Keywords: neurocysticercosis - periventricular demyelination - parenchymal demyelination - Taenia
crassiceps
Palavras-chave: neurocisticercose - desmielinização periventricular - desmielinização parenquimal
- Taenia crassiceps
Amongst the tropical neglected diseases, neurocysticercosis (NCC) emerges as a parasitic
public health infection in regions where pigs roam freely, with clandestine slaughtering
and deficient basic sanitation[1 ],[2 ]. NCC afflicts around 2.5 to 8.3 million people including symptomatic and asymptomatic
cases throughout the world[2 ]. Its prevalence is related to socio-economic cultural factors such as eating and
hygienic habits and pig farming in developing countries. On the other hand, the increase
in the immigrant population from endemic areas in developed countries is accounted
responsible for the expansion in the NCC cases[3 ].
The cysticerci promote an influx of inflammatory cells that vary according to the
encephalic location of the parasite and to the time of infection starting from the
moment they penetrate into the central nervous system. When it reaches the ventricles,
an obstruction of the cerebrospinal fluid may occur leading to hydrocephaly, intracranial
hypertension, ventricular dilatation, headache, inflammatory reactions, epilepsy,
cerebral vascular accident, and radicular compression, presenting or not fibrosis[4 ],[5 ],[6 ].
Experimental models have been useful in the understanding of the complex host-parasite
interaction of NCC. Up to the moment, there are five experimental models used in NCC
studies: Mesocestoides corti in BALB/c mice[7 ], Taenia crassiceps in both BALB/c and C57BL/6 mice[8 ], and inoculation of T. solium active oncospheres in rats[9 ] and piglets[10 ].
The aggregation of parasites and inflammatory cells in extraparenchymal regions, inducing
an initial Th1 immune response followed by a Th2 one leading to the final destruction
of the parasites in a late stage of the infection, has been reported[7 ],[11 ]. It has been suggested that the parasites induce a balance with the initial immune
response, which is lost due to the shift of the immune profile throughout the infection
time leading to the destruction of the parasite[12 ]. The role of the foamy macrophages is controversial. However, it is believed that
they are responsible for the phagocytosis of the destroyed cell membranes, accumulating
cholesterol within their cytoplasm, which gives their aspect of foam[13 ].
The idiopathic demyelinating inflammatory diseases of the CNS constitute a heterogenous
group of injuries, which differ in their clinic course, pathology, and immunopathogenesis.
In spite of the wide variety of differences between these diseases, what they present
in common is the inflammation of the CNS, demyelination, and varied axonal loss[14 ]. They may occur in two distinct phases: an initial one characterized by the inflammatory
process and a latter one with a progressive neurodegeneration[15 ]. It has been suggested that the oxidative stress generated by the inflammation may
be the cause or at least play an auxiliary role in the pathogenesis of the main neurodegenerative
diseases[16 ],[17 ],[18 ],[19 ]. The demyelination may be induced by soluble factors produced by the inflammatory
cells, which diffuse through the parenchyma in a direct form or through the microglia
activation[19 ],[20 ].
Demyelination is widely studied in patients and in experimental models of Multiple
Sclerosis (MS), which is an inflammatory demyelinating disease of the CNS and causes
synaptic alterations in the hippocampus[21 ]. It is also described the dual role of nitric oxide (NO)[22 ] leading to the diffuse regional atrophy of the brain[23 ]. These characteristics in the axonal pathology were experimentally reproduced[24 ] showing a heterogenous pathogenesis[25 ] and that the cortical demyelination occurs precociously[26 ].
Amongst the literature, there are several hypothesis trying to explain the relation
between inflammation and demyelination in several neurodegenerative diseases, but
not in a parasitic one. Therefore, the aim of this study was to describe the influence
of the parasite in the periventricular and parenchymatous demyelination throughout
the infection period using the intracranial inoculation of T. crassiceps cysticerci, i.e., experimental NCC.
METHODS
Experimental design
This study was approved by the Ethics Committee in Animal Use from the Federal University
of Goiás (CEUA/UFG), protocol number 010/11.
The NCC experimental infection was performed using female BALB/c mice from 8 to 12
weeks old (n=40). The use of female mice is indicated because estradiol and progesterone
stimulate reproduction and infectivity of the cysticerci. Initial stage T. crassiceps cysticerci[27 ] were removed from one intraperitoneal infected mouse, washed twice with PBS for
posterior intracranial inoculation[28 ].
The mice were anesthetized (0.01 mL/g of 2% Xilazine and 10% Ketamine); afterwards,
the tricotomy of the superior region of the head was performed in order to expose
the cranial surface. A longitudinal incision was performed in the skin and subcutaneous
tissue, which allowed the visualization of the bone junctions and the bregma point.
The cranial perforation was performed with a micromotor (LB100-Beltec) and diamond
drill (44.5 × 2 mm) according to the stereotaxic coordinates antero-posterior =-2.0
and lateral =+3.0. Three cysticerci were inoculated in each animal with a 1 mL syringe
with an adapted 25G needle (2 mm length) which enabled to reach the brain ventricles.
After the surgical procedures, the incisions were closed with acrylic resin and sutured.
The inoculation of 10 µl of physiologic solution (NaCl 0.9%) was inoculated in the
control animals[8 ]. No animal died after the intracranial inoculation.
The euthanasia of the animals was performed at 7, 30, 60, and 90 days after the inoculation
(DAI) through cervical dislocation. The brains were removed for the histopathologic
analysis. Each experimental day was evaluated with five infected animals and five
control ones.
Histopathologic analysis
The removed brains were fixed in 10% buffered formalin for 24 hours, processed for
inclusion in paraffin, sliced in 5 µm width, and stained with hematoxylin and eosin
(HE). Three serial cuts with 50-µm spacing were performed from each animal.
The pathologic processes analyzed in the brain tissues were: ventriculomegaly, polymorphonuclear
cells inflammatory infiltration (PMN), mononuclear cells inflammatory infiltration
(MN), foamy macrophages, meningitis, ependimitis, microgliosis (increase in the glial
cellularity, especially astrocytic and microglial), and injuries in granular cells
(components of the hippocampus). These pathologic processes were classified in a semi-quantitative
form as follows: absente, discrete with up to 25% of area commitment, moderate with
26 to 50% of area commitment, and accentuated with more than 50% of area commitment[29 ]. The ventriculomegaly was analyzed in a semi-quantitative form using the same parameters
described above, but using as comparison the contra-lateral non-infected ventricle
from the same animal[30 ].
Demyelination and axonal injurie analyzes
The demyelination was evaluated through the Luxol Fast Blue staining technique. The
Nissl staining was performed in order to verify the axonal disorganization or injury
in demyelinated areas. The demyelination was evaluated in a qualitative and semi-quantitative
form according to the following criteria:
Location - periventricular or parenchymatous.
Intensity - restricted to the periventricular demyelination cases: absent (score 0),
discrete (demyelination sites, score 1), moderate (demyelinating areas up to 50% of
the periventricular area, score 2), and accentuated (demyelinating areas greater than
50% of the periventricular area, score 3).
Distribution - restricted to the parenchymatous demyelinating areas: focal or diffuse.
The axonal disorganization was evaluated according to the fibers disposition, lamellar
or irradiated, in a descriptive form.
Statistical analysis
The statistical analysis was performed with the Sigma Stat 3.5 software. All variables
were tested as to normal distribution and homogenous variation. The pathologic processes
were evaluated through the Mann-Whitney test and Student’s t-test, according to the
parametric and non-parametric distribution. The demyelination was evaluated through
mean and mean standard error. The differences were considered significant when p≤0.05.
RESULTS
All mice from the infected groups presented the cysticerci located within one of the
lateral ventricles inducing inflammation, expansion of the ventricular cavity, and,
in some cases, deviation of the brain midline. All results obtained after the analyses
are described in [Table 1 ] and illustrated in [Figures 1 ], [2 ], and [3 ].
Table 1
Analysis of the general pathologic processes in the brain of female BALB/c mice intracranially
inoculated with Taenia crassiceps cysticerci.
Microscopic alterations
DAI
Control group (n=5)
median (min-max)
Infected group (n=5)
median (min-max)
p-value
Ventriculomegaly
7
0 (0‒0)
0 (0‒0)
1.000
30
0 (0‒0)
3 (1‒3)
0.008
60
0 (0‒0)
1 (0‒3)
0.151
90
0 (0‒0)
2 (1‒3)
0.008
PMN inflammatory infiltration
7
0 (0‒0)
0 (0‒0)
1.000
30
0 (0‒0)
1 (1‒2)
0.008
60
0 (0‒0)
0 (0‒1)
0.690
90
0 (0‒1)
1 (0‒2)
0.310
MN inflammatory infiltration
7
1 (1‒1)
0 (0‒2)
0.690
30
1 (0‒1)
2 (2‒2)
0.008
60
0 (0‒1)
1 (0‒2)
0.151
90
0 (0‒1)
2 (1‒3)
0.008
Foamy macrophages
7
0 (0‒0)
0 (0‒0)
1.000
30
0 (0‒0)
1 (1‒1)
0.008
60
0 (0‒0)
0 (0‒1)
0.310
90
0 (0‒0)
1 (0‒1)
0.032
Meningitis
7
0 (0‒0)
0 (0‒0)
1.000
30
0 (0‒1)
0 (0‒1)
0.690
60
0 (0‒0)
0 (0‒0)
1.000
90
0 (0‒0)
1 (1‒1)
0.008
Ependimitis
7
0 (0‒0)
0 (0‒0)
1.000
30
0 (0‒1)
1 (0‒2)
0.222
60
0 (0‒1)
0 (0‒1)
0.690
90
0 (0‒0)
1 (0‒2)
0.032
Microgliosis
7
0 (0‒1)
0 (0‒0)
0.690
30
0 (0‒0)
0 (0‒0)
1.000
60
0 (0‒0)
0 (0‒0)
1.000
90
0 (0‒0)
0 (0‒1)
0.690
Hippocampus injury CA2 and CA3 areas
7
2 (1‒2)
1 (1‒1)
0.151
30
0 (0‒2)
2 (1‒3)
0.095
60
1 (0‒2)
2 (2‒3)
0.032
90
1 (1‒2)
2 (1‒3)
0.548
DAI: days after the inoculation; PMN: polymorphonuclear cells; MN: mononuclear cells;
CA: Cornu Ammonis; bold: statistical difference performed by the Mann-Whitney test.
Figure 1 Photomicrograph of the brain of mice infected with Taenia crassiceps cysticerci and control groups, intracranially inoculated with saline physiologic
solution (NaCl 0.9%). (A) microgliosis (insert) in control group 7 days after the
inoculation (DAI), scale 100 µm. (B) presence of cysticercus (arrow) surrounded by
inflammatory process in the ventricle, 30 DAI, scale: 100 µm. (C) presence of foamy
macrophages (arrow) in infected animals 90 DAI, scale 20 µm. (D) ventriculomegaly
and presence of cysticercus (arrow), 90 DAI, scale: 10 µm. Staining: H&E.
Figure 2 Comparison of the intensity of periventricular and parenchymatous demyelination in
Taenia crassiceps intracranially infected mice and the control group inoculated with saline physiologic
solution (NaCl 0.9%). The results are expressed in mean and mean standard error of
the scores as follows: absent - score 0; discrete - score 1; moderate - score 2; accentuated
- score 3. *: statistical difference. CT: control group, INF: infected group, DAI:
days after the inoculation.
Figure 3 Photomicrograph of brains from mice intracranially infected with Taenia crassiceps cysticerci and control groups, intracranially inoculated with saline physiologic
solution (NaCl 0.9%), stained with Luxol Fast Blue and Nissl. (A) normal pattern of
myelination in laminar form, control group, 60 days after the inoculation (DAI), Luxol
Fast Blue stain. (B) normal pattern of axons, control group, 90 DAI, Nissl stain.
(C) accentuated ventriculomegaly due to the mass effect and presence of cysticercus
(arrow) in Cornu Ammonis areas (CA2 and CA3) of the hippocampus, causing ischemia and adjacent parenchymal
demyelination, 90 DAI, Luxol Fast Blue stain. (D) parenchymatous demyelination in
pyramidal hippocampus area due to inflammatory focus, 90 DAI, Luxol Fast Blue stain.
(E) demyelinating area (arrow and insert) associated to loss of hippocampus cells,
60 DAI, Luxol Fast Blue stain. (F) irradiated axon pattern (arrow and insert) next
to the inflammatory process, 60 DAI, Nissl stain. Scale: 200 µm.
At 7 DAI, the control group presented MN inflammatory infiltration, microgliosis,
and dispersion of the hippocampus cells. In the infected group, it was also observed
the presence of MN inflammatory infiltration, but with greater intensity. There was
no periventricular nor parenchymatous demyelination in both groups, control and infected.
However, the infected group presented axonal disorientation in a parenchymatous inflammation
site ([Table 1 ], [Figure 1 ]).
At 30 DAI, the alterations observed in the infected group were greater when compared
to the ones observed in the control group. It was possible to detect ventriculomegaly
in different intensities, mixed moderate inflammatory infiltration, discrete presence
of foamy macrophages, discrete meningitis, ependimitis, and hippocampus injury. In
the control group, it was possible to observe MN inflammatory infiltration, discrete
meningitis, ependimitis, and injury in the hippocampus. In the infected group, the
demyelination was discrete in 100% of the analyzed animals and only 40% of them presented
focal parenchymal demyelination. In addition, one infected animal presented axonal
disorganization in a parenchymatous inflammatory focus at 30 DAI ([Table 1 ], [Figures 1 ] and [3 ]).
At 60 DAI, the mice from the control group presented MN inflammatory infiltration,
discrete ependimitis, and hippocampus injury, while the infected mice presented ventriculomegaly
of the lateral ventricle, mixed profile of inflammatory infiltration, discrete presence
of foamy macrophages, ependimitis, and injury in the hippocampus cells. In the infected
group, the periventricular demyelination was present in 80% of the animals. The parenchymal
demyelination in CA2 and CA3 hippocampus areas was observed in 20% of the cases in
a focal form ([Table 1 ], [Figure 3 ]).
At 90 DAI, the infected mice presented discrete intensity of foamy macrophages and
microgliosis, ventriculomegaly, mixed profile of inflammatory infiltration, ependimitis,
and varied intensity of alterations in the hippocampus cells, while in the control
group it was possible to observe discrete intensity of mixed inflammatory infiltration
and hippocampus alterations. The periventricular demyelination was observed in 40%
of the analyzed animals and 80% of them presented focal parenchymal demyelination
([Table 1 ], [Figures 1 ] and [3 ]).
DISCUSSION
The present study used the NCC experimental model of female BALB/c mice intracranially
inoculated with initial stage T. crassiceps cysticerci. This parasite is the most used as T. solium experimental model as they belong to the same genus, presenting antigenic and epitope
similarities. They are very rare in human infections, which make its handling very
safe. In addition, its capability of asexual reproduction through budding provides
a great source of biological material[28 ].
In the initial phase of the infection, it was possible to observe an influx of inflammatory
cells. These cells belonging to the glial group of cells are responsible for the normal
functioning of the CNS and are able to communicate between themselves, as well as
with neurons and vascular cells[30 ]. Bernardinelli et al.[31 ] described that glial cells participate in the maintenance of neurons in homeostasis
and when there are alterations, they release Ca+2 leading to neuron death through deficiency in glucose uptake and catabolism. Our
results corroborate with this description because it was possible to observe glial
cell alterations in both groups, control and infected, probably due to the reception
of stimuli, which provoked morphology changes, and acquisition of new phagocytic effector
functions capable of inducing inflammation and contributing to the disease pathogenesis[32 ]. Matos-Silva et al.[8 ] observed that at 7 days of neuro-infection with T. crassiceps in BALB/c mice it was possible to observe gliosis, edema, hyperemia, ependimitis,
perivasculitis, and inflammatory infiltration of polymorphonuclear cells and mononuclear
cells, all in discrete intensity. Additionally, the analysis of another experimental
model of neurocysticercosis using Mesocestoides corti in BALB/c mice indicated that after two days of infection it is possible to detect
an inflammatory infiltration of macrophages, neutrophils, γδ T cells, and NK cells
in the cerebral parenchyma. In addition, the presence of parasite’s antigens throughout
the parenchyma lead to disseminated encephalitis[7 ].
At 30 DAI, the cysticerci presence within the ventricles, leading to ventriculomegaly
and ependimitis, was probably the cause of the local inflammatory process, allowing
the influx of mixed profile of inflammatory cells. Among these cells, it was possible
to observe foamy macrophages, which accumulate cholesterol, and its esters derived
from cell debris and the parasite itself. The inclusion of lipids bodies in immune
cells has been recognized as fundamental, as they are important organelles responsible
for lipids metabolism, trafficking, and signaling. They present the enzymatic arsenal
associated to conversion of arachidonic acid into eicosanoids. They are also responsible
for the production of cytokines such as Interleukin-1 (IL-1), IL-6, IL-8, IL-12, IL-10,
tumoral necrosis factor-alpha (TNF-α), and transforming growth factor beta (TGF-β)[32 ]. In addition, gliosis has been linked to the presence of foamy macrophages in infections
and inflammatory diseases of the central nervous system[33 ].
After the implantation of the parasite in the CNS, several mechanisms are initially
observed such as the blocking and inhibition of complement activation through taeniastatin
and paramyosin, degradation of immunoglobulins, altering cytokine production, i.e.,
decreasing IL-1 and IL2, and increasing IL-10 and IL-4. Therefore, it is possible
to observe the inhibition of the Th1 immune profile which is replaced by Th2[34 ],[35 ]. The parasite’s tegument is composed by glycoconjugates with high antigenic variability,
enabling the immune evasion because they are dynamically responsive to the alterations
in the hosts microenvironment, favoring the presence of alternatively activated macrophages
(AAMo or M2), which enable the parasite’s proliferation[34 ],[35 ],[36 ],[37 ],[38 ]. At this point, the parasite has established the ideal survival environment, which
is seen in our results in the latter phase of the infection, during which it was possible
to observe a predominance of MN cells in the inflammatory infiltration, characteristic
of the Th2 profile.
Helminths are uncapable of lipid synthesis and therefore must acquire it exogenously.
After the lipid absorption, it is incorporated into the parasite’s tegument and cell
membranes[34 ]. The lipidic corpuscles within macrophages are related to several inflammatory conditions
and may function as storage sites and for the synthesis of inflammatory mediators[32 ]. On the other hand, the first line of defense against microorganism composed by
PMN cells are attracted to the infection site and produce chemokines when activated,
attracting other immune cells[33 ],[36 ]. This local inflammation may have been the cause of the periventricular and parenchymatous
demyelination observed in our results. This is in accordance to the literature reports
that show that inflammatory mechanism and the oxidative stress generated by the inflammation
are the cause or an auxiliary factor in the pathogenesis of the main neurodegenerative
diseases[16 ],[17 ],[18 ]. However, this is the first description of this pathogenic effect in the NCC process.
Most of the cases of demyelination observed in our results were focal and parenchymatous,
while only 40% were periventricular. These results corroborate with other studies,
which reported that demyelination might be a consequence of a local inflammatory process[26 ]. This is the first description of this process in experimental NCC. Lin et al.[39 ] confirmed the destruction of the myelin sheath induced by a local inflammatory process
related to the presence of a parasite, Angiostrongylus cantonensis . Also, the maintenance of the NCC inflammatory process has direct relation to the
increase in the expression of VEGF (vascular endothelial growth factor), which facilitates
the influx of immune cells[9 ],[10 ].
The role of the inflammatory cells in the tissue injury has been studied in several
other diseases[13 ],[16 ],[26 ]. However, this role in the neuroinflammation and demyelination in NCC is still an
enigma. The experimental models for NCC have contributed to unveil part of this process.
Cardona et al.[7 ] have described the initial immune response and the predominance of the Th1 profile
with Tγδ (gama delta) lymphocites. Other authors described the inflammatory profile
composed by mononuclear cells, T lymphocytes, plasmocytes, macrophages, and eosinophils
at late stages of the infection[8 ],[9 ],[10 ]. These data corroborate with our results, in which was also possible to observer
MN cell inflammatory infiltration at a later stage of infection. One of the probable
causes of the demyelination observed after 30 DAI is the presence of Tγδ lymphocites
throughout the experimental infection, which induced an increase in the pro-inflammatory
cytokines production and greater local cellular immune response[7 ],[12 ].
An experimental study described the infection of mice with Trichobilharzia regent (Schistosomatidae) and the histologic and immunohistochemical consequences of this
infection. The authors described the axon injuries and demyelination to mechanical
injury caused by the parasite’s migration through the nervous tissue and not to the
host’s immune response[40 ], while the experimental neurological infection with Angiostrongylus cantonensis (Strongylida) induces the release of inflammatory mediators, leading to intracerebral
myelin breakdown, and increase in myelin proteins in the cerebrospinal fluid[39 ].
The proper understanding of the neuroinflammation / neurodegeneration / demyelination
cycle may help in the treatment of several neurodegenerative diseases, improving the
clinical symptoms caused by the neuronal loss. The mechanism by which the immune response
reacts to several different stimuli is still being clarified. The study and understanding
of the multiple factors of the teniasis/cysticercosis complex and the role of inflammatory
cells in CNS may help in its treatment, improving symptoms related to neuron injury.
In our study, little alterations were observed in histopathologic analyzes of the
control groups. The surgical procedure promoted the brain blood barrier (BBB), allowing
the initial influx of inflammatory cells leading to the hippocampus alterations observed
in the late phase of infection. When the BBB integrity is lost, there is an overflow
of molecules and cells, which may help to understand some of the mechanisms involved
in the cell activation in NCC[30 ].
In conclusion, the experimental model of NCC used in this study was able to demonstrate
ventriculomegaly and periventricular demyelination at 30 DAI, probably due to the
inflammatory process. In the late phase of the infection, it was possible to observe
the chronic inflammatory process and the mass effect causing ventricle distension,
ependimitis, meningitis, and glial and hippocampus alterations. This is the first
description of parenchymatous demyelination in an experimental model of NCC.
