging from 42 to 58%).
Similarly, in the depressed group, the intratracheal
injection of OVA caused a higher cell influx in
sensitized rats compared with non-sensitized animals,
irrespective of the day of application of the inescapable
shock. However, no statistical differences in the total
cell counts of OVA-sensitized rats were found between
depressed and non-depressed groups (Table 1).
In the non-depressed group, a significant (p < 0.05)
eosinophil recruitment in BAL fluid was observed in
sensitized rats, as compared with the non-sensitized
animals (Fig. 1A). A similar result was observed in the
depressed group when both non-sensitized and sensi-
tized animals were compared. However, no statistical
differences in sensitized animals where observed
between depressed and non-depressed groups, irre-
spective of the day of application of the inescapable
shock (Fig. 1A).
received only 8mg of Al(OH) . On day 14, both
3
sensitized and non-sensitized rats were anaesthetized
with chloral hydrate (300 mg/kg, intraperitoneally
(
i.p.)) and the trachea was exposed through a midline
ventral incision of approximately 0.5 cm length in the
neck. With the aid of a 26.5 gauge needle, 0.4 ml of
0.25% solution of OVA was injected into the airways.
Immediately after this procedure, the animals were
sutured and allowed to recover from the anesthesia.
Leukocyte number in bronchoalveolar lavage
BAL fluid was performed 48 h after OVA challenge.
Briefly, the animals were again anaesthetized with
chloral hydrate (300 mg/kg, i.p.) and exsanguinated by
aortic puncture. The trachea was exposed and cannu-
lated with a polyethylene tube (1 mm diameter)
connected to a syringe. The lungs were washed by
flushing with phosphate-buffered saline (PBS) solution
containing heparin (20IU/ml) and 0.03% serum
albumin. The PBS buffer was instilled through the
trachealcannula asone 10 ml aliquot followed by three
5
ml aliquots. The BAL recovered after each aliquot
instillation was combined and centrifuged at 1000 ´ g
for 10 min in a Hermle centrifuge (Z360 K, Berthold
Hermle AG, Gosheim, Germany). The cell supernatant
was discarded and the cell pellet was resuspended in
In the non-depressed group, the number of mono-
nuclear cells in BAL fluid from OVA-sensitized rats was
significantly higher than from non-sensitized rats (Fig.
1B). Similarly, in the depressed group, the mononuclear
cell counts were significantly higher in OVA-sensitized
rats compared with non-sensitized rats;except at day 0,
2
ml of PBS buffer.Thetotalcell numbers were counted
in T u¨ rk’s solution while differential counts were
230
Mediators of Inflammation · Vol 10 · 2001

Behavioral depression and airway leucocyte influx
Table 1. Total leukocyte counts in BAL fluid from both non-depressed (non-sensitized and sensitized) and depressed (non-
sensitized and sensitized) rats in response to intratracheal injection of OVA (0.4 ml of 0.25% ovalbumin solution)
6
Leucocyte number (´10 /BAL)
Non-depressed
Depressed
Non-sensitized
OVA-sensitized
Non-sensitized
OVA-sensitized
Day 0
Day 7
Day 13
3.2 ± 0.6
2.2 ± 0.3
2.8 ± 0.2
10.1 ± 1.6*
7.5 ± 2.8*
11.0 ± 2.2*
4.1 ± 0.5
2.4 ± 0.4
3.2 ± 0.6
9.9 ± 1.6*
6.5 ± 0.6*
16.3 ± 3.8*
BAL fluid was evaluated at 48h post-OVA injection. * p < 0.05 compared with respective non-sensitized groups. Results are mean values ± SEM
of data from seven or eight rats.
where no statisticaldifferences between non-sensitized
and sensitized rats were observed (Fig. 1B). When the
mononuclear cell counts in sensitized rats in both the
non-depressed and depressed groups were compared,
no statistical differences were found.
The neutrophil counts in BAL fluid of OVA-sensi-
tized rats were low both in the non-depressed
(2–13%) or depressed (2–12%) groups, irrespective
of the day of application of inescapable shock (not
shown).
Discussion
The present study clearly demonstrates that beha-
vioral depression does not change the pattern of total
and differential leukocyte infiltration into the airways
of allergen-induced airway inflammation in rats,
irrespective of the day of application of inescapable
electric footshocks before ovalbumin challenge (1, 7
or 15 days).
Accumulation of eosinophils cells into tissues is a
feature of a variety of allergic diseases, including
1
5
asthma. The sequence of molecular events that lead
mature eosinophils to mobilize from bone marrow to
peripheral blood and to airways are poorly under-
stood, but it is well established that these events are
modulated by certain cytokines, including inter-
leukin-5 and eotaxin, and by the adhesion molecules
1
6
VLA-4 and VCAM-1. Our findings that inescapable
electric footshocks had no effect on the pattern of
eosinophil infiltration in BAL fluid of allergen-sensi-
tized rats rule out that behavioral depression affects
mechanisms that contribute to lung eosinophil migra-
tion in allergic rats. A similar conclusion could be
drawn with respect to mononuclear cell counts in
OVA-sensitized rats, where the inescapable electric
footshocks failed to affect the counts of these cells in
BAL fluid. Interestingly, chronic mild unpredictable
prenatal stress has been shown to increase the
number of eosinophils in BAL fluid of offspring rats
5
sensitized against OVA, possibly by mechanisms
involving inhibition of development and/or activity of
the hypothalamic-pituitary-adrenal (HPA) axis in the
FIG. 1. Effects of inescapable shock applied on day 0, day 7 or
day 13 on the eosinophil (A) and mononuclear cell (B) counts
in BAL fluid after 48h following intratracheal injection of
OVA. Each column represents the mean ± SEM (n = 7–8). * p
17–20
fetuses or adult animals,
in central corticosteroid receptors. Furthermore, a
as well as by a decrease
2
1
correlation between inflammatory (and/or immuno-
<
0.05 compared with respective non-sensitized groups. ND-
22
logical) hyperactivity and depression
as conse-
NS, Non-depressed, non-sensitized; ND-S, non-depressed,
sensitized; D-NS, depressed, non-sensitized; D-S, depressed,
sensitized.
quence of HPA dysfunction has been reported in the
2
3
endogenous depression.
It is well known that
Mediators of Inflammation · Vol 10 · 2001
231

M. A. Varriano et al.
7
8
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2
4
are importantmodulators of theimmune system, and
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2
5
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1
1
1
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1
1
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6
,7
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1
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1
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ACKNOWLEDGMENT. Marcos A. Varriano was supported by CNPq.
2
2
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Received 15 May 2001;
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