INTRODUCTION
Human neurotoxocarosis is a zoonotic infection caused by neuroinvasive larvae of Toxocara canis (Werner, 1782) or Toxocara cati (Schrank, 1788), the common roundworms of dogs and cats, respectively (Finsterer and Auer, Reference Finsterer and Auer2007). After infection, larvae enter the host's tissue by penetrating the intestinal wall. Ultimately, passive blood transport and active migration may take larvae to the central nervous system (CNS). Such neuroinfections might manifest clinically in motor impairments such as ataxia, paresis or paralysis (Russegger and Schmutzhard, Reference Russegger and Schmutzhard1989; Villano et al. Reference Villano, Cerillo, Narciso, Vizioli and Del Basso De Caro1992; Sommer et al. Reference Sommer, Ringelstein, Biniek and Glockner1994; Moreira-Silva et al. Reference Moreira-Silva, Rodrigues, Pimenta, Gomes, Freire and Pereira2004). Furthermore, strong associations with epilepsy (Quattrocchi et al. Reference Quattrocchi, Nicoletti, Marin, Bruno, Druet-Cabanac and Preux2012) as well as cases of neuropsychological disturbances, such as dementia or depression, have been described (Fortenberry et al. Reference Fortenberry, Kenney and Younger1991; Rüttinger and Hadidi, Reference Rüttinger and Hadidi1991; Richartz and Buchkremer, Reference Richartz and Buchkremer2002).
Migrating Toxocara larvae may remain viable for years in host tissues and produce Toxocara excretory/secretory (TES) antigen (reviewed by Maizels, Reference Maizels2013). Despite remarkable worldwide prevalence of anti-TES antibodies (reviewed by Maizels, Reference Maizels2013), the well-characterized clinical syndromes (reviewed by Strube et al. Reference Strube, Heuer and Janecek2013) and suggested implications of this parasite infection in allergic conditions (Pinelli and Aranzamendi, Reference Pinelli and Aranzamendi2012) as well as neurodegenerative diseases (Söndergaard and Theorell, Reference Söndergaard and Theorell2004; Liao et al. Reference Liao, Fan, Kao, Ji, Su, Lin and Cho2008), the true pathogenic potential of Toxocara larvae is still poorly understood. Particularly, the underlying cause for parenchymal damage and demyelination in chronic CNS infections, as demonstrated in mice (Epe et al. Reference Epe, Sabel, Schnieder and Stoye1994; Heuer et al. Reference Heuer, Beyerbach, Lühder, Beineke and Strube2015), remains unclear. Mechanical effects of larvae perforating the brain tissue as well as exposure to larval TES and immune-mediated processes might play a role.
To address this question in vitro, co-cultures of viable T. canis and T. cati larvae with different CNS cell populations as well as a CNS tissue culture, so-called brain slices, were established. Neuronal, oligodendrocytal and microglial murine cell lines were employed to monitor consequences of cellular exposure to larvae. Additionally, suitability of brain slice co-culture as a replacement for in vivo studies on neurotoxocarosis was assessed by histological analysis.
MATERIALS AND METHODS
Animals
Experimental infection of dogs and cats for maintenance of T. canis and T. cati was approved by the ethics commission of the State Office for Consumer Protection and Food Safety of the German federal state of Lower Saxony under reference number 33.9-42502-05-01A038. For brain slice generation, adult C57Bl/6J mice were housed in Makrolon cages in a 12 h light/dark cycle. Standard rodent diet and water were supplied ad libitum. For organ removal, mice were euthanized by cervical dislocation. According to the German Animal Welfare Act, euthanasia of mice for brain removal was reported to the University's animal welfare officer.
In vitro hatching of Toxocara larvae
T. canis and T. cati eggs were isolated from feces of experimentally infected dogs and cats by standard sedimentation–flotation technique using saturated sodium chloride solution. Afterwards, eggs were allowed to embryonate for 4–6 weeks in tap water at 25 °C in an incubator (BT5C42E, Heraeus, Hanau, Germany). Larval hatching in vitro was performed according to Rajapakse et al. (Reference Rajapakse, Vasanthathilake, Lloyd and Fernando1992) with slight modifications. Briefly, eggs were concentrated in 1 mL tap water followed by adding 10 mL sodium hypochlorite (Carl Roth, Karlsruhe, Germany). The mixture was incubated at room temperature for 19 min. Afterwards, 40 mL of deionized water were added and the solution was centrifuged at 100 g for 5 min. Furthermore, eggs were washed eight times with 0·85% sterile NaCl solution with subsequent centrifugation at 1500 g for 5 min each. After the last centrifugation step, saline solution was removed to 1 mL of total volume and 10 mL phosphate-buffered saline (PBS) supplemented with 1% penicillin/streptomycin (GE Healthcare, Freiburg, Germany) were added. Eggs were gassed for 5 min with 100% CO2 from a gas cylinder and then centrifuged at 3000 g for 5 min. Subsequently, 10 mL of supernatant were removed carefully and 5 mL of larval culture medium [RPMI 1640 with 1% (w/v) glucose, 0·85% (w/v) sodium hydrogen carbonate, 10 U mL−1 penicillin/streptomycin (GE Healthcare) and 0·25 μg per mL amphotericin B (Genaxxon BioScience, Ulm, Germany)] were added. Larvae were then allowed to migrate through a gauze-covered Baermann-funnel overnight to eliminate any remaining egg shells, dead larvae and faeces particles from the medium (de Savigny, Reference de Savigny1975). Funnels were placed in a standard cell culture incubator (NU-4950E, NuAire, Plymouth, Minnesota, USA) at 37 °C with 5% CO2 supply. Larvae collected at the bottom of the funnel were transferred into six-well plates and fresh larval culture medium was added. Larval culture medium was replaced by the respective cell line medium (see section below) after 24 h of incubation. Both larval species (T. canis and T. cati) were maintained in media of the respective cell lines with daily media changes for at least 2 days before use in cell culture experiments.
Maintenance of central nervous cell cultures
CAD cells (Qi et al. Reference Qi, Wang, McMillian and Chikaraishi1997), a murine neuronal cell line, were obtained from Sigma-Aldrich (Taufkirchen, Germany) and maintained as described by Qi et al. (Reference Qi, Wang, McMillian and Chikaraishi1997). Briefly, undifferentiated CAD cells were grown in 25 and 75 cm2 tissue culture flasks (Sarstedt, Nürnbrecht, Germany) in Dulbecco's modified Eagle's medium (DMEM)/F12 medium supplemented with 1% GlutaMAX™ (Life Technologies, Darmstadt, Germany), 8% fetal bovine serum (FBS; GE Healthcare) and 2% penicillin/streptomycin (GE Healthcare). CAD cells differentiate into a cell division arrested primary neuron-like phenotype under conditions of serum deprivation. Differentiation was induced by switching to DMEM/F12 medium (Life Technologies) with 50 ng mL−1 sodium selenite (Sigma-Aldrich) and 2% penicillin/streptomycin after plating the cells in serum-containing medium 24 h before. Cells were incubated at 37 °C and 5% CO2 supply. Undifferentiated CAD cells were passaged at confluence every 3–4 days by gently rinsing them off the cell culture flask bottom with 5–10 mL of fresh medium and re-plating them at a 1:5–1:10 dilution.
BV-2 cells (Blasi et al. Reference Blasi, Barluzzi, Bocchini, Mazzolla and Bistoni1990), which are considered a valid model for the brain's resident microglia (Henn et al. Reference Henn, Lund, Hedtjärn, Schrattenholz, Pörzgen and Leist2009), were purchased from Banca Biologica e Cell Factory (ICLC ATL03001, Interlab Cell Line Collection, IRCCS Azienda Ospedaliera Universitaria San Martino – IST Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy). Cells were maintained in 25 cm2 tissue culture flasks in RPMI 1640 medium supplemented with 2 mm L-glutamine (Life Technologies), 10% FBS and 2% penicillin/streptomycin at 37 °C and 5% CO2 supply. Every 3–4 days, cells were passaged at a 1:5–1:10 dilution after trypsination and centrifugation at 300 g for 5 min.
As oligodendrocytal cells, BO-1 cells (Pringproa et al. Reference Pringproa, Kumnok, Ulrich, Baumgärtner and Wewetzer2008) were cultured in 25 cm2 tissue culture flasks pre-coated with poly-L-lysine (Sigma-Aldrich). Cells were maintained in B104-conditioned medium containing the N1 supplements as described by Louis et al. (Reference Louis, Magal, Muir, Manthorpe and Varon1992) and passaged at confluence, every 2–5 days at a 1:2–1:5 dilution after trypsination and centrifugation at 300 g for 5 min.
Co-culture of central nervous cell lines and Toxocara larvae
Co-culture experiments were run in six-well plates and repeated at least once. Seeding densities were 1 × 104 cells per well for undifferentiated CAD- and BV-2 cells, 2 × 104 cells per well for BO-1 cells and 6 × 104 cells per well for differentiated CAD cells. After seeding into six-well plates, cells were allowed to attach for at least 24 h before addition of larvae. Per experiment and group, six wells of cells each were exposed to 10, 50, 100 or 500 T. canis or T. cati larvae resulting in n = 12 replicates per larval quantity of each parasite species (exposure group). Undifferentiated CAD cells and BV-2 cells in co-culture with larvae are depicted in Fig. 1. Wells with unexposed cells served as controls. Viability of cells in culture after exposure to larvae was assessed using two different methods. On day 5 after addition of T. canis and T. cati larvae, cells were harvested to assess cell viability via trypan blue exclusion assay. This dye accumulates in damaged or dead cells. Assay performance and microscopic counting were carried out following a standard protocol (Strober, Reference Strober, Coligan, Bierer, Margulies, Shevach and Strober2001). Additionally, cytotoxicity was evaluated enzymatically. As the cytoplasmic enzyme lactate dehydrogenase (LDH) is released into the supernatant if the cell membrane's integrity is impaired, it is a reliable marker for natural cytotoxicity (Korzeniewski and Callewaert, Reference Korzeniewski and Callewaert1983). Thus, cell culture supernatants were assayed using a commercial LDH assay [CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Mannheim, Germany)] twice and in triplicates (n = 72 per exposure group) according to the manufacturer's instructions. The respective media as well as supernatants of T. canis and T. cati larval cultures without central nervous cells served as negative controls.
Toxocara canis larvae and undifferentiated CAD neuronal cells (A) or BV-2 microglial cells (B), respectively, after 5 days in co-culture. Microscopically, both larvae and cells appear viable. Scale bars represent 200 μ m (A) and 50 μ m (B).
Brain slices
Generation of brain slices was based on methods published previously (Stoppini et al. Reference Stoppini, Buchs and Muller1991; Daza et al. Reference Daza, Englund and Hevner2007; Kim et al. Reference Kim, Kim, Park, Lee and Namkoong2013). Briefly, adult C57Bl/6J mice (>6 months of age) were euthanized by cervical dislocation, decapitated and brains were removed under sterile conditions. Cerebella were disconnected and cerebra were halved vertically and immediately embedded in liquid 4% low melting agarose (Sigma-Aldrich) using a 24-well tissue culture plate kept on ice. After approximately 5 min, agarose had hardened and was removed from the tissue culture plate in one piece. The embedded brain tissue was then attached to the vibratome block (Lancer® vibratome series 1000 sectioning system, The Vibratome Company, St. Louis, Missouri, USA) using cyanoacrylate adhesive and vibratome block and attached tissue were entirely submerged in ice-cold PBS with 1% antibiotics (GE Healthcare) in the vibratome basin. Slices of approximately 200 μ m were cut (Fig. 2), placed in ice-cold dissection medium [Hibernate A with 2% Gibco® B27® supplement (Life Technologies, Darmstadt, Germany), 1% GlutaMAX™ supplement, 1% penicillin/streptomycin and 0·25 μg mL−1 amphotericin B (Genaxxon BioScience, Ulm, Germany)] according to Kim et al. (Reference Kim, Kim, Park, Lee and Namkoong2013) for at least 5 min and then transferred onto cell culture inserts (PICM0RG50, Millipore, Molsheim, France) insix-well plates with 1 mL of serum-free brain slice culture medium [Neurobasal A with 2% Gibco® B27® supplement, 1% GlutaMAX™ supplement, 1% penicillin/streptomycin and 0·25 μg mL−1 amphotericin B (Genaxxon BioScience, Ulm, Germany)] according to Kim et al. (Reference Kim, Kim, Park, Lee and Namkoong2013). Initially, slices were incubated at 37 °C with 5% CO2 supply for 24 h. After media change, duplicate slices of six different animals per group were incubated for 6 days with 10, 50 or 100 T. canis or T. cati larvae. Daily media changes were performed by carefully removing 0·5 mL of medium from each well and adding 0·5 mL of fresh brain slice medium. Control slices of all animals, at least one slice per animal, were not exposed to larvae but otherwise treated identically. Supernatants of day 6 after addition of T. canis and T. cati larvae were analysed for LDH by CytoTox 96® Non-Radioactive Cytotoxicity Assay twice and in triplicates (n ≥ 36 per exposure group) as described above.
Generation of adult mouse brain slices using the Lancer® vibratome series 1000 sectioning system (The Vibratome Company).
For histological analysis, brain slices were submerged in 10% phosphate-buffered formalin (Roti®-Histofix, Carl Roth) and embedded in paraffin wax. Slices were stained with haematoxylin and eosin (H&E). In H&E stains, damaged and viable neurons were counted in three fields of vision per slice at 40× magnification. Thus, between 430 and 1430 cells were evaluated per group. Subsequently, numbers of damaged and viable neurons were expressed as percentage of total cells per group.
Statistical analyses
Control and exposure groups were compared by Mann–Whitney U tests and subsequent Bonferroni–Holm corrections using the GraphPad Prism 6 software (version 6.03; GraphPad Software, La Jolla, CA, USA). Differences were considered significant, if P ≤ 0·05.
RESULTS
Trypan blue exclusion assay of central nervous cell–Toxocara co-cultures
Microscopic identification and enumeration of viable and dead cells showed that cell death was increased significantly in undifferentiated CAD cells compared with unexposed controls when co-cultivated with 100 and 500 T. canis larvae (Fig. 3A). Toxocara cati larvae did not increase cell death in undifferentiated CAD cells. Successful differentiation of CAD cells was confirmed by growth of long, axon-like processes. In these cells, only addition of 100 T. cati larvae induced a significant effect on cell viability (Fig. 3C). Proportions of dead cells in differentiated CAD cells were markedly higher than in undifferentiated CAD cells. In BO-1 cells, death rates were close to 0% and only 500 T. canis larvae caused a significantly higher degree of cell death (Fig. 3E). In BV-2 cells, less dead cells were detected in setups exposed to 10 and 50 T. canis larvae as well as 10, 100 and 500 T. cati larvae compared with controls (Fig. 3G).
Trypan blue exclusion assay (A, C, E, G) and LDH release assay (B, D, F, H) of undifferentiated CAD cells (A, B), differentiated CAD cells (C, D), BO-1 cells (E, F) and BV-2 cells (G, H) 5 days after exposure to 10, 50, 100 and 500 viable T. canis and T. cati larvae, respectively. Differentiated CAD cells appear to be most affected by larval presence of both T. canis and T. cati. Error bars indicate standard deviation (s.d.), asterisks statistically significant differences (P ≤ 0·05).
LDH activity assay of central nervous cell–Toxocara co-cultures
In undifferentiated CAD cells, LDH activity was enhanced compared with unexposed controls by co-cultivation with 500 viable T. canis larvae (Fig. 3B). Less than 500 T. canis or T. cati larvae did not have a significant effect on LDH release of undifferentiated CAD cells. In contrast, a significantly higher LDH activity compared with controls was detected in supernatants of differentiated CAD cell co-cultures when incubated with 50, 100, 500 T. canis larvae or 10, 50, 100 or 500 T. cati larvae (Fig. 3D). In supernatants of BO-1 cell–Toxocara co-cultures, LDH activity of control and exposure groups was very low (Fig. 3F). Thus, statistical analysis was not feasible. In supernatants of BV-2 cell co-culture setups, LDH activity was not significantly affected by presence of either larval species (Fig. 3H). Larval viability monitored by microscopic inspection did not appear to be influenced by co-culture conditions (Fig. 1). Additionally, LDH activity of T. canis- and T. cati larval culture supernatants was measured and did not differ from the negative control.
Brain slice–Toxocara co-cultures
LDH activity of brain slice–Toxocara co-culture media on day 6 of co-incubation did not differ significantly from controls. Histological analysis of H&E stained slices showed medium to high numbers of damaged or dying neurons (Fig. 4), macro-and microglia cells, gliosis, proliferation of endothelial cells and single gitter cells in Toxocara-exposed and control slices. Larvae were not detected in the tissue. Microscopic evaluation of neuron viability and LDH measurements did not reveal any significant differences between control and exposure groups. LDH activity in brain slices and proportions of damaged neurons in these slices are depicted in Fig. 5.
Viable and damaged neurons in control (A) and brain slices infected with 100 T. cati larvae (B) and 100 T. canis larvae (C) (H&E stain). Dashed arrows indicate viable neurons with round cores and nuclei. Continuous arrows indicate damaged neurons with swollen or shrunken eosinophilic cytoplasm. Scale bars represent 50 μ m (A–C).
Analysis of cell death and damage in brain slice–Toxocara co-cultures after 6 days of incubation with 10, 50 and 100 T. canis or T. cati larvae, respectively. Microscopic evaluation of damaged neurons in H&E stained slides (A) and LDH activity in co-culture media (B) did not reveal any significant differences of T. canis- or T. cati-exposed brain slices compared with controls. Error bars indicate s.d..
DISCUSSION
Severe histopathological changes have been described in brains of mice infected with T. canis, while animals present with central nervous impairments (Epe et al. Reference Epe, Sabel, Schnieder and Stoye1994; Heuer et al. Reference Heuer, Beyerbach, Lühder, Beineke and Strube2015). Although lesions have been reported to be predominantly located in the white matter (Summers et al. Reference Summers, Cypess, Dolinsky, Burright and Donovick1983; Dolinsky et al. Reference Dolinsky, Hardy, Burright and Donovick1985) and demyelination as well as indication of axonal damage have been documented in T. canis-infected animals (Epe et al. Reference Epe, Sabel, Schnieder and Stoye1994; Janecek et al. Reference Janecek, Beineke, Schnieder and Strube2014; Heuer et al. Reference Heuer, Beyerbach, Lühder, Beineke and Strube2015), limited information about the susceptibility of individual CNS cell types to Toxocara-mediated damage is available to date. Cell culture-based approaches to unravel neuropathology caused by this parasite are scarce. The only CNS cell type investigated in the context of neurotoxocarosis is the astrocyte, where T. canis-TES has been found to trigger apoptosis (Hsiao et al. Reference Hsiao, Liao, Lin, Lee, Fan, Liao, Lin, Hwu and Chang2013). The impact of T. cati as well as the reaction of other CNS cell types to TES or Toxocara larvae themselves is completely unknown.
Focusing on the histological damage described above oligodendrocytal (BO-1), neuronal (CAD) and microglial (BV-2) cell lines as well as brain slice cultures were selected for co-cultivation with viable Toxocara larvae. With the successful establishment of a co-culture of T. canis or T. cati larvae with murine CNS cell lines or brain slices, respectively, a tool for research on cell-specific effects of larval presence in vitro is provided. Brain slice cultures in particular offer the possibility to investigate parasite-host interplay in three-dimensional, organotypic tissue organization (Stoppini et al. Reference Stoppini, Buchs and Muller1991, Reference Stoppini, Buchs, Brun, Muller, Duport, Parisi and Seebeck2000). Typically, perinatal brain tissue is used for cultivation as it shows enhanced viability (Kim et al. Reference Kim, Kim, Park, Lee and Namkoong2013). Nevertheless, this study aimed to further simulate natural conditions by use of adult mouse brain slices, as neurotoxocarosis in paratenic hosts is a chronic disease process resulting generally from postnatal infections. Thus, the use of perinatal brain slices for researches of the adult brain is controversial (Kim et al. Reference Kim, Kim, Park, Lee and Namkoong2013).
To investigate potential cytotoxic effects of larval presence, release of LDH into the culture medium was employed in brain slice and CNS cell co-cultures. Additionally, trypan blue staining were performed on CNS cell co-cultures as usage of more than one assay method has been recommended in cytotoxicity testing to avoid experimental artefacts (Ciapetti et al. Reference Ciapetti, Granchi, Verri, Savarino, Stea, Savioli, Gori and Pizzoferrato1998). Trypan blue exclusion stain was chosen in addition to the LDH assay as sometimes simple, inexpensive manual techniques are less prone to interferences than enzymatic kits (Weyermann et al. Reference Weyermann, Lochmann and Zimmer2005). Indeed, results differed between the two methods used in all the examined cell types. Although the trypan blue exclusion test is commonly used for routine viability checks in cell culture, its downfalls are time-consuming manual counting, investigator's bias and sampling errors (Uliasz and Hewett, Reference Uliasz and Hewett2000). For brain slices, LDH activity coincided with microscopic evaluation of neurons in H&E stains. Furthermore, LDH activity measurement has previously been described as a reliable method to quantify cell death in neurons (Koh and Choi, Reference Koh and Choi1987), oligodendrocytes, microglia (Lyons and Kettenmann, Reference Lyons and Kettenmann1998) and brain slices (Mozes et al. Reference Mozes, Hunya, Posa, Penke and Datki2012). Therefore, LDH assay results were considered sustainable.
Depending on cell lines and media used, results from trypan blue staining differed significantly from those obtained by LDH activity measurement. In differentiated CAD cells, for example, the groups T. canis 50, 100, 500 and T. cati 10, 50 and 500 did not show statistically significant differences to controls in the dye exclusion test, whereas they did in LDH measurements. Such false negative results of trypan blue stains have been reported previously (Krause et al. Reference Krause, Carley and Webb1984). Cell type-dependent differences and staining of serum protein in the media have been described (Black and Berenbaum, Reference Black and Berenbaum1964; Strober, Reference Strober, Coligan, Bierer, Margulies, Shevach and Strober2001) and may explain why results of manual cell counts for BV-2 cells, a model for the brain's resident microglia (Henn et al. Reference Henn, Lund, Hedtjärn, Schrattenholz, Pörzgen and Leist2009), and undifferentiated neuronal CAD cells, grown in serum-containing media, cannot or just partially be reproduced in the LDH assay. Furthermore, LDH tests have been shown to detect early events of cell injury, which cannot be detected by trypan blue stain (Mitchell et al. Reference Mitchell, Kenneth and Acosta1980). This may serve to explain why in differentiated CAD cells almost all parasite exposed groups showed a significantly higher LDH level than controls, whereas in the dye exclusion test only one group (exposed to 100 T. cati larvae) revealed significantly more stained cells. Overall, the LDH assay appears to be a feasible test for parasite-induced cytotoxicity.
Looking at the effect of viable T. canis or T. cati larvae on the different cell types in detail, viability of microglial and oligodendrocytal cells did not appear to be significantly affected. As in vivo oligodendrocytal damage occurs weeks after larvae have entered the brain, the lack of an effect may be due to the short incubation period of 5 days. On the other hand, the relation of larvae to cells and TES concentration were certainly higher in this in vitro assay than in an in vivo infection. Eight weeks post infection (pi) with 1000 T. canis larvae, Epe et al. (Reference Epe, Sabel, Schnieder and Stoye1994) reported a maximum of 110 larvae in the entire brain of C57Bl6 mice. Janecek et al. (Reference Janecek, Beineke, Schnieder and Strube2014) detected a maximum of 36·3 T. canis larvae (day 42 pi) and 9·4 T. cati larvae (day 98 pi) in the CNS of C57Bl6 mice in an infection setting with 2000 embryonated T. canis or T. cati eggs. Therefore, the lack of compromised viability in BO-1 cells could also indicate, that oligodendrocytal damage in vivo is not the primary result of TES exposure. However, the methods used only detect cellular damage associated with impairments of membrane integrity. Further research is needed to evaluate potential intracellular effects, which might lead to apoptosis in the long term. In contrast, the lack of an effect on microglial (BV-2) cells and on brain slices appears to demonstrate that the histopathological picture presented in vivo with gitter cell accumulations and demyelination (Janecek et al. Reference Janecek, Beineke, Schnieder and Strube2014; Heuer et al. Reference Heuer, Beyerbach, Lühder, Beineke and Strube2015) is neither a direct effect of TES nor a very local primary immune response to such. In the light of these findings, in vivo pathology appears to be the result of other processes, e.g. chronic trauma responses to migrating larvae as well as more extended or even global immune-mediated brain damage. Another explanation might be that TES-mediated neuronal damage and axonal loss could lead to secondary demyelination and subsequent activation of microglia and this process might just take more than 6 days.
Although histological examination of brain slices co-cultured with Toxocara larvae did not reveal a clear predilection of any cell type to TES-mediated cytotoxicity and evaluation of neuronal survival did not reveal differences to controls, further research is needed to understand the mechanisms of a possible Toxocara-associated neurotoxicity. As the histopathological alterations observed in vivo (Epe et al. Reference Epe, Sabel, Schnieder and Stoye1994; Janecek et al. Reference Janecek, Beineke, Schnieder and Strube2014; Heuer et al. Reference Heuer, Beyerbach, Lühder, Beineke and Strube2015) could not be reproduced in the brain slice model, the system cannot serve as a full replacement for animal trials in neurotoxocarosis research. Additionally, it cannot be determined whether the neuronal damage observed is due to brain slice generation or parasitic effects. In contrast, other studies using neonatal hippocampal rat brain slices and protozoan parasites, such as Toxoplasma gondii (Scheidegger et al. Reference Scheidegger, Vonlaufen, Naguleswaran, Gianinazzi, Müller, Leib and Hemphill2005), Neospora caninum (Vonlaufen et al. Reference Vonlaufen, Gianinazzi, Müller, Simon, Björkman, Jungi, Leib and Hemphill2002) and Trypanosoma brucei brucei (Stoppini et al. Reference Stoppini, Buchs, Brun, Muller, Duport, Parisi and Seebeck2000), highlight the usefulness of the model for the study of parasite–host interactions. Stoppini et al. (Reference Stoppini, Buchs, Brun, Muller, Duport, Parisi and Seebeck2000) propose incubation of organotypic brain slices in the presence of proinflammatory agents for trypanosomiasis research, as the CNS pathology is predominantly an inflammatory process. Molecular and histopathological examinations on neurotoxocarosis draw a similar picture with immune-mediated factors and traumatic injuries playing an important part in pathogenesis (Epe et al. Reference Epe, Sabel, Schnieder and Stoye1994; Janecek et al. Reference Janecek, Beineke, Schnieder and Strube2014, Reference Janecek, Wilk, Schughart, Geffers and Strube2015). Therefore, further optimization of the Toxocara brain slice co-culture with injection of larvae into the slice and administration of proinflammatory agents might aid in simulating a broader spectrum of the disease's aspects and natural route of exposure to TES.
However, the presence of 10, 50, 100 and 500 T. cati as well as 50, 100 and 500 T. canis larvae has a cytotoxic effect on differentiated CAD cells. Undifferentiated CAD cells are only affected by 500 T. canis larvae, suggesting an enhanced susceptibility of TES-mediated cytotoxicity of differentiated over undifferentiated CAD cells and overall, of the neuronal cell line over microglial and oligodendrocytal ones. Interestingly, T. cati and T. canis larvae reveal a similar cytotoxic potential in differentiated, primary neuron-like, CAD cells, although T. canis larvae cause more extensive parenchymal brain damage in vivo (Janecek et al. Reference Janecek, Beineke, Schnieder and Strube2014; Heuer et al. Reference Heuer, Beyerbach, Lühder, Beineke and Strube2015). Therefore, long-term effects of T. cati larvae in the host's brain should not be underestimated as they appear to mediate neurotoxic effects.
Concluding remarks
The successful establishment of a co-culture system of Toxocara larvae with different murine cell lines and brain tissue culture enables further molecular and cell type-specific research on this zoonotic pathogen. Although the brain slice co-culture does not appear to be a satisfying substitute for in vivo experiments to date, optimization of this system might lead to its employment for specific questions regarding neurotoxocarosis. Observed cytotoxic effects of both T. canis and T. cati on murine neuronal cells highlight their destructive potential and raise more questions about consequences of their presence in the human brain. Therefore, in vitro approaches leading to a deeper understanding of the molecular mechanisms of this disease are highly desirable as long-term effects of human infections with Toxocara spp. as well as their impact on public health are still poorly understood.
ACKNOWLEDGEMENTS
The authors would like to thank Bettina Buck and Danuta Waschke for excellent technical assistance and both Annika Lehmbecker, PhD and Ingo Spitzbarth, PhD for advice on brain slice culture generation and LDH assays.
FINANCIAL SUPPORT
A PhD grant of the ‘Hannover Graduate School of Veterinary Pathobiology, Neuroinfectiology and Translational Medicine’ for L.H. is gratefully acknowledged.
