Introduction

Neurocysticercosis (NCC) is a common parasitic disease of the central nervous system in developing countries. Despite the development of tools and knowledge to control the disease, it remains a neglected disease with a significant burden in endemic countries. In contrast, it reappears in developed countries because of migratory flows (Bhattarai et al. 2019; Singh et al. 2017; Abraham et al. 2020). Data from Latin America suggest that the prevalence of the disease is decreasing in some countries while it persists in others (Rodríguez-Rivas et al. 2022). In Africa and parts of Asia, the situation is difficult to assess owing to the lack of diagnostic tools; however, NCC is still an important cause of seizures in these regions (Stelzle et al. 2022; Sahu et al. 2017).

NCC is a heterogeneous disease. Infected patients may remain asymptomatic or experience mild symptoms (such as headaches or single seizures) for long periods. Severe symptoms with increased intracranial pressure may appear suddenly in some patients, probably triggered by some damage caused by the parasite with the release of cysticercal antigens that may trigger an exacerbated neuroinflammation (Fleury et al. 2010). This heterogeneity is linked to several factors, such as the number and location of cysts within the central nervous system, the genetic particularities of the parasite and patients across different geographical regions, and the differences in infection pressure in different populations (Ito & Budke 2021; Hamamoto Filho et al. 2020). Regarding the location of cysts, the presence of cysts within the cerebrospinal fluid (CSF) compartments (extraparenchymal NCC) is the most severe form of the disease. It may cause vasculitis, hydrocephalus, and increased intracranial pressure. In addition, this form of disease is less responsive to medical treatment and has higher mortality rates (Fleury et al. 2011; Marcin-Sierra et al., 2017; Hamamoto Filho et al. 2019a).

Another factor contributing to NCC heterogeneity is the patient’s sex. When parasites are present in the parenchyma, the female sex is associated with more intense pericystic oedema and contrast enhancement on neuroimaging examinations, and worse outcomes (Brutto et al. 1988; Kelvin et al., 2009). Additionally, in one study including patients with parenchymal and extraparenchymal NC, higher CSF cytokine levels (interleukin [IL]-6, IL-10) were observed among women (Chavarría et al. 2005), while a higher lymphocyte CSF count was reported among female patients with extraparenchymal NC (Marcin-Sierra et al. 2017).

Hosts’ sexual dimorphism in cysticercosis has been largely explored in the murine model of intraperitoneal Taenia crassiceps cysticercosis. These studies showed that cysticerci grow in larger numbers in female mice than males. Sex steroids play a significant role in regulating these differences in the parasite load (Huerta et al. 1992; Escobedo et al. 2009). Moreover, drastic endocrinological changes occur during the infection suggesting the complexity of the host-parasite relationship (Gomez et al. 2000).

In this context, we aimed to gain further insights into sexual dimorphism in the inflammatory responses accompanying the murine extraparenchymal experimental NCC caused by T. crassiceps.

Materials and methods

Animals

Wistar rats (Rattus norvegicus, 25 females and 22 males) aged 6 to 7 weeks were used. The animals were handled according to ethical guidelines and current legislation. The research project and all protocols were approved by the Ethics Committee on the Use of Animals (CEUA) of the Botucatu Medical School (CEUA 1318/2019). During the 90 days of the experiment, the animals were kept in polyethylene boxes (40 × 30 × 15 cm), under controlled conditions of light (12 h light/12 h dark) and temperature (24 ± 2 °C), with water and food provided ad libitum. They were examined weekly for signs of illness or altered behaviour.

Experimental design

According to a previously described technique, the animals were subjected to cisternal inoculation of T. crassiceps (Hamamoto Filho et al. 2019b). Ninety days post-inoculation, the rats were euthanized to harvest the brain. Animals without visible cysts during euthanasia were discarded. By simple randomization, half of the animals were subjected to morphological analysis and immunohistochemistry, and the other half were subjected to enzyme-linked immunosorbent assay (ELISA) to measure some cytokines.

Sample size

According to a pilot study, with 5–6 animals in each experimental group, it is possible to determine statistically significant differences between the groups. As each animal was subjected to morphological analysis or measurement of inflammatory features, each group should have a minimum of 10–12 animals. Furthermore, based on our experience, considering a 66% success rate in inducing the disease, 22 animals would be needed in each experimental group. This sample size was estimated assuming simple random sampling; type I and II errors were equal to 0.05 and 0.20, respectively. Additional inoculations were performed in cases of deaths or a higher number of uninfected animals. Therefore, 25 female and 22 male rats were used.

Parasites and inoculations

Cysts of T. crassiceps (ORF strain), maintained in the peritoneal cavity of mice, were aseptically removed and selected according to their size (0.5 mm in diameter) and viability (intact membrane).

For inoculation, Wistar rats were anaesthetized with ketamine (87 mg/kg) and xylazine (13 mg/kg). Inoculation was performed after a 1-cm skin incision at the occipito-cervical transition, with the suboccipital puncture of the cisterna magna using a 24-G needle and inoculation of 50 viable T. crassiceps cysts in 0.2 ml of saline. The skin was then sutured with 4.0 mononylon thread.

Histological analysis

After euthanasia with an overdose of ketamine and intraperitoneal xylazine, cardiac perfusion was performed with a peristaltic pump for perfusion and fixation (30 ml/min), initially with 0.9% saline and then 4% paraformaldehyde. Subsequently, the brains were removed and sectioned in the transverse plane in the topography of the optic chiasm with a brain matrix. The sections were kept in paraformaldehyde overnight at room temperature and dehydrated in increasing concentrations of alcohol baths, clarified in xylene, and paraffinized.

The paraffin blocks were sectioned at 5-μm slices stained with haematoxylin and eosin. Morphometric analysis was performed to quantify immune cells (lymphocytes and plasmacytes) in the basal arachnoid and periventricular regions (striatum and lateral septal nucleus).

The glial fibrillary acid protein (GFAP) expression level was estimated by immunohistochemistry to evaluate astrocytes’ activation within the periventricular zone. The blocks were sectioned at 3-μm slices, and the slices were dewaxed in xylene and rehydrated in decreasing concentrations of alcohol. For antigen recovery, the slides were heated at 98 °C with citrate buffer (pH 6.0) for 30 min and then incubated in 0.3% hydrogen peroxide at room temperature for endogenous tissue peroxidase block. Furthermore, they were washed with Tris-buffered saline/Tween 20 (TBST), pH 7.5, and incubated with primary antibodies against GFAP (mouse monoclonal IgG1 [2A5] to GFAP, Abcam, Cambridge, MA, USA) in a humid chamber at room temperature overnight. The slides were washed again with TBST, incubated with a secondary peroxidase horseradish polymer-conjugated antibody for 30 min at room temperature, washed with TBST, incubated with 3,3′ diaminobenzidine stain for 5 min, and counterstained with haematoxylin.

The morphometric analysis was based on Weibel counting reticle (Weibel et al. 1966), a mathematical model that quantitatively transforms two-dimensional to three-dimensional acquisition. The studied structures intersecting with the reticle lines were counted and divided by the total number of lines.

Cytokine measurement

After euthanasia with an overdose of xylazine and ketamine intraperitoneally, a craniectomy was performed to remove the brains, which were then transported in liquid nitrogen and frozen at −80 °C. The brains were thawed under temperature control, and the cerebral hemispheres were dissected. The latter were then sonicated with a solution of Tris and HCl, 1% NP40, and protease inhibitor. IL-6, IL-10, and interferon-gamma (IFN-γ) levels were quantified in the homogenized solution of brain tissues using the sandwich ELISA according to the manufacturer’s instructions (BD OptEIATM). IL-6 and IL-10 expression levels were determined using IL-6 ELISA - BD OptEIATM (BD Biosciences, San Diego, CA) and IL-10 ELISA - BD OptEIATM, respectively. IFN-γ assay was performed using high-binding ELISA microplates sensitized with 80 μl of IFN-γ monoclonal antibody (5 μg/μl of clone XMG in phosphate-buffered saline). The measurement was determined using a Thermo/Labsystems microplate reader with specific filters for each cytokine. The assays were performed in duplicate.

Magnetic resonance imaging

For illustrative purposes, five male and five female rats were randomly selected to undergo magnetic resonance imaging (MRI) to assess ventricle enlargement. The animals were anaesthetized with 1.5% inhaled isoflurane. MRI was performed using 7-T equipment (Siemens Magnetom scanner, Siemens, Erlanger, Germany), with a T2-TSE sequence (parameters: plane resolution = 270 μm, FOV = 64 × 128 mm, matrix = 240 × 480 mm, thickness = 700 μm, TR/TE = 6000/60 ms, eco train length = 5, total time of acquisition = 4 min 43 s). A normal ventricle size was considered at 4.5 mm of the distance between the frontal horns of the lateral ventricles at the level of the interventricular foramen. Mild ventricle dilatation was considered for a distance between 4.5 and 5.5 mm, and severe dilatation was considered when the distance was higher than 5.5 mm.

Statistical analysis

To determine the normality of the data, the Shapiro–Wilk test was used. The Mann–Whitney test was used to compare two independent groups with non-parametric data. For variables with normal distribution, Student’s t-test was used to compare the groups. Differences with p < 0.05 were considered statistically significant. Statistical Package for the Social Sciences (SPSS) v. 24.0 (IBM Corp, Armonk, NY, USA) and GraphPad Prism v. 8.2.0 (GraphPad Software, La Jolla, CA, USA) were used for the analyses.

Results

Experimental infection

Based on the observation of cysts during necropsy, 13/22 animals developed neurocysticercosis (59.1%) in the male group, and 15/25 animals developed neurocysticercosis (60%) in the female group. Seven males and eight females were used for morphological study, and six males and seven females were used to measure cytokine levels. Throughout the observation period, no animal developed signs of illness, discomfort, or abnormal behaviour.

Female rats presented more inflammatory infiltrate in the arachnoid than male rats

On histologic examination, lymphocytes and plasmacytes infiltrating the basal arachnoid region (near the optic chiasm) and periventricular zone (nearby the basal ganglia) were observed (Fig. 1). The ratio of lymphocytes/μm2 was higher among female than male rats in the arachnoid (p=0.023), but there was no statistically significant difference in the periventricular zone (Fig. 2). The ratio of plasmacytes/area was not different between the groups either in the arachnoid or the periventricular zone (p = 0.093 and 0.102, respectively). The ratio of reactive astrocytes (immunopositive cells) in the periventricular zone was also higher in the female group, with a statistically significant difference (p=0.001).

Fig. 1
figure 1

Inflammatory infiltrates (dashed lines) within the arachnoid-brain interface (A, B) and the periventricular zone (C, D). A higher number of inflammatory cells were observed in the female rats. In the periventricular zone, there was also a higher immune positivity for GFAP (arrowheads, E, F) in female rats. Forty times magnification

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Fig. 2
figure 2

Comparison of histologic patterns between the groups of male and female rats. The ratio of lymphocytes/μm2 within the basal arachnoid-brain interface was higher in the female group (A). The ratio of lymphocytes/μm2 within the periventricular zone (B) and the ratio of plasmocytes/μm2 in the arachnoid-brain interface (C) and the periventricular zone (D) did not reach a statistically significant difference. The immune positivity of astrocytes for GFAP was higher in the female group (E)

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IL-6 and IL-10 levels were significantly higher in females than in male rats

The levels of IL-6, IL-10, and IFN-γ are presented in Table 1. A significantly higher level of IL-6 and IL-10 was observed in females than in males. The numerical mean level of IFN-γ was also higher in females, but the difference was not statistically significant.

Table 1 Cytokine levels
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Detection of hydrocephalus (ventricle enlargement) was more frequent in females

On MRI, marked ventricle enlargement was observed in 2/5 males (40%) and 3/5 females (60%). No ventricle enlargement was observed in 2/5 males (40%) and 1/5 females (20%, Fig. 3).

Fig. 3
figure 3

Seven-tesla MRI T2-weighted images from animals in the two experimental groups. The images to the left correspond to animals with ventricle enlargement, whereas those to the right correspond to animals without ventricle enlargement, i.e. without hydrocephalus

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Discussion

This study presents the first evidence of sexual dimorphism in the neuroinflammation accompanying murine experimental extraparenchymal NCC (ExP-NCC). In humans, some differences between men and women in ExP-NCC have been previously reported, particularly the higher cellularity in the cerebral spinal fluid in women (Marcin-Sierra et al. 2017). However, it is difficult to describe intracranial inflammation in humans except for autopsy studies. In contrast, in experimental models, it is possible to evaluate the presence of inflammatory cells in different parts of the central nervous system using histopathological analysis, the status of astrocytes, and the profile of pro- and anti-inflammatory cytokines (Alvarez et al. 2010; Moura et al. 2016; de Lange et al. 2019; Sitali et al. 2022). In our study, we observed that the inflammatory reaction associated with subarachnoid infection with T. crassiceps was more severe in female than in male rats. Female rats presented a higher concentration of immune cells in the arachnoid, higher reactive astrogliosis in the periventricular region, higher in situ pro-inflammatory cytokine (IL-6) and anti-inflammatory cytokine (IL-10), and more intense hydrocephalus on MRI. The higher inflammatory profile in females resembles what was reported in human NCC (Fleury et al. 2004). This finding should be further explored in ExP-NCC in humans. In addition, these findings provide new alternatives to modulate neuroinflammation through hormones that can be systematically explored using the experimental model of ExP-NCC.

There was no significant difference in inflammatory cells in the periventricular zone. This is probably because the cysts were placed in the subarachnoid space without direct contact with the ventricles. We have previously observed the migration of subarachnoid cysts to the ventricles in our model (Hamamoto Filho et al. 2017); however, this was not observed in the present study.

The observation of more intense immune positivity to GFAP, i.e. reactive astrogliosis, in the periventricular area in female rats is noteworthy. GFAP is overexpressed in animals with hydrocephalus according to the degree of ventricle enlargement (Del Bigio et al., 2003; Santos et al. 2016). In addition to the MRI results in 10 animals, this finding suggests that hydrocephalus is more intense in females than males, probably due to the more intense inflammatory reaction.

Higher levels of IL-6 were observed in females, demonstrating a more pro-inflammatory pattern. Accordingly, IL-10 levels increased in females to control the inflammatory reaction (Sciutto et al. 2013). This mixed response may be related to endocrinal regulation, since cytokine production can be regulated by hormones, with direct implications to central nervous system diseases (Bornstein et al. 2004; Morales-Montor & Larralde 2005; Członkowska et al. 2006). However, the levels of IFN-γ were not different between the groups. Future studies with larger setting of cytokines may help understand the balance of pro- and anti-inflammatory cytokines involved in this infection model. The cytokine profile accompanying this murine model closely resembles that reported in severe human ExP-NCC (Chavarría et al. 2005). This is another similarity between a human and murine infection that consolidates the use of this model to study the modulation of neuroinflammation.

Regarding the clinical consequences of the infection, we did not observe any signs consistent with intracranial hypertension, such as abnormal behaviour, malaise, or anorexia/vomiting, throughout the study period. However, we have previously demonstrated that this model of neurocysticercosis-induced hydrocephalus requires a long time to cause changes in behavioural patterns (Hamamoto Filho et al. 2019b). A longer follow-up period can verify whether females will present more intracranial hypertension symptoms than males.

This sexual dimorphism of inflammation in extraparenchymal neurocysticercosis seems to be related to the endocrine status, particularly the steroid levels. Experimentally, it has been proposed that cysticerci may use steroids to improve their ability to synthesize androgens and oestrogens, thereby improving their reproductive capacity (Toledo et al. 2018; Romano et al. 2015; Hinojosa et al. 2012). Furthermore, in vivo studies with T. crassiceps demonstrated that the cysts may cause changes in the host’s neuroimmunoendocrine profile as a survival strategy (Morales-Montor & Larralde 2005; Arteaga-Silva et al. 2009; Nava-Castro et al. 2022). Recently, sexual hormones were shown to change the expression and morphology of T. crassiceps’ flame cells, thereby determining the survival (oestrogens and progesterone) or death (androgens) of the parasites (Ambrosio et al. 2014; Ambrosio et al. 2015).

As a limitation of the present study, it is worth mentioning that we did not include a sham group to exclude the possible effect of the surgical procedure on inflammation, even though it was not observed previously (Hamamoto Filho et al. 2019b). Besides, future investigations with systematic evaluation of the cysts’ developmental stages (viable, degenerating, or dead) will provide relevant information about the characteristics of inflammatory infiltrates, as well as the role of intrinsic differences between male and female brain structures on the susceptibility to infection.

In conclusion, inflammatory responses are more severe in female than male rats infected with T. crassiceps in the subarachnoid space. Sexual dimorphism is a critical variable in infectious diseases, and a better comprehension of this difference is a critical step for developing personalized approaches (Gay et al. 2021; Wesołowska 2022). The mechanisms underlying sexual dimorphism in the neuroinflammation accompanying murine ExP-NCC, including the relevance of endocrinological components, will be further studied to identify new therapeutic targets for immunomodulation. This is particularly important considering that the potent steroid anti-inflammatory agents currently used to treat patients with ExP-NCC decrease the effectiveness of the cysticidal treatment in approximately 70% of the patients.