Cognitive dysfunction is sustained after rescue therapy in experimental cerebral malaria, and is reduced by additive antioxidant therapy.

Abstract

Neurological impairments are frequently detected in children surviving cerebral malaria (CM), the most severe neurological complication of infection with Plasmodium falciparum. The pathophysiology and therapy of long lasting cognitive deficits in malaria patients after treatment of the parasitic disease is a critical area of investigation. In the present study we used several models of experimental malaria with differential features to investigate persistent cognitive damage after rescue treatment. Infection of C57BL/6 and Swiss (SW) mice with Plasmodium berghei ANKA (PbA) or a lethal strain of Plasmodium yoelii XL (PyXL), respectively, resulted in documented CM and sustained persistent cognitive damage detected by a battery of behavioral tests after cure of the acute parasitic disease with chloroquine therapy. Strikingly, cognitive impairment was still present 30 days after the initial infection. In contrast, BALB/c mice infected with PbA, C57BL6 infected with Plasmodium chabaudi chabaudi and SW infected with non lethal Plasmodium yoelii NXL (PyNXL) did not develop signs of CM, were cured of the acute parasitic infection by chloroquine, and showed no persistent cognitive impairment. Reactive oxygen species have been reported to mediate neurological injury in CM. Increased production of malondialdehyde (MDA) and conjugated dienes was detected in the brains of PbA-infected C57BL/6 mice with CM, indicating high oxidative stress. Treatment of PbA-infected C57BL/6 mice with additive antioxidants together with chloroquine at the first signs of CM prevented the development of persistent cognitive damage. These studies provide new insights into the natural history of cognitive dysfunction after rescue therapy for CM that may have clinical relevance, and may also be relevant to cerebral sequelae of sepsis and other disorders.

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Cognitive Dysfunction Is Sustained after Rescue Therapy
in Experimental Cerebral Malaria, and Is Reduced by
Additive Antioxidant Therapy
Patricia A. Reis1, Clarissa M. Comim2, Fernanda Hermani2, Bruno Silva2, Tatiana Barichello2, Aline C.
Portella3, Flavia C. A. Gomes3, Ive M. Sab1, Valber S. Frutuoso1, Marcus F. Oliveira4, Patricia T. Bozza1,
Fernando A. Bozza5, Felipe Dal-Pizzol6, Guy A. Zimmerman7, Joa ˜o Quevedo2, Hugo C. Castro-Faria-
Neto1*
1Laborato ´rio de Imunofarmacologia, Instituto Oswaldo Cruz, Fundac ¸a ˜o Oswaldo Cruz, Rio de Janeiro, Brazil, 2Laborato ´rio de Neurocie ˆncias and Instituto Nacional de
Cie ˆncia e Tecnologia Translacional em Medicina, Universidade do Extremo Sul Catarinense, Criciu ´ma, Brazil, 3Instituto de Cie ˆncias Biome ´dicas Universidade Federal do Rio
de Janeiro, Rio de Janeiro, Brazil, 4Instituto de Bioquı ´mica Me ´dica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, 5Instituto de Pesquisa Clı ´nicas Evandro
Chagas, Fundac ¸a ˜o Oswaldo Cruz, Rio de Janeiro, Brazil, 6Laborato ´rio de Fisiopatologia Experimental, Universidade do Extremo Sul Catarinense, Criciu ´ma, Brazil,
7Department of Medicine and Program in Human Molecular Biology and Genetics, University of Utah, Salt Lake City, Utah, United States of America
Abstract
Neurological impairments are frequently detected in children surviving cerebral malaria (CM), the most severe neurological
complication of infection with Plasmodium falciparum. The pathophysiology and therapy of long lasting cognitive deficits in
malaria patients after treatment of the parasitic disease is a critical area of investigation. In the present study we used several
models of experimental malaria with differential features to investigate persistent cognitive damage after rescue treatment.
Infection of C57BL/6 and Swiss (SW) mice with Plasmodium berghei ANKA (PbA) or a lethal strain of Plasmodium yoelii XL (PyXL),
respectively, resulted in documented CM and sustained persistent cognitive damage detected by a battery of behavioral tests
after cure of the acute parasitic disease with chloroquine therapy. Strikingly, cognitive impairment was still present 30 days after
the initial infection. In contrast, BALB/c mice infected with PbA, C57BL6 infected with Plasmodium chabaudi chabaudi and SW
infectedwithnonlethalPlasmodiumyoeliiNXL(PyNXL)didnotdevelopsignsofCM,werecuredoftheacuteparasiticinfectionby
chloroquine, and showed no persistent cognitive impairment. Reactive oxygen species have been reported to mediate
neurological injury in CM. Increased production of malondialdehyde (MDA) and conjugated dienes was detected in the brains of
PbA-infected C57BL/6 mice with CM, indicating high oxidative stress. Treatment of PbA-infected C57BL/6 mice with additive
antioxidants together with chloroquine at the first signs of CM prevented the development of persistent cognitive damage.
These studies provide new insights into the natural history of cognitive dysfunction after rescue therapy for CM that may have
clinical relevance, and may also be relevant to cerebral sequelae of sepsis and other disorders.
Citation: Reis PA, Comim CM, Hermani F, Silva B, Barichello T, et al. (2010) Cognitive Dysfunction Is Sustained after Rescue Therapy in Experimental Cerebral
Malaria, and Is Reduced by Additive Antioxidant Therapy. PLoS Pathog 6(6): e1000963. doi:10.1371/journal.ppat.1000963
Editor: Jean Langhorne, National Institute for Medical Research, United Kingdom
Received September 18, 2009; Accepted May 25, 2010; Published June 24, 2010
Copyright: ? 2010 Reis et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The present work was supported by CNPq (www.cnpq.br), FAPERJ (www.faperj.br), PRONEX (www.cnpq.br/programasespeciais/pronex/index.htm),
PAPES (www.castelo.fiocruz.br/vppdt1/papes.php) and NIH/NINDS (RO3 NS04512). The funders had no role in study design, data collection and analysis, decision
to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: hugocfneto@gmail.com
Introduction
Malaria, together with tuberculosis and human immunodefi-
ciency virus/acquired immunodeficiency syndrome (HIV/AIDS),
is one of three most important infectious diseases worldwide, with
devastating morbidity and mortality and deleterious economic
consequences [1]. More than 400 million people suffer from
malaria, which causes over two million deaths annually, mainly
among African children [2]. Cerebral malaria (CM) is the most
severe neurological complication of infection with Plasmodium
falciparum and is the main cause of acute non-traumatic
encephalopathy in tropical countries. Mortality is high. In
addition, physical and neurologic deficits are frequently seen at
the time of hospital discharge in children surviving CM, although
most resolve within 6 months after discharge [3]. Nevertheless,
several retrospective studies suggest that cognitive deficits in
children with CM are more frequent, and persist far longer than
physical and neurologic deficits [4,5,6,7,8]. Boivin et al. [4]
reported that 21% of children .5 years old with CM have
cognitive deficits 6 months after discharge, and that increased
seizure frequency and prolonged coma duration are associated
with persistent cognitive deficits. Desruisseaux and coworkers [9]
reported cognitive dysfunction in the acute phase of experimental
infection with Plasmodium berghei ANKA in mice. A test of work
memory performed at the 7th day of infection demonstrated
significant impairment in visual memory in C57BL/6 mice
associated to significant histological alterations as well as
hemorrhage and inflammation [9].
Although the pathogenesis of CM has been extensively
investigated, many aspects of the cellular and molecular
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pathogenesis remain incompletely defined [10]. This is in part due
to the complexity of the host-pathogen interaction, which includes
intricate biologic and inflammatory responses, variations in
immune status and genetic background of the host, and factors
unique to the malarial parasites [1]. This complexity has been
revealed by clinical and experimental observations that have
recently included informative mouse models [11,12,13]. Biochem-
ical features also influence the natural history and complications of
CM [14]. For example, there is evidence that oxidative stress
mediates some of the tissue damage caused by experimental
malarial infection and in cultured human cells [15,16].
Until recently physicians have focused on survival of patients with
CM and not on long-term outcomes and sequellae and, as a result,
the incidence and impact of chronic neurocognitive dysfunction have
been underestimated and underreported [17]. Similarly, there has
been little inquiry into these issues in experimental CM. Here, we
establish and characterize permissive and resistant murine models
that clearly demonstrate sustained cognitive dysfunction due to CM.
In addition, we demonstrate that a component of the cognitive
dysfunction is related to oxidative stress and that this can be favorably
modified by an interventional strategy that includes antioxidants in
addition to specific rescue chemotherapy aimed at the malarial
pathogen. Because oxidative stress is a pathogenetic mechanism in
othersyndromesofneurocognitiveinjuryandneurodegeneration,the
findings may also be relevant to other systemic inflammatory
syndromes with cerebral involvement.
Results
Characterization of different models of malaria in mice
In order to establish the clinical course of neurobehavioural
complications of CM, mice from diverse genetic backgrounds were
infected with different strains of Plasmodium. Mortality, parasitemia
and behavior alterations (detected by the SHIRPA protocol – see
below) were recorded. First, we compared C57BL/6 and BALB/c
mice infected with PbA (Figure 1A, C). Ninety five percent of
C57BL/6 mice died between 7 and 10 days (with an average of 7.7
days of survival, Figure 1C) after infection, with cerebral manifesta-
tions including convulsion, paralysis, and coma. Mean parasitaemia
was 23%. Seventy percent of BALB/c mice died within 15 days after
infection with severe anemia and overwhelming parasitemia(,80%),
but no signs of cerebral malaria. C57BL/6 mice had 40% mortality
within 15 days after infection with Pch (Figure 1C). These animals
showed high parasitemia (average of 46%) on day 7 and profound
anemia, but no signs of CM were observed at anytime during the
experiment.Inthismodel,parasitemiaatday10postinfectionwasan
averageof11%insurvivingmice.Inanadditionalmodelofinfection,
used to examine the effect of genetic background and parasite
variables, SW mice infected with PyXL had 100% lethality
(Figure 1D) within 8 days, surviving an average of 7.25 days and
displaying clear signs of CM at day 6 associated with substantial
parasitemia (approximately 32%). In contrast, when SW mice were
infected with the non-lethal PyNXL, 100% of the animals survived
for at least 15 days post infection with parasitemia over 20% and no
signs of CM. These results are in agreement with others in the
literature [12,13,18] and confirm that C57BL/6 and SW mice are
susceptible to CM when infected with PbA or the lethal strain of Py,
respectively.
Early neurological alterations of CM in susceptible strains
are detected by screening with the SHIRPA protocol
The summary results of primary screening by SHIRPA on days
3 and 6 post-infection are shown in Table 1. On day 3 post-
infection, no alterations were observed in any of the groups tested
(C57BL/6 versus BALB/c infected with PbA; C57BL/6 infected
with PbA versus Pch; or SW mice infected with PyNXL versus
PyXL) (Table 1). On day 6, however, C57BL/6 mice infected with
PbA and SW infected with PyXL displayed significant alterations
of reflex and sensory function, motor behavior, and autonomic
function. On day 7, the animals demonstrated additional
alterations of muscle tone and strength (not shown). BALB/c
mice showed minor alterations of motor behavior and autonomic
function (only in two tests in this protocol, while susceptible
C57BL6 infected with PbA and SW mice infected with PyXL
displayed positive findings in three and four assessments,
respectively). Previously, Lackner et al. [19] reported that
alterations of autonomic function and muscle tone and strength
are specific and early signs of CM. Using these criteria, the
SHIRPA protocol was prospectively applied on day 6 to identify
CM. Positive results diagnosing CM were then taken as an
indication to start chloroquine treatment, and to conduct further
assessment of cognitive function in CM-positive animals. Interest-
ingly, when we started treatment with chloroquine at 25 mg/kg on
day 6, signs of neurological involvement were rapidly responsive
and were abolished by day 7 post infection (data not shown).
Long-lasting cognitive impairment is present in animals
that displayed initial neurologic signs of CM
To investigate the occurrence of late cognitive impairment,
PbA-infected C57BL/6 mice that had early signs of CM as
detected by the SHIRPA protocol were treated from day 6 to 12
with chloroquine and submitted to the open field-task analysis at
day 15 post infection. Chloroquine treatment was very effective in
controlling parasitemia, since infected red blood cell counts were
reduced to 0.6660.6% at day 16 and parasites were not recovered
at day 30 (1.160.56%) post infection. There were no differences in
the numbers of crossings and rearings observed when groups of
Author Summary
Cerebral malaria (CM) is a deadly consequence of
Plasmodium falciparum infection. Severe neurologic defi-
cits are frequent during CM. Although most resolve within
6 months, several retrospective studies have described
high frequencies of long-lasting cognitive impairment after
an episode of CM. We developed behavioral tests to
identify cognitive impairment due to experimental CM.
During infection with Plasmodium berghei ANKA (PbA),
mice susceptible to CM (C57BL/6) developed long-lasting
cognitive impairment in contextual and aversive memory.
The same profile was seen in Swiss Webster mice infected
with Plasmodium yoelii XL, a lethal strain that also induces
neurological dysfunctions in susceptible mice strains,
confirming that the cognitive dysfunction is closely
associated to the development of CM. Reactive oxygen
species are described as mediators of neurological and
cognitive impairment associated to sepsis and Alzheimer’s
disease. Here we found enhanced production of mal-
ondialdeyde and conjugated dienes in brains of PbA-
infected C57BL/6 mice, indicating oxidative stress. Antiox-
idant therapy with N-acetylcisteine and desferroxamine, as
an additive to chloroquine, prevented the cognitive
impairment, confirming the importance of oxidative stress
in CM-associated cognitive sequellae. Administration of
additive antioxidants may be a successful therapeutic
strategy to control long-lasting consequences of CM and
in other severe systemic inflammatory syndromes with
neurological involvement.
Antioxidants Abolished Malaria’s Cognitive Sequela
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PbA-infected C57Bl/6 and BALB/c mice subjected to the same
rescue treatment with chloroquine were studied in the training
session (Figure 2). In the test session, non-infected C57BL/6 mice
treated with chloroquine or saline demonstrated a significant
decrease in the numbers of crossings and rearings, indicating intact
cognitive skills. In contrast, there was no reduction in crossings or
rearings in PbA-infected C57BL/6 mice rescued with chloroquine
(Figure 2A, B; right bars), indicating diminished cognitive capacity
[20]. Importantly, the decrease in cognitive ability was persistent
for at least 30 days indicating a long lasting dysfunction (Figure 2C,
D). In parallel, PbA-infected BALB/c mice that did not have CM
based on SHIRPA analysis (Table 1), but were, nonetheless,
treated with chloroquine showed a significant reduction in both
crossings and rearings (Figure 2E, F, p,0.05, Student’s T Test)
that was not different from what was observed in non-infected
controls. Thus, despite being infected with PbA, as confirmed by
parasitological examinations, BALB/c mice do not develop CM
and its sequelae, i.e., late cognitive impairment.
Importantly, when C57BL6 mice were infected with Pch, a
Plasmodium strain that does not induce CM [13] (Table 1), the
pattern was similar to that of uninfected animals and CM-resistant
BALB/c mice (Figure 3A, B). Therefore, even though C57BL/6
mice are susceptible to CM, when animals of this genetic
background are infected with a Plasmodium strain that does not
cause central nervous system involvement they do not develop
signs of CM or consequent cognitive impairment based on our
tools of detection. Conversely, cognitive impairment identified by
our analytic instruments was not restricted to the C57BL/6
background since it was also observed in SW mice. SW mice
infected with lethal strain PyXL [21] developed early signs of
cerebral dysfunction that was not detected after infection with a
non-lethal PyNXL strain (Table 1). SW mice infected with PyNXL
showed a significant reduction in the numbers of test events when
training and testing sessions were compared and the pattern was
not different from non-infected control animals (Figure 3C, D).
Nevertheless, when SW mice infected with PyXL were subjected
to testing there was no reduction in test events in training and
testing sessions (Figure 3C, D, right bars). A similar pattern was
observed in PbA-infected C57BL/6 animals. Finally, we also
performed experiments on PbA infected C57BL/6 animals that
were depleted of CD8+lymphocytes by treatment with anti-CD8
monoclonal antibody. CD8+cells were previously shown to have
an important role in CM [22]. In agreement with previous reports,
single dose treatment with anti-CD8 temporarily reverse or
stabilize the progression of CM [22,23]. However, parasitemia
and, consequently, anemia, are persistent in anti-CD8 treated
mice and probably contribute to late deaths observed in these
animals [22,23]. In our hands, the first death in the anti-CD8
treated group was observed on day 13, but the majority of deaths
occurred later on days 16–18. Importantly, the results from an
open-field test can be altered if the mice are seriously ill, since the
motor activity and general behavior are usually affected under this
condition, interfering with the performance of the animals during
the test. Therefore, to ensure that the results of the cognitive tests
were not reflecting compromised behavior due to an ongoing
severe systemic illness we decided to perform the experiments on
Figure 1. Time-course of parasitemia and survival rate of C57BL6, BALB/c and Swiss Webster (SW) mice after infection with PbA,
Pch, PyNXL or PyXL (n=12–20/group). Date on parasitemia (A and B) are shown as mean 6 SEM. Comparisons of C57BL6-PbA versus C57BL6-
Pch (#), C57BL6-PbA versus BALB/c-PbA (*) and C57BL6-Pch versus BALB/c-PbA (+), were significant by Tukey’s Multiple Comparison Test (A). (h) and
(‘) indicate p,0.05 in relation to day 3 post-infection during PyNXL and PyXL infection, respectively (B). Survival curves (C and D) were evaluated by
Log-rank (Mantel-Cox) and Gehan-Breslow-Wilcoxon tests.
doi:10.1371/journal.ppat.1000963.g001
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Table 1. Summary of primary screening results.
Tests
C57/BL6
Balb/c
Swiss webster
RBC
PbA
Pch
RBC
PbA
RBC
PyNXL
Py XL
3D
6D
3D
6D
3D
6D
3D
6D
3D
6D
3D
6D
3D
6D
3D
6D
Reflex and sensory function
Visual placing
3 (3/3)
3 (3/3)
3 (3/3)
3 (3/3)
3 (3/3)
3 (3/3)
3 (3/3)
3 (3/3)
3 (3/3)
3 (3/3)
3 (3/3)
3 (3/3)
3 (3/3)
3 (3/3)
3 (3/3)
3 (3/3)
Pinna reflex
1 (1/2)
1 (1/2)
1 (1/2)
1 (0/2)
2 (1/2)
1.25 (1/2)
1 (1/2)
1 (1/2)
1 (1/2)
1 (0/2)
1 (1.2/2)
1 (1/1)
1 (1/1)
1 (0/2)
1 (11/1
1 (0/1)
Toe pinch
3 (3/3)
3 (1/3)
3 (1/3)
3 (1/3)
3 (2/3)
3 (1/3)
3 (2/3)
3 (1/3)
3 (3/3)
2 (3/3)
3 (3/3)
3 (1/3)
3 (2/3)
3 (2/3)
3 (0/3)
3 (0/3)
Corneal reflex
1 (1/2)
1 (1/2)
1 (1/2)
1 (0/2)
1 (1/2)
1 (1/2)
2 (1/2)
1.6 (1/2)
1 (1/2)
1 (0/2)
1.4 (1/2)
1.8 (1/2)
1 (1/2)
1.8 (1/2)
2 (1/2)
1 (1/2)
Contact reflex
1 (1/1)
1 (1/1)
1 (1/1)
1 (0/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (0/1)
1 (1/1)
1 (0/1)
Positional reflex
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
Neuropsychiatric state
Spontaneous activity
2 (2/3)
2 (223)
2 (2/2)
2 (1/2)*
2 (2/2)
2 (2/2)
2 (2/2)
2 (2/2)
2 (1/2)
2 (1/2)
2 (2/2)
2 (2/2)
2 (2/2)
2 (2/2)
2 (2/3)
1 (0/2)*
Transfer arousal
3 (3/5)
3 (3/5)
3 (3/5)
3 (2/5)*
5 (3/5)
4 (3/5)
3 (3/5)
3 (3/5)
3 (3/3)
3 (2/5)
3 (3/3)
3 (3/3)
3 (3/3)
3 (3/4)
3 (3/3)
3 (1/3)
Touch escape
3 (2/3)
3 (1/3)
3 (2/3)
3 (1/3)
3 (3/3)
3 (3/3)
3 (2/3)
2 (1/3)
2.5 (2/3)
2 (1/3)
3 (3/3)
3 (3/3)
3 (3/3)
3 (3/3)
3 (3/3)
3 (1/3)
Positional passivity
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/1)
Fear
1 (0/1)
1 (0/1)
1 (0/1)
1 (0/1)
1 (0/1)
0.5 (0/1)
1 (0/1)
1 (0/1)
1 (0/1)
1 (0/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
Biting
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
Irritability
1 (1/1)
1 (1/1)
1 (1/1)
1 (0/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
Vocals
1 (1/1)
1 (1/1)
1 (1/1)
1 (0/1)*
1 (1/1)
1 (1/1)
1 (0/1)
1 (0/1)
1 (0/1)
1 (0/1)
1 (0/1)
0 (0/1)
0 (0/0)
1 (0/1)
0 (0/0)
1 (0/1)*
Motor behavior
Locomotor activity
17.6 (5.7)
18.0 (6.1)
16.3 (4.9)
14.2 (4.6)*
17.6 (6.5)
21.3 (6.4)
16.1 (5.3)
19.9 (11.2)
11.6 (4.7)
6.5 (4.5)* 13.9 (9.0)
11.8 (5.5)
13.6 (5.2)
9.2 (4.6)
10.2 (6.0)
4.7 (3.8)*
Body position
3 (3/3)
3 (3/3)
3 (3/3)
3 (2/3)
3 (3/3)
3 (3/3)
3 (3/3)
3 (3/3)
3 (3/3)
3 (3/3)
3 (3/3)
3 (3/3)
3 (3/3)
3 (3/3)
3 (3/3)
3 (2/3)
Shivering
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/1)*
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/1)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
Gait
0 (0/0)
0 (0/0)
0 (0/0)
1 (0/1)*
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/1)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/1)
0 (0/1)
0.8 (0/3)*
Pelvic elevation
2 (2/2)
2 (2/2)
2 (2/2)
2 (0/3)
2 (2/2)
2 (2/2)
2 (2/2)
2 (2/2)
2 (2/2)
2 (2/2)
2 (1/2)
2 (2/2)
2 (2/2)
2 (2/3)
2 (2/2)
2 (0/3)
Tail elevation
1 (1/2)
1 (1/2)
1 (1/1)
1 (1/2)*
1 (0/2)
1 (1/2)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/2)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/2)
1 (1/1)
1 (0/1)
Trunk curl
1 (1/1)
1 (1/1)
1 (1/1)
1 (0/1)
1 (1/1)
1 (0/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (0/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
0 (0/1)
0 (0/1)*
Limb grasping
0 (0/0)
0 (0/1)
0 (0/1)
0 (0/1)
0 (0/0)
0.5 (0/1)
0 (0/0)
0 (0/1)
0 (0/0)
0 (0/1)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/1)
Wire manoeuvre
0 (0/1)
0 (0/1)
1 (0/1)
1 (0/3)*
0 (001)
0 (0/0)
0 (0/1)
0 (0/0)
0 (0/0)
0 (0/1)
0 (0/0)
0 (0/0)
0 (0/1)
0.8 (0/1)
0 (0/1)
1 (0/3)*
Negative geotaxis
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/2)
Autonomous function (early CM parameter)
Respiration rate
2 (2/2)
2 (2/2)
2 (2/2)
2 (0/2)*
2 (2/2)
2 (2/2)
2 (2/2)
2 (2/2)
2 (0/2)
0 (0/2)*
2 (2/2)
2 (2/2)
2 (2/2)
2 (2/2)
2 (0/3)
0 (0/2)*
Palpebral closure
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/1)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/1)
Ruffled fur
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/1)*
0 (0/0)
0 (0/0.12)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/1)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/1)
0 (0/0)
0 (0/1)*
Skin color
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
0 (0/1)*
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animals that were treated both with chloroquine and anti-CD8. In
fact, combined treatment with chloroquine and anti-CD8
monoclonal antibody prevented the occurrence of cognitive
damage in these animals (reduction in crossings/rearings between
training and testing sessions in untreated animals 34.0/32.5%
versus reduction in crossings/rearings between training and testing
sessions in anti-CD8 treated animals 13.0/0.0%). Together, these
results indicate a clear correlation between the occurrence of CM
and the development of late cognitive impairment.
Different memory tasks are affected after CM
To determine if CM differentially influences memory skills, we
submitted mice to different cognitive tasks including step-down
latency and inhibitory avoidance, continuous multiple-trials step-
down inhibitory avoidance and object recognition task. For this
purpose, we elected to use PbA infection in C57BL/6 and BALB/
c strain as positive and negative comparative models, respectively.
The step-down latency and inhibitory avoidance in the test
session at day 15 post infection (Figure 4) was not different from
training and test in PbA-infected C57BL6 mice treated with
chloroquine (mean of latency of 9 and 9.5 s, training and test
sessions respectively; Z=21.075; p=0.282, Wilcoxon’s Test),
suggesting impairment in aversive memory. On the contrary, non-
infected mice treated with chloroquine or saline showed an
increase in step-down latency, indicating intact aversive memory,
when comparing their behavior in training and test sessions. A
similar pattern was seen with Pb-infected BALB/c mice, where
comparisons between infected and non-infected mice were not
statistically different (Figure 4).
When we applied the continuous multiple trials step-down
inhibitoryavoidance taskanalysis(Figure5),we observedasignificant
increase in the number of training trials required to reach the
acquisition criterion (50 sec on the platform) with PbA-infected
C57Bl/6 mice treated with chloroquine as compared to the non-
infected controls (f(5–54)=8; p=0.0001, Wilcoxon’s Test). The results
of this task suggest that PbA-infected C57Bl/6 mice required
approximately two times more stimulation to reach the acquisition
criterion compared to non-infected animals receiving the same
treatment, indicating learning impairment after recovery from CM
[24]. As expected, PbA-infected BALB/c mice did not show any
differences in the number of training trials required to reach the
acquisition criterion when compared to non-infected controls. In the
retention test, there was no difference between groups at all the time
points tested. Therefore, learning ability, but not long term aversive
memory retention skills, is impaired in PbA-infected C57BL/6 mice.
PbA-infected C57Bl/6 mice treated with chloroquine showed
an impairment of novel object recognition memory, i.e., they did
not spend a significantly higher percentage of time exploring the
novel object during short (Z=21.782; p=0.075, Kruskal-Wallis’s
Test) or long-term (Z=21.753; p=0.080, Kruskal-Wallis’s Test)
retention test sessions in comparison to the training trial (Figure 6).
In contrast, this pattern was not reproduced in PbA-infected
BALB/c mice (Figure 6). This result indicates that, as in other
memory tasks, CM is associated with late deficits in cognition and
memory skills that are not shared by infected animals that did not
have clinical or neurobehavioural evidence for CM.
CM is associated with increased oxidative stress in brain
tissues
Oxidative stress is thought to be an important mechanism in the
pathogenesis of neurodegenerative diseases and in sepsis-associated
encephalopathy [25,26]. To examine this issue in experimental CM,
we measured lipid peroxidation by the production of MDA, and the
formation of diene conjugated species. On day 3 post infection, no
Table 1. Cont.
Tests
C57/BL6
Balb/c
Swiss webster
RBC
PbA
Pch
RBC
PbA
RBC
PyNXL
Py XL
3D
6D
3D
6D
3D
6D
3D
6D
3D
6D
3D
6D
3D
6D
3D
6D
Heart rate
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (0/1)
Tears
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
Salivation
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
0 (0/0)
Muscle tone and strength (early CM parameter)
Grip strength
2 (2/2)
2 (2/2)
2 (2/2)
2 (2/2)
2 (2/2)
2 (2/2)
2 (2/2)
2 (2/2)
2 (2/2)
2 (2/2)
2 (2/2)
2 (2/2)
2 (2/2)
2 (2/2)
2 (2/2)
2 (2/2)
Body tone
1 (1/1)
1 (1/1)
1 (1/1)
1 (0/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (0/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (0/1)
Limb tone
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (0/1)
Abdominal tone
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (1/1)
1 (0/1)
Values of individual parameters of Cerebral Malaria (CM) at days 3 and 6 post-infection are shown. Animals infected with malarial parasite (PbA, Pch, PyNXL and PyXL) were compared to animals of the same background inoculated
with uninfected RBC. Data are shown as median (upper/lower quartile) or mean 6 SD when appropriated. N=12–15/group.
*p,0.05 or less by Wilcoxon Signed Rank Test.
doi:10.1371/journal.ppat.1000963.t001
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Figure 2. Open field task analysis identifies mouse strains that are susceptible to CM after PbA infection. C57BL/6 (A–D) and BALB/c (E
and F) mice (n=12–20/group) were infected with PbA (106PRBC i.p.) and treated with chloroquine (25 mg/kg) starting at day 6 post-infection. Data
are expressed as mean 6 S.E.M. of crossings (A, C, and E) and rearings (B, D and F) during training (gray bars) and test (black bars) sessions performed
at days 15 and 16 (A,B,E, and F) or 30 and 31 (C and D) respectively; * significant difference between groups in training and test sessions (Student’s T
test, p,0.05 A and B, E and F; Mann Whitney test, p,0.05 to C and D).
doi:10.1371/journal.ppat.1000963.g002
Figure 3. Impaired performance in the open field task is associated with development of CM. C57BL/6 mice (n=12–20/group) were
infected with PbA or Pch (106PRBC, A and B), or SW mice (n=10–12/group) were infected with PyNXL or PyXL (106PRBC, C and D) and treated with
chloroquine (25 mg/kg) starting at day 6 post-infection. Control groups were inoculated with the same number of uninfected RBC. Data are
expressed as mean 6 S.D. of crossings (A and C) and rearings (B and D) of training (gray bars) and test (black bars) sessions performed on days 15 and
16 post-infection respectively; *significant difference between groups in training and test studies (Student’s T test, p,0.05).
doi:10.1371/journal.ppat.1000963.g003
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significant differences in lipid peroxidation were detected in brains of
C57BL/6 mice infected with PbA compared to those inoculated with
control RBC (Figure 7A, C). On day 6 post infection, however, the
amount of both MDA and diene conjugates (Figure 7B, D, p,0.05,
Student’s T Test) were increased in brain tissue from PbA-infected
mice when compared to the RBC group.Conversely,C57BL/6 mice
infected with Pch and BALB/c mice infected with PbA, which do not
develop CM, did not show increased production of MDA (Figure 7E,
F) or diene conjugates (data not shown). These data identify oxidative
stress in the brains of C57BL/6 mice infected with PbA but not in
non-infected controls or mice infected with Pch, a Plasmodium strain
that does not cause CM, suggesting that oxidative injury is a
component of neurological impairment and, potentially, cognitive
dysfunction in murine CM.
Figure 4. Aversive memory is also affected by CM as detected by the inhibitory avoidance task. C57BL/6 and BALB/c mice (n=12–20/
group) were infected with PbA (106PRBC) and treated with chloroquine starting at day 6 post-infection. Control groups were inoculated with the
same number of uninfected RBC. (A) On day 15 all animals were subjected to a training session of inhibitory avoidance task, where the latency time
on the platform is recorded and an electrical shock is given immediately after the mice step on the bars. (B) 24 h later, aversive memory was tested by
recording the latency time on the platform (with a cut-off of 180 sec). Data are expressed as individual values and horizontal lines represent the
median latencies, in seconds; *Significant difference compared with uninfected controls (comparisons among groups were performed by Mann-
Whitney U test; individual groups were analyzed by Wilcoxon tests, p,0.05).
doi:10.1371/journal.ppat.1000963.g004
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Figure 5. Long term aversive memory is not affected by CM as detected by continuous multiple-trials step-down inhibitory
avoidance. C57BL/6 and BALB/c mice (n=12–20/group) were infected with PbA (106PRBC) and treated with chloroquine starting at day 6 post-
infection. Control groups were inoculated with the same number of uninfected RBC. On day 15 all the animals were submitted to a training session
that consisted of a 0.3 mA 2.0 sec foot shock at the time that the animal stepped down on the grid. (A) Number of trials needed to achieve
acquisition criterion (180 sec on the platform) one hour and thirty minutes after the training session. (B) Latency period on the platform 24 hours
after the training session (cut-off at 180 seconds). Data are expressed as mean 6 S.E.M. of the number of trials required to reach acquisition criterion
(50 sec on the platform) in (A) and as individual values with median represented by a horizontal line in (B). *Significant difference between groups
(comparisons among groups were performed by Mann-Whitney U test, the within individual groups were analyzed by Wilcoxon tests, p,0.05).
doi:10.1371/journal.ppat.1000963.g005
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Treatment with desferoxamine and N-acetylcysteine
prevent late cognitive impairment after CM
Taoufiq and coworkers [27] proposed that the protection of the
endothelium by antioxidant delivery may constitute a relevant
strategy in CM. Therefore, we asked if antioxidants used as an
additive together with antimalarials therapy would reduce
subsequent cognitive impairment in mice that developed early
clinical signs of CM. We treated PbA-infected C57BL/6 that
showed signs of CM, detected by the SHIRPA protocol, with
chloroquine plus a combination of desferoxamine and N-
acetylcysteine treatment starting when antimalarial treatment
was initiated on day 6 post-infection and continuing for 7 days. As
Figure 6. The ability to recognize new objects is impaired in mice after CM. C57BL/6 and BALB/c mice (n=12–20/group) were infected with
PbA (106PRBC) and treated with chloroquine starting at day 6 post-infection. Control groups were inoculated with the same number of uninfected
RBC. On day 15 all the animals were submitted to memory recognition task. Results are shown as mean 6 S.E.M. of the object recognition index
calculated as described in Materials and Methods. *p,0.05 Kruskal–Wallis analyses of variance followed by Mann–Whitney U-tests.
doi:10.1371/journal.ppat.1000963.g006
Figure 7. Oxidative stress is increased in the brains of mice with CM. Oxidative damage was assayed by measuring TBARS (A, B, E and F) and
conjugated diene (C and D) formation in brains on days 3 (A and C) and 6 (B, D, E and F) post-infection of C57BL/6 with PbA or Pch (106PRBC, n=10/
group, A–E) or BALB/c with PbA (106PRBC, n=6, F). Control groups received the same number of uninfected RBC (106). Results are expressed as mean
6 S.E.M. and * represents p, 0.05 compared to RBC group according to Student’s t-test.
doi:10.1371/journal.ppat.1000963.g007
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described previously, treatment with chloroquine alone dramati-
cally reduced mortality and parasitemia, but did not prevent
cognitive damage (Figure 2). On the other hand, treatment with
desferoxamine or N-acetylcysteine alone or in combination had no
effect on the parasitemia curve (data not shown). We found that
the treatment with a combination of chloroquine, desferoxamine
and N-acetylcysteine ameliorated cognitive impairment in infected
mice. Importantly, combination of chloroquine, desferoxamine
and N-acetylcysteine was equally effective in controlling parasit-
emia as the treatment with chloroquine alone (0.66%60.65 in
chloroquine treated animals vs 0.71%60.49 in animals with
combination treatment, ns). Figure 8 shows that there was a
significant reduction in numbers of crossing and rearing events
when analysis in test and training sessions of mice treated with
anti-parasitic and an antioxidant drugs (p,0.05) was compared to
analysis of animals given chloroquine alone. The combined
administration of desferoxamine and N-acetylcysteine is a
necessary condition, since when chloroquine was given with either
desferoxamine or N-acetylcysteine we did not see protection
against the cognitive damage (Figure 8A, B). Combination therapy
was also able to abolish microvascular congestion and plugging
detected by histological examinations of the cortex, hippocampus
and cerebellum of treated mice (Figure 8, panels G, J and M) at
day 7 post-infection, histologic features that were present in
untreated mice with clinical signs of CM (Figure 8, panels F,I and
L). Administration of desferoxamine plus N-acetylcysteine without
chloroquine did not protect animals from early death with high
parasitemia and therefore could not be tested as a treatment for
cognitive impairment. The protection of cognitive function by
chloroquine together with desferoxamine and N-acetylcysteine was
seen both in C57BL/6 mice infected with PbA (Figure 8A, B) and
SW mice infected with PyXL and (Figure 8C, D), indicating that
the additive therapy with antioxidants is able to prevent cognitive
impairment due to CM in relevant models of the disease and
diverse genetic backgrounds. Because artesunate has become the
standard therapy to treat P. falciparum malaria in humans [28], we
also performed an experiment in which the animals were treated
with a combination of artesunate (100 mg/kg, b.w., p.o.) plus
desferoxamine and N-acetylcysteine following the same protocol
described above. As seen with chloroquine, combination therapy
with artesunate was able to prevent the cognitive damage observed
in untreated C57BL/6 mice infected with PbA (reduction in
crossings/rearings between training and testing sessions in
untreated animals 14.0/13.3% versus reduction in crossings/
rearings between training and testing sessions in artesunate
together with deferoxamine and N-acetylcysteine treated animals
32.8/23.8%).
Discussion
More than 500,000 children develop CM in sub-Saharan Africa
each year, of whom 110,000 die [29]. Additionally, survivors may
not fully recover from CM since long-term cognitive impairment is
observed in 12–14% of those individuals [6]. In a study conducted
by Dugbartey and coworkers [7], children with a history of CM
performed significantly poorer than those without previous CM in
bimanual tactile discrimination, accuracy of visual scanning, visual
memory, perceptual abstraction and rule learning skill, right ear
auditory information processing, and dominant-hand motor
speed. The social and economic burden of persistent cognitive
dysfunction is not yet fully clear. Nevertheless, these residual
deficits may affect future cognitive development in children, and
this establishes the potential for devastating impact in adulthood.
CM may thus be the chief cause of cognitive impairment in
children in Sub-Saharan Africa and an important cause of
cognitive impairment in adults in this region. Additional insights
regarding the pathogenesis of cognitive deficits in CM and
strategies for effective therapy to prevent this devastating
complication are urgently required.
The natural history of cognitive dysfunction in experimental CM
and its response to rescue therapy with antimalarial are unknown.
Here we addressed these issues and provide new insights that may
have clinical relevance. In the present work we demonstrated
cognitive damage in animalsrescued from CM by treatment with the
antimalarial drugs chloroquine and artesunate in the early phase of
the disease. In addition, we found that antioxidant agents that have
previously been used in clinical regimens reduce cognitive dysfunc-
tion when given as additive to antimalarial therapy.
Experimental CM is characterized by brain edema, parenchy-
mal lesions, blood brain barrier breakdown, and reduced cerebral
blood flow. These pathophysiological responses are associated with
impaired brain metabolism reflecting cellular injury and bioener-
getic disturbances [30]. Magnetic resonance imaging studies
suggest lesions in the corpus callosum and striatum [30]. The
corpus callosum is one of the most prominent fiber systems of the
mammalian brain. Patients with callosal damage cannot read text
presented in the left visual field, and animals in which the callosum
is divided, and sensory input restricted to one hemisphere, fail to
show interhemispheric transfer of learning [31]. Taken together,
these date suggest that damage in specific regions of the brain due
to CM could generate cognitive damage as well as lack of memory
or learning, similar to what was observed in neurocognitive
impairment following CM in African children [4]. Additional
studies are required to elucidate the mechanisms of central
nervous system injury in children with CM as a necessary
precursor to the development of interventions to prevent
consequent long-term cognitive impairment [32]. We developed
surrogate models that mimic clinical CM and its cognitive
sequelae after parasitic cure by chloroquine, establishing invalu-
able tools to study mechanisms and consequences of cerebral
involvement in malaria. We found that distinct cognitive abilities
are affected in this condition, and that the use of antioxidant
therapy concomitant with anti-malarial drugs was an effective
therapy to prevent late cognitive damage to the host.
In experimental malaria, infection can vary in severity
depending on the species and strain of Plasmodium, the dose of
parasites and the mouse genetic background. We chose our
innoculum based on previous work on experimental CM in the
literature [12,19,33,34,35], but we recognize the possibility that
different results could have been obtained if we had used a mild
infection model. In non-lethal infections, such as those caused by
Pch and Py 17XNL, resolution generally results in immunity to a
second challenge with the same strain, but not to a heterologous
parasite. Some parasite strains are lethal only to a particular strain
of mice (for instance Pch to 129sv, A/J and DBA/2 mice) and some
are uniformly lethal (P. berghei ANKA, Py 17XL or YM), indicating
that parasite associated factors as well as the host genetic
background interact to determine lethality [13]. In the PbA
model, the genetic background of the murine host is extremely
important and modulates the disease outcome. For instance, the
Th-1 biased C57BL/6 mouse is susceptible to the development of
CM, whereas the Th-2 biased BALB/c mouse is resistant [12].
Although PbA infection is regarded as a standard model of
experimental CM, there have been conflicting results using the Py
17XL parasite as a CM model. Contrary to PbA, Py 17XL has
been described to induce high parasitemia, massive anemia and
kidney failure without CM (for review see Engwerda et al., [11]).
On the other hand, other studies report that Py 17XL induces
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clear signs of CM and is a useful model of this condition in the
laboratory setting [13,21,36]. We detected high parasitemia (32%)
at day 7 after Py 17XL infection and these animals exhibited signs
of cerebral dysfunction when submitted to the SIRPA protocol.
Because we were able to establish sensitive and reproducible
methods by which CM could be unequivocally demonstrated by
performing tests described in the SHIRPA protocol [19,37], our
findings are consistent with previous literature indication that Py
17XL induces important dysfunctions in the central nervous
system.
Based on previous studies [19], C57BL/6 mice with CM
develop a wide range of behavioral and functional alterations as
the syndrome progresses, and significant impairment in all
functional categories when assessed 36 hours prior to death.
Reflex, sensory function and neuropsychiatric state are altered in
the early phase of malaria infection, and muscle tone, strength and
autonomic functions are affected in animals with CM exclusively.
We confirmed these findings in several models of CM. We also
observed that C57BL/6 mice treated with chloroquine are rescued
to basal locomotor activity when tested by the SHIRPA protocol
(data not shown). Nevertheless, a cognitive deficit persists and was
clearly demonstrated when the animals were subjected to specific
tasks, such as the memory habituation open-field test performed
15 and 30 days after CM, indicating that cure of the parasitic
infection does not prevent the development of late cognitive
sequelae once CM is established. Furthermore, C57BL/6 mice
infected with Pch developed clinical signs of infection but failed to
develop CM and cognitive damage, indicating that cognitive
impairment is not an unavoidable consequence of systemic
malarial infection in C57BL/6 mice, but rather is associated with
the development of clinically detected CM. We also found that
severe infection without clinically established CM is not sufficient
to trigger cognitive impairment using the PbA-infected BALB/c
mice model. Taken together, these data document a strict
correlation between development of CM and long-lasting
cognitive impairment in surrogate models of malaria. It is our
working hypothesis that acute cerebral malaria that leads to death
in the absence of rescue therapy and long term cognitive
dysfunction in animals that have been rescued with chloroquine
share some of the same cellular and molecular mechanisms, and
that the substrate for long term cognitive dysfunction is initiated by
cerebral injury during the acute period of untreated cerebral
malaria. We do not exclude, however, the possibility that long
term cognitive dysfunction may also have complex mechanisms
that are independent of those that cause neurologic injury and
death during acute cerebral malaria, and that these undefined
mechanisms only operate if the animal survives. Future investiga-
tions are aimed to clarify this point.
Memory function is vulnerable to a variety of pathological
process including neurodegenerative diseases, strokes, tumors,
head trauma, hypoxia, cardiac surgery, malnutrition, attention-
deficit disorder, depression, anxiety, medications, and normal
aging [38]. One of the most elementary nonassociative learning
tasks is that of behavioral habituation to a novel environment [39].
We identified deficits in memory habituation in open-field test
analysis, which revealed long-term memory defect in mice with
experimentally-induced CM. This deficit was unrelated to changes
in basic exploratory or motor processes. Rather, it is likely to be
directly related to impaired hippocampus-dependent memory
processes [40,41]. Additional target areas such as prefrontal cortex
could also be involved, since reduced density of neuronal cells in
this area is known to lead to orientation disturbances and memory
problems in complex tasks [42,43].
Memory habituation impairment was not the only late
consequence of CM in our models as, in fact, several other
cognitive deficits were documented in PbA-infected C57BL/6
mice. Step-down inhibitory avoidance learning triggers biochem-
ical events in the hippocampus that are necessary for the retention
of this task. The events are similar in many ways to those described
for different types of long-term potentiation and other forms of
neural plasticity [44,45]. They are triggered by glutamate receptor
activation and involve at least four different cascades led by
different protein kinases (PK), including protein kinase G, PKC,
calcium-calmodulin-dependent protein kinase II (CaMKII), and
PKA. Several steps in these cascades have been implicated in other
forms of learning that also involve the hippocampus (reviewed by
Izquierdo & Medina [45]).
Step-down inhibitory avoidance involves learning, acquired
generally in one single trial, and long-term aversive memory
retention. C57BL/6-infected mice lack long-term memory reten-
tion ability (24 hours post-stimulus) (Figure 4B) and have deficits in
learning even when they are submitted to multiple trials
(Figure 5A). The inhibitory avoidance task relies heavily on the
dorsal hippocampus, but also depends on the entorhinal and
parietal cortex and is modulated by the amygdala [44,45]. CM
may, therefore, be affecting distinct areas in the brain to interrupt
different facets of memory and task performing ability.
We found that object recognition is also impaired after CM.
This task, originally developed by Ennaceur and Delacour [46], is
based on the tendency of rodents to explore a novel object more
than a familiar one. Because no rewarding or aversive stimulation
is used during training, the learning occurs under conditions of
relatively low stress or arousal [46]. We observed that PbA-
infected C57BL/6 mice rescued from CM with chloroquine had
significant impairment in novel object recognition memory
compared with sham-infected mice. These findings are important
since the novel object recognition task in rodents is a nonspatial,
nonaversive memory test, in contrast to other tests performed in
this study (habituation and aversive memories) [47]. Recognition
of objects is thought to be a critical component of human
declarative memory that is mainly dependent on the hippocam-
pus. Object recognition is commonly impaired in human patients
affected by neurodegenerative diseases, or who have suffered brain
injury [47,48].
Figure 8. Additive antioxidant treatment prevents cognitive impairment after CM. C57BL/6 (A and B) and SW mice (C and D) (n=12–20/
group) were infected with PbA or PyXL, respectively (106PRBC). As a control, one group was inoculated with the same number of uninfected RBC
(n=6/group). Starting on day 6 post infection, infected mice were divided into 2 groups and treated orally with chloroquine (25 mg/kg b.w.),
chloroquine plus desferoxamine (DFX, 20 mg/kg b.w., i.p.), chloroquine plus N-acetylcysteine (NAC, 20 mg/kg b.w., i.p.) or with the combination of
chloroquine/DFX/NAC for 7 days. On day 15 and 16 post-infection all the animals were submitted to open field task training and test sessions,
respectively. Data are expressed as mean 6 S.E.M. of crossings (A and C) and rearings (B and D) in training (gray bars) and test (black bars) sessions;
*significant difference between groups in training and test sessions (p,0.05, Student’s T test). Panels E–M ilustrate histological examinations (H&E
staining) of different brain regions in non-infected (RBC), infected (PRBC, PbA 106) and mice treated with the combination of chloroquine/DFX/NAC
(PbA+Cq+AOX). E–G) cerebral cortex; H–J) hippocampus; K–M) and cerebellum. Microvascular congestion and plugging (arrows) were detected in all
analyzed regions in PbA-infected mice, but were not seen in controls or treated animals rescued with chloroquine and additive antioxidants. Scale
bar, 100 mm.
doi:10.1371/journal.ppat.1000963.g008
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We do not know if the cognitive defects are reversible, but our
experiments indicate that they persist for at least 30 days after
rescue from CM with chloroquine alone. Experiments are in
progress to determine the duration of CM induced cognitive
deficiency imposed by CM in these models.
The mechanisms for cognitive impairment in CM are not
completely characterized, but inflammation and vascular dysfunc-
tion appear to be the basis of cerebral involvement [9]. During
malarial infection, the host and parasites are under severe
oxidative stress with increased production of reactive oxygen
species (ROS) and NO by activated cells in the host [14]. When
produced in large amounts, ROS and nitrogen intermediates may
cause damage to the host tissue including the vascular endothe-
lium. Endothelial damage may lead to increased vascular
permeability and leukocyte and platelet adherence, all seen in
cerebral malaria [49]. Despite being generally accepted, this view
has been challenged by observations showing that gp91phox
deficient mice and inhibitors of iNOS fail to modify the
development of cerebral malaria in appropriate murine models
[50,51]. We have performed preliminary experiments using NOS
deficient mice and observed that those animals, despite being
susceptible to high parasitemia and early death with CM
symptoms, were protected from the cognitive damage if treated
with chloroquine at day 6 post infection. Together, these
observations may suggest that the pathology leading to mortality
during CM may occur via different mechanisms than that leading
to cognitive dysfunction after successful rescue therapy. In this
pathophysiologic milieu, antioxidants may be an effective strategy
to counteract damage in CM, and metal chelators may be of
particular interest [52].
Products of lipid peroxidation are markers for oxidative stress
in several diseases and experimental models [53]. To charac-
terize the oxidative damage during early events of CM we
measured TBARS and conjugated diene formation on days 3
and 6 post infection. Our findings indicate a significant increase
in oxidative stress in the brains of PbA-infected mice on day 6
post-infection, further suggesting antioxidants as a potential
additive therapy to reduce cerebral damage and cognitive
dysfunction during CM. Oxidative stress is associated with the
development of neurodegenerative diseases and is important to
the development of multiple organ dysfunction syndromes
during sepsis [25,26], providing a precedent for this approaches.
In fact, combined antioxidant therapy with N-acetylcysteine and
desferoxamine improves survival in sepsis induced by cecal
ligation and puncture (CLP) in rats by decreasing oxidative
stress and limiting mitochondrial dysfunction [54]. Barichello
and coworkers [55] showed that the combined therapy also
prevents late memory impairment in experimental sepsis. N-
acetylcysteine is a well-known thiolic antioxidant that acts as a
precursor for glutathione synthesis [56]. The reducing thiol
group in N-acetylcysteine also reacts directly with ROS, leading
to cellular protection against oxidative damage in vitro and in vivo
[57]. Desferoxamine is a powerful iron chelator that can inhibit
iron dependent free radical reactions and has been shown to
diminish oxidant damage in several animal model of human
disease [58,59]. Previous studies have demonstrated that
desferoxamine protects against brain ischemic injury in neonatal
rats when administered after an ischemia-reperfusion insults
[60]. In adult rats, desferoxamine protects against focal cerebral
ischemia when given as a preconditioning stimulus 72 h before
the ischemic insult [61]. In agreement with the protective effect
of antioxidants in sepsis-induced brain dysfunction, we found
that combined treatment with N-acetylcysteine, desferoxamine
and chloroquine in PbA-infected C57BL/6 mice or Swiss mice
infected with PyXL prevented cognitive damage as detected by
the open-field task test, further indicating a role for oxidative
stress in the development of cognitive dysfunction in experi-
mental CM and providing an approach to modify this
consequence of cerebral injury. In addition, our initial
experiments indicate that antioxidants are effective as additive
treatment in combination with artesunate as well. Because N-
acethylcysteine and desferoxamine have been used in clinical
treatment of human subjects and their pharmacologic profile
and side effects are known, we suggest that these drugs should
be examined as additive therapy for antimalarial drugs in
clinical trials to investigate their potential to decrease, or
prevent, cognitive damage after CM.
Methods
Animals
6–8 weeks old C57BL/6, BALB/c (n=10/group per experi-
ment) and Swiss webster (SW, n=8/group) mice from the Oswaldo
Cruz Foundation breeding unit, weighing 20 to 25 g, were used
for the studies. The animals were kept at constant temperature
(25uC) with free access to chow and water in a room with a
12 hour light/dark cycle. The experiments in these studies were
approved by the Animal Welfare Committee of the Oswaldo Cruz
Foundation under license number L033/09 (CEUA/FIOCRUZ).
The guidelines followed by this Committee were created by the
same institution that provided ethical approval.
Drugs
N-acetylcysteine (Zambom Group S.p.A., Italy), desferoxamine
(Novartis Bioscience S.A., Brazil), and chloroquine (Farmanguin-
hos, Oswaldo Cruz Foundation, Brazil) were directly dissolved in
saline solution (NaCl 0.9%, w/v). The solutions were prepared
immediately before use and were protected from the light before
administration to the animals.
Parasites, infection and disease assessment
Uncloned parasite lines of Plasmodium berghei ANKA, Plasmodium
chabaudi chabaudi and Plasmodium yoelii were used in this study.
Plasmodium berghei ANKA (PbA) parasitized red blood cells (PRBC)
from BALB/c or C57BL/6 mice, Plasmodium chabaudi chabaudi (Pch)
in C57BL/6 PRBC, Plasmodium yoelii non-lethal (PyNXL), and
Plasmodium yoelii lethal (PyXL) in Swiss Webster PRBC donor
strains were kept in liquid nitrogen and were thawed and passed
into normal mice that served afterwards as parasite donors. 6–8
weeks old C57BL/6, BALB/c and Swiss webster (SW) mice were
inoculated intraperitoneally with 0.2 mL suspension of 106
parasitized red blood cells (n=8–10/group). As a control group
for infection, mice were inoculated with 106non parasitized red
blood cells (RBC). Parasitaemia on days 3, 5, 7 and 10 and survival
rate were recorded.
Histopathology
On day 7 post-infection, animals of different groups (control,
PbA-infected, and PbA-infected rescued with chloroquine and
antioxidant; n=3 per group) were transcardially perfused with
0.9% saline solution and 4% paraformaldehyde in PBS. The
brains were carefully dissected, cryoprotected in 10%, 20%, and
30% sucrose at 4uC, and embedded in O.C.T. (Tissue-Tek) for
frozen sectioning on a cryotome (Leica Microsystems). Parasag-
ittal sections were cut at 12 mm and placed on slides for staining
with haematoxylin and eosin (H&E – VETEC, Rio de Janeiro)
for histological analysis by a blinded expert pathology. Sections
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were examined on an Axioplan light microscope (Zeiss,
Germany).
Experimental design
Mice were infected as described above. On day 3 and 6, they
were subjected to SHIRPA protocol testing (see below) to identify
neurobehavioral signs of CM. Animals that were positive for
clinical signs of CM detected in this fashion were immediately
started on chloroquine treatment (25 mg/kg b.w., orally) and were
treated daily for 7 days (15 days analisis) or 21 days (30 days
analysis). At day 15 post infection, the animals were subjected to a
battery of behavioral tests to access cognitive function. As a
control, uninfected mice received saline (when indicated) or
chloroquine. An additional group of animals received additive
antioxidant therapy with desferoxamine and N-acethylcisteine,
(each 20 mg/kg b.w., intraperitoneally) from day 6 to 12 post
infection, concomitant with chloroquine, and then were subjected
to behavioral tasks on day 15.
In an additional experiment, mice were infected with 106PbA-
PRBC. At day 6 mice were intraperiotoneally treated with a single
dose of 0.5 mg anti-CD8 Mab obtained from Hybridomas 53-6.7
[62] and orally treated with chloroquine (25 mg/kg b.w.) during 7
days.
Behavioral analysis by SHIRPA protocol
The behavioral testing was performed according to the
SHIRPA protocol [63], 1997). The primary screen was performed
as described for detection of CM by Lackner and coworkers [19].
Individual tests are described in Table 1.
Open field task
Habituation to an open-field was carried out as described
by Vianna and coworkers [39]. Animals were gently placed
on an open field apparatus and allowed to explore the arena
for 5 minutes (training session). 24 h later they were submitted
to a similar open-field session (test session). Crossing of
the black lines and rearing performed in both sessions were
counted.
Step-down inhibitory avoidance test
The step-down inhibitory avoidance test was performed as
described by Quevedo et al., [64]. In the training trial, animals were
placedontheplatformandtheirlatencytostepdownonthegridwith
all four paws was measured with an automatic device. Immediately
after stepping down on the grid, the animals received a 0.4 mA,
2.0 secondsfoot shock.Aretentiontest trialwasperformed 24 hafter
training and permanence on the grid is recorded.
Multiple-trials step-down inhibitory avoidance task
Continuous multiple-trials step-down inhibitory avoidance task
testing was performed in the same step-down inhibitory avoidance
apparatus, however, in the training session, animals were placed
on the platform and immediately after stepping down on the grid,
received a 0.3 mA, 2.0 seconds foot shock. 1 h 30 min later, this
procedure was repeated until the mice remained on the platform
for 50 seconds and the number of training trials required was
recorded. On the following day the retention test was performed
and the result was given by latency period on the platform, with a
cut-off at 180 seconds [65,66].
Object recognition task
The object recognition task was carried out as described in
previous studies [67]. Briefly, animals had the opportunity to
explore the open field for 5 min. On the following day, a training
session was conducted by placing individual mice for 5 min into
the field in the center of the arena, in which two identical objects
(object A1 and A2; Double Lego Toys) were positioned in two
adjacent corners at 10 cm from the walls. In a short-term
memory (STM) test (1.5 h after training), the mice explored the
open field for 5 min in the presence of one familiar (A) and one
novel (B) object. In a long-term memory (LTM) test (24 h after
training), the mice explored the field for 5 min in the presence of
the familiar (A) and different novel (C) object. Objects had only
distinction in shape. The exploratory preference was defined as
percentage of the total exploration time animal spent investigat-
ing the object A or the novel object and calculated for each
animal by the ratio TB or C/(TA+TB or C) [TA=time spent
exploring the familiar object A; TB or C=time spent exploring the
novels objects B or C).
Assessment of oxidative stress
To characterize the oxidative stress in murine brains, lipid
peroxidation levels were measured by assays of thiobarbituric acid
reactive species - TBARS [68] - and the formation of diene-
conjugated species [69]. Brains from animals dying of CM were
homogenized in cold phosphate buffer, pH 7.4 with BHT (final
concentration 0.2%). Briefly, the samples (0.5 mL) were mixed with
equal volume of thiobarbituric acid 0.67% (Sigma Chemical, St.
Louis, MO) and then heated at 96uC for 30 min. TBARS were
determined by the absorbance at 535 nm. To analyze diene-
conjugate formation, lipids were extracted by partition on chlor-
oform:methanol (2:1, v:v) and the organic phase was submitted to
espectrophotometric analysis at 234 nm. Results were expressed as
malondialdehyde (MDA, e=1,566105M21cm21) and diene equiv-
alents (e=2,956104M21cm21) per milligram of protein (BCA assay)
[68].
Statistical analysis
Data were expressed as mean 6 SEM. Statistical significance
of survival curves were evaluated by Log-rank (Mantel-Cox) and
Gehan-Breslow-Wilcoxon tests.
SHIRPA was performed by nonparametric test (Wilcoxon
rank-sum test). Data from the open-field task were analyzed
with ANOVA followed by Tukey post hoc and Student’s T tests
and expressed as mean 6 SEM. Data from the inhibitory
avoidance task, object recognition task and the number of
training trials from continuous multiple trials step-down
inhibitory avoidance are reported as median and interquartile
ranges and comparisons among groups were performed using
Mann–Whitney U tests. The variations within individual groups
were analyzed by Wilcoxon’s test. Difference in amounts of
MDA and diene-conjugates were evaluated by Student’s T test.
In all comparisons, p,0.05 or less was taken as statistical
significance.
Statistical analysis from
Acknowledgments
The authors are grateful to Dr. Dalma Maria Banic for kindly providing
the P. chabaudi and P. yoelii strains and Dr. Mirian B. F. Werneck for kindly
providing the anti-CD8 moAb.
Author Contributions
Conceived and designed the experiments: ACP FCAG MFO FDP JQ
HCCFN. Performed the experiments: PAR CMC FH BS ACP IMS.
Analyzed the data: PAR CMC FH BS TB ACP FCAG FAB FDP.
Contributed reagents/materials/analysis tools: TB ACP FCAG VSF MFO
PTB FAB FDP GAZ JQ HCCFN. Wrote the paper: PAR CMC GAZ JQ
HCCFN.
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