Keywords
hydrocephalus - neurocysticercosis - albendazole - experimental design
Palavras-chave
hidrocefalia - neurocisticercose - albendazol - desenho experimental
Introduction
Neurocysticercosis (NCC) is the most common parasitic disease of the central nervous
system (CNS) worldwide.[1]
[2] Although it is potentially eradicable, it remains endemic in developing countries,
and migratory flows have reintroduced the disease in Europe and the USA.[3]
[4]
Neurocysticercosis is a pleomorphic disease because Taenia solium cysts may lodge anywhere in the CNS and the parasite-host interaction is highly heterogeneous.[5] The extraparenchymal form of NCC has a more aggressive course because cysts in the
cerebrospinal fluid (CSF) compartments can elicit an intense inflammatory reaction,
leading to vasculitis, hydrocephalus, and increased intracranial pressure.[6]
[7]
Hydrocephalus is a major concern in extraparenchymal NCC, as patients with NCC-induced
hydrocephalus have higher rates of morbidity and mortality. The management of NCC-related
hydrocephalus is also more challenging than that of hydrocephalus of other etiologies
because of the higher rates of shunt malfunction, infection, and the need for reoperation.[8]
[9]
[10]
[11]
Albendazole is a well-established drug for the treatment of neurocysticercosis. Although
some controversy regarding the safety of albendazole in the treatment of extraparenchymal
NCC has arisen in the last decades, albendazole remains the drug of choice even for
giant extraparenchymal cysts with a mass effect. However, caution in the use of albendazole
is needed because the drug accelerates the death of the parasites in the CSF compartments,
worsening the clinical symptoms and eventually deteriorating the course of hydrocephalus.[12]
[13]
The pathophysiologic mechanisms of NCC-induced hydrocephalus are not fully understood,
although experimental models have helped solve some questions regarding leukocyte
infiltration and inflammatory reactions—mainly for the parenchymal form of the disease.[14]
[15]
[16]
[17]
[18]
[19] Recently, we developed an experimental model of NCC-induced hydrocephalus that reproduces
magnetic resonance imaging (MRI) and histologic findings of human NCC.[20] In the present study, we aimed to analyze the effects of albendazole treatment in
a rat model of NCC with concomitant hydrocephalus to evaluate the correlation between
the course of hydrocephalus and animal behavior.
Methods
Animals
The Institutional Animal Care and Use Committee analyzed and approved the project.
The animals were handled according to ethical guidelines and current legislation.
Eighteen adult Wistar rats weighing ∼ 150 g were used. The rats were kept in rooms
with controlled humidity and temperature (21°C) under a regular light cycle of 12/12
hours. Food and water were available ad libitum.
Sample size
According to previous studies, five animals are sufficient to determine statistical
differences between groups in ventricle volume. Assuming a mortality rate of 30% immediately
after the inoculation procedure and during the observational period, we used 9 animals
for each group (18 in total). This sample size was estimated assuming a random sample
with type I and II errors of 0.05 and 0.02, respectively, and a normal distribution
of ventricle volumes without potential confounding factors.
Experimental Design
Eighteen rats weighing 150–200 g were inoculated with 50 cysts of Taenia crassiceps (a T. solium analog cestode). Two animals died immediately after the inoculation. Thus, 16 rats
were kept for observation. Three months after the inoculation, the rats underwent
MRI and the open field test (OFT) to evaluate behavior patterns. The rats were then
randomly divided into two groups: treatment with albendazole and no treatment (control
group). One week after the treatment, the animals again underwent MRI and the OFT,
and were euthanized for histologic assessment. [Fig. 1] shows a flowchart of the experimental design.
Fig. 1 Flowchart of the experimental design.
Parasites and Inoculation
The methods for the maintenance and inoculation of T. crassiceps have been described previously.[21] Briefly, after general intraperitoneal anesthesia with ketamine and xylazine, the
rats were inoculated with 50 metacestodes of T. crassiceps, each one, removed from the peritoneal cavities of infected mice, into the subarachnoid
space (cisterna magna).
MRI
An MRI was performed using a 0.25 Tesla equipment, model Vet-MR (Esaote, Genoa, Italy).
Improved visualization of ventricles and cysts was achieved using a T2-weighted gradient-echo
sequence (0.6 mm thickness; T eco 5 ms; T repetition 10 ms). An experienced team analyzed
the MRI findings focusing on cyst distribution. Volumetric analysis was performed
using the ITK-SNAP software, version 3.0.0 (Penn Image Computing and Science Laboratory,
Pennsylvania, USA).
Open Field Test
We used a wood chamber of 72 × 72 cm gridded with 9 equally sized squares (18 × 18
cm). Rats were placed in the chamber, and their behavior was recorded using a GoPro
digital camera for 5 minutes. The records were analyzed by two independent observers
registering the number of crossed lines, center time, stretch attend posture, and
defecation. When the disagreement between the observers was lower than 10%, we used
the higher value. When the disagreement was higher than 10%, the observers reexamined
the record together to determine a final consensus value.
Histologic Assessment
After the second OFT, the animals were euthanized with an overdose of pentobarbital
for encephalon removal. We used heart perfusion with, and overnight immersion in,
10% neutral-buffered formalin for brain fixation. The next day, slices were cut at
the level of the optic chiasm, dehydrated in increasing concentrations of alcohol,
diafanized in xylene, and embedded in paraffin. The paraffin blocks were cut into
3-μm sections, and the sections were stained with hematoxylin-eosin. For histological
assessment, we used the criteria of Matos-Silva for experimental encephalitis, focusing
on meningeal, perivascular, ependymal, and choroid plexus leukocyte infiltration,
periventricular gliosis and edema, choroid plexus edema, ependymitis and ependymal
destruction and hyperemia. These parameters were graded semiquantitatively (absent:
0 points, light: 1 point, moderate: 2 points, and severe: 3 points). The total score
of tissue damage was the sum of the scores of each parameter.
Statistical Analysis
The BioEstat 5.3 software (BioEstat Software, Belém, PA, Brazil) was used to assess
the normal distribution of variables (Shapiro-Wilk test), differences between pre
and posttreatment ventricle size (t-test), correlation between OFT results and ventricle
size (Pearson correlation), and correlation between histologic grades and ventricle
size (Spearman correlation). Statistical significance was set at p = 0.05.
Results
Of the 16 inoculated rats, nine developed hydrocephalus (the cutoff value for a normal
ventricle volume was 5.0 mm3). [Fig. 2] shows an example of a hydrocephalic animal with cysts in the CSF compartments. Eight
of these nine hydrocephalic animals were in the treatment group at randomization.
Because the groups were not comparable, we excluded the control group from the ventricle
and behavioral analyses, and focused our assessment on pre- and post- treatment differences.
Fig. 2 Magnetic resonance imaging showed hydrocephalus with ventricular enlargement (A, arrow) and cysts in the basal cistern (B, arrowhead) and in the cisterna magna (B, asterisk) in a T. crassiceps-inoculated mouse.
Ventricle volumes before and after treatment showed a slight but non-significant difference
(168.11 mm3 versus 184.98 mm3, p = 0.45). The distribution and location of cysts did not change, except in one animal
that had no cysts within the basal cisterns pre-treatment but showed cysts after treatment.
The results are summarized in [Table 1].
Table 1
Ventricle volume of each animal in the treatment group before and after the treatment,
and the distribution of cysts in the main cerebrospinal fluid (CSF) compartments
|
Animal
|
Pretreatment
|
Posttreatment
|
|
Ventricle volume (mm3) *
|
Cisterna magna
|
Brain convexity
|
Basal cisterns
|
Intra-ventricular
|
Ventricle volume (mm3) *
|
Cisterna magna
|
Brain convexity
|
Basal cisterns
|
Intra-ventricular
|
|
1
|
107.20
|
Yes
|
No
|
Yes
|
No
|
26.25
|
Yes
|
No
|
Yes
|
No
|
|
2
|
46.35
|
Yes
|
Yes
|
No
|
No
|
43.53
|
Yes
|
Yes
|
Yes
|
Yes
|
|
3
|
291.70
|
Yes
|
No
|
Yes
|
Yes
|
324.0
|
Yes
|
No
|
Yes
|
Yes
|
|
4
|
68.08
|
No
|
No
|
Yes
|
No
|
88.17
|
No
|
No
|
Yes
|
No
|
|
5
|
177.10
|
No
|
No
|
No
|
No
|
165.4
|
No
|
No
|
No
|
No
|
|
6
|
17.80
|
No
|
No
|
Yes
|
No
|
29.81
|
No
|
No
|
Yes
|
No
|
|
7
|
69.34
|
Yes
|
No
|
Yes
|
No
|
101.20
|
Yes
|
No
|
Yes
|
No
|
|
8
|
567.30
|
Yes
|
No
|
Yes
|
No
|
701.50
|
Yes
|
No
|
Yes
|
No
|
*The mean ventricle volume before and after treatment was 168.11vmm3 (±183.46) and 184.98vmm3 (±230.58), respectively. This difference did not reach statistical significance (p = 0.45). Only animal #2 showed a difference in the presence of cysts in the basal
cisterns pre and posttreatment.
Regarding the behavioral pattern in the OFT, we found no differences before and after
treatment (line crossing: p = 0.73; center time: p = 0.35; stretch attend posture: p = 0.73; defecation: p = 0.45). In addition, no correlation was found between the ventricle volume and OFT
pattern variations (p > 0.05).
On histologic assessment, we were able to verify the occurrence of mononuclear leukocyte
infiltration in diverse sites, such as the perivascular and peri-ependymal region,
choroid plexus, and meningeal membranes ([Fig. 3]). To determine whether ventricle volume was associated with the histologic damage
score, we considered all 16 animals and found a positive correlation (rho coefficient = 0.53;
p = 0.04).
Fig. 3 Histologic assessment showed meningeal mononuclear leukocyte infiltration (A, arrow), a pattern of chronic ependymitis (B, dotted lines), and intense perivascular leukocyte mononuclear infiltration (C).
Discussion
Neurocysticercosis is a pleomorphic disease, and the full understanding of its pathophysiologic
mechanisms is hindered by the heterogeneity of the parasite-host interaction and by
the interference of drugs commonly employed to treat NCC in clinical practice, such
as cysticides and corticosteroids.[22] For this reason, many investigators have attempted to establish experimental models
of the disease.[23]
The use of albendazole for extraparenchymal NCC remained controversial for some time;
however, it is currently well-accepted and recommended.[24] Nevertheless, the risk of exacerbation of symptoms, notably deterioration of hydrocephalus,
should be always borne in mind when prescribing albendazole.[25]
[26] We aimed to examine whether the use of albendazole in an experimental model of extraparenchymal
NCC could reproduce this potential adverse effect.
We found that neither the hydrocephalus nor behavioral patterns changed significantly
in the short term after the albendazole treatment. However, we cannot state that albendazole
was a safe drug in our experimental model since the drug was not effective either,
as the cysts remained visible in the CSF spaces in the MRI analyses. Thus, the drug
was not sufficiently effective to completely eliminate the parasite in the short term.
Similarly, in clinical practice, the analysis of a subgroup from a large randomized
controlled trial did not show increased rates of disappearance of extraparenchymal
cysts in patients even 12 months after albendazole treatment.[27] In fact, few controlled trials of medical treatment for extraparenchymal NCC have
been published, and most of the current knowledge is based on case series.[28] Experimental models can contribute to filling this evidence gap.
In addition, albendazole dosage should also be considered. Góngora-Rivera et al (2006)
proposed that a higher dose (30 mg/kg/day) would be more effective for larger cysts
in subarachnoid and intraventricular cysticercosis.[29] The concomitant use of steroids is also recommended to reduce inflammatory reactions.[30] To date, we have observed inflammatory cells in different analyzed brain regions,
and we believe that future experiments using steroids may be useful to demonstrate
the reduction of inflammation.
Finally, the present study adds new information on the experimental model of NCC-induced
hydrocephalus. We have previously observed inflammation, edema, gliosis, and ependymal
destruction in this model. In the present study, we were able to demonstrate that
the degree of hydrocephalus was associated with the severity of tissue lesions.
Some limitations must be pointed out. First, we observed high heterogeneity of the
disease between the control and treatment groups. Future studies should only use animals
with verified hydrocephalus for randomization. Second, it is not clear whether the
parasite mass-effect or the inflammation was more important in the development of
hydrocephalus. Demonstrating that hydrocephalus can occur even with the parasites
destroyed will guide the understanding of the role of inflammation in this model of
extraparenchymal NCC.
Conclusion
In the short term, albendazole did not deteriorate the course of hydrocephalus and
behavioral patterns in a model of neurocysticercosis-induced hydrocephalus.
Erratum: The name of author Pedro Tadao Hamamoto Filho has been corrected as per Erratum published.
DOI of the Erratum is 10.1055/s-0039-1683954.
