Hostname: page-component-8448b6f56d-dnltx Total loading time: 0 Render date: 2024-04-20T01:16:51.128Z Has data issue: false hasContentIssue false

Effects of Toxocara larvae on brain cell survival by in vitro model assessment

Published online by Cambridge University Press:  17 June 2015

LEA HEUER
Affiliation:
Institute for Parasitology, University of Veterinary Medicine Hannover, Buenteweg 17, 30559 Hannover, Germany
SABINE HAENDEL
Affiliation:
Institute for Parasitology, University of Veterinary Medicine Hannover, Buenteweg 17, 30559 Hannover, Germany
ANDREAS BEINEKE
Affiliation:
Department of Pathology, University of Veterinary Medicine Hannover, Buenteweg 17, 30559 Hannover, Germany
CHRISTINA STRUBE*
Affiliation:
Institute for Parasitology, University of Veterinary Medicine Hannover, Buenteweg 17, 30559 Hannover, Germany
*
*Corresponding author. Institute for Parasitology, University of Veterinary Medicine Hannover, Buenteweg 17, 30559 Hanover, Germany. Tel: +49 511 953 8711. Fax: +49 511 953 8870. E-mail: christina.strube@tiho-hannover.de

Summary

Neuroinvasive larvae of the common dog and cat roundworms, Toxocara canis and Toxocara cati, may cause severe neurological and neuropsychological disturbances in humans. Despite their pathogenic potential and high prevalence worldwide, little is known about their cell-specific influences and cerebral host–pathogen interactions in neurotoxocarosis. To address this discrepancy, a co-culture system of viable larvae with murine neuronal (CAD), oligodendrocytal (BO-1) and microglial (BV-2) cell lines has been established. Additionally, murine adult brain slices have been co-cultured with Toxocara larvae to consider complex organotypic cell–cell interplay. Cytotoxicity of larval presence was measured enzymatically and microscopically. Microscopic evaluation using trypan blue exclusion assay revealed to be less reliable and sensitive than the lactate dehydrogenase activity assay. Ultimately, even low numbers of both T. canis and T. cati larvae have impaired survival of differentiated CAD cells, which morphologically resemble primary neurons. In contrast, viability of oligodendrocytal and microglial cells as well as brain slices was not impaired by larval presence. Therefore, immune-mediated mechanisms or trauma by migrating larvae presumably induce the in vivo pathology rather than acute cytotoxic effects. Conclusively, the helminthic larvae co-culture system presented here is a valuable in vitro tool to study cell-specific effects of parasitic larvae and their products.

Type
Research Article
Copyright
Copyright © Cambridge University Press 2015 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

Black, L. and Berenbaum, M. C. (1964). Factors affecting the dye exclusion test for cell viability. Experimental Cell Research 35, 913.Google Scholar
Blasi, E., Barluzzi, R., Bocchini, V., Mazzolla, R. and Bistoni, F. (1990). Immortalization of murine microglial cells by a v-raf/v-myc carrying retrovirus. Journal of Neuroimmunology 27, 229237.Google Scholar
Ciapetti, G., Granchi, D., Verri, E., Savarino, L., Stea, S., Savioli, F., Gori, A. and Pizzoferrato, A. (1998). False positive results in cytotoxicity testing due to unexpectedly volatile compounds. Journal of Biomedical Materials Research 39, 286291.3.0.CO;2-B>CrossRefGoogle ScholarPubMed
Daza, R. A., Englund, C. and Hevner, R. F. (2007). Organotypic slice culture of embryonic brain tissue. Cold Spring Harbor Protocols 2007, pdb.prot4914.Google Scholar
de Savigny, D. H. (1975). In vitro maintenance of Toxocara canis larvae and a simple method for the production of Toxocara ES antigen for use in serodiagnostic tests for visceral larva migrans. Journal of Parasitology 61, 781782.Google Scholar
Dolinsky, Z. S., Hardy, C. A., Burright, R. G. and Donovick, P. J. (1985). The progression of behavioral and pathological effects of the parasite Toxocara canis in the mouse. Physiology and Behavior 35, 3342.Google Scholar
Epe, C., Sabel, T., Schnieder, T. and Stoye, M. (1994). The behavior and pathogenicity of Toxocara canis larvae in mice of different strains. Parasitology Research 80, 691695.Google Scholar
Finsterer, J. and Auer, H. (2007). Neurotoxocarosis. Revista do Instituto de Medicina Tropical de São Paulo 49, 279287.Google Scholar
Fortenberry, J. D., Kenney, R. D. and Younger, J. (1991). Visceral larva migrans producing static encephalopathy in an infant. Pediatric Infectious Disease Journal 10, 403406.CrossRefGoogle ScholarPubMed
Henn, A., Lund, S., Hedtjärn, M., Schrattenholz, A., Pörzgen, P. and Leist, M. (2009). The suitability of BV-2 cells as alternative model system for primary microglia cultures or for animal experiments examining brain inflammation. ALTEX 26, 8394.Google Scholar
Heuer, L., Beyerbach, M., Lühder, F., Beineke, A. and Strube, C. (2015). Neurotoxocarosis alters myelin protein gene transcription and expression. Parasitology Research 114, 21752186. doi: 10.1007/s00436-015-4407-1.Google Scholar
Hsiao, W. W., Liao, H. S., Lin, H. H., Lee, Y. L., Fan, C. K., Liao, C. W., Lin, P. Y., Hwu, E. T. and Chang, C. S. (2013). Biophysical analysis of astrocytes apoptosis triggered by larval E/S antigen from cerebral toxocarosis-causing pathogen Toxocara canis . Analytical Sciences 29, 885892.Google Scholar
Janecek, E., Beineke, A., Schnieder, T. and Strube, C. (2014). Neurotoxocarosis: marked preference of Toxocara canis for the cerebrum and T. cati for the cerebellum in the paratenic model host mouse. Parasites and Vectors 7, 194.CrossRefGoogle Scholar
Janecek, E., Wilk, E., Schughart, K., Geffers, R. and Strube, C. (2015). Microarray gene expression analysis reveals major differences between Toxocara canis and Toxocara cati neurotoxocarosis and involvement of T. canis in lipid biosynthetic processes. International Journal for Parasitology 45, 495503.Google Scholar
Kim, H., Kim, E., Park, M., Lee, E. and Namkoong, K. (2013). Organotypic hippocampal slice culture from the adult mouse brain: a versatile tool for translational neuropsychopharmacology. Progress in Neuro-Psychopharmacology and Biological Psychiatry 41, 3643.CrossRefGoogle Scholar
Koh, J. Y. and Choi, D. W. (1987). Quantitative determination of glutamate mediated cortical neuronal injury in cell culture by lactate dehydrogenase efflux assay. Journal of Neuroscience Methods 20, 8390.Google Scholar
Korzeniewski, C. and Callewaert, D. M. (1983). An enzyme-release assay for natural cytotoxicity. Journal of Immunological Methods 64, 313320.Google Scholar
Krause, A. W., Carley, W. W. and Webb, W. W. (1984). Fluorescent erythrosin B is preferable to trypan blue as a vital exclusion dye for mammalian cells in monolayer culture. Journal of Histochemistry and Cytochemistry 32, 10841090.CrossRefGoogle ScholarPubMed
Liao, C. W., Fan, C. K., Kao, T. C., Ji, D. D., Su, K. E., Lin, Y. H. and Cho, W. L. (2008). Brain injury-associated biomarkers of TGF-beta1, S100B, GFAP, NF-L, tTG, AbetaPP, and tau were concomitantly enhanced and the UPS was impaired during acute brain injury caused by Toxocara canis in mice. BMC Infectious Diseases 8, 84.CrossRefGoogle ScholarPubMed
Louis, J. C., Magal, E., Muir, D., Manthorpe, M. and Varon, S. (1992). CG-4, a new bipotential glial cell line from rat brain, is capable of differentiating in vitro into either mature oligodendrocytes or type-2 astrocytes. Journal of Neuroscience Research 31, 193204.CrossRefGoogle ScholarPubMed
Lyons, S. A. and Kettenmann, H. (1998). Oligodendrocytes and microglia are selectively vulnerable to combined hypoxia and hypoglycemia injury in vitro . Journal of Cerebral Blood Flow and Metabolism 18, 521530.Google Scholar
Maizels, R. M. (2013). Toxocara canis: molecular basis of immune recognition and evasion. Veterinary Parasitology 193, 365374.CrossRefGoogle ScholarPubMed
Mitchell, D. B. S., Kenneth, S. S. and Acosta, D. (1980). Evaluation of cytotoxicity in cultured cells by enzyme leakage. Journal of Tissue Culture Methods 6, 113116.Google Scholar
Moreira-Silva, S. F., Rodrigues, M. G., Pimenta, J. L., Gomes, C. P., Freire, L. H. and Pereira, F. E. (2004). Toxocariasis of the central nervous system: with report of two cases. Revista da Sociedade Brasileira de Medicina Tropical 37, 169174.CrossRefGoogle ScholarPubMed
Mozes, E., Hunya, A., Posa, A., Penke, B. and Datki, Z. (2012). A novel method for the rapid determination of beta-amyloid toxicity on acute hippocampal slices using MTT and LDH assays. Brain Research Bulletin 87, 521525.Google Scholar
Pinelli, E. and Aranzamendi, C. (2012). Toxocara infection and its association with allergic manifestations. Endocrine, Metabolic and Immune Disorders – Drug Targets 12, 3344.CrossRefGoogle ScholarPubMed
Pringproa, K., Kumnok, J., Ulrich, R., Baumgärtner, W. and Wewetzer, K. (2008). In vitro characterization of a murine oligodendrocyte precursor cell line (BO-1) following spontaneous immortalization. International Journal of Developmental Neuroscience 26, 283291.Google Scholar
Qi, Y., Wang, J. K., McMillian, M. and Chikaraishi, D. M. (1997). Characterization of a CNS cell line, CAD, in which morphological differentiation is initiated by serum deprivation. Journal of Neuroscience 17, 12171225.Google Scholar
Quattrocchi, G., Nicoletti, A., Marin, B., Bruno, E., Druet-Cabanac, M. and Preux, P. M. (2012). Toxocariasis and epilepsy: systematic review and meta-analysis. PLOS Neglected Tropical Diseases 6, e1775.Google Scholar
Rajapakse, R. P., Vasanthathilake, V. W., Lloyd, S. and Fernando, S. T. (1992). Collection of eggs and hatching and culturing second-stage larvae of Toxocara vitulorum in vitro . Journal of Parasitology 78, 10901092.CrossRefGoogle ScholarPubMed
Richartz, E. and Buchkremer, G. (2002). Cerebral toxocariasis: a rare cause of cognitive disorders. A contribution to differential dementia diagnosis. Der Nervenarzt 73, 458462.CrossRefGoogle ScholarPubMed
Russegger, L. and Schmutzhard, E. (1989). Spinal toxocaral abscess. Lancet 2, 398.Google Scholar
Rüttinger, P. and Hadidi, H. (1991). MRI in cerebral toxocaral disease. Journal of Neurology, Neurosurgery, and Psychiatry 54, 361362.Google Scholar
Scheidegger, A., Vonlaufen, N., Naguleswaran, A., Gianinazzi, C., Müller, N., Leib, S. L. and Hemphill, A. (2005). Differential effects of interferon-gamma and tumor necrosis factor-alpha on Toxoplasma gondii proliferation in organotypic rat brain slice cultures. Journal of Parasitology 91, 307315.Google Scholar
Sommer, C., Ringelstein, E. B., Biniek, R. and Glockner, W. M. (1994). Adult Toxocara canis encephalitis. Journal of Neurology, Neurosurgery, and Psychiatry 57, 229231.Google Scholar
Söndergaard, H. P. and Theorell, T. (2004). A putative role for Toxocara species in the aetiology of multiple sclerosis. Medical Hypotheses 63, 5961.Google Scholar
Stoppini, L., Buchs, P. A. and Muller, D. (1991). A simple method for organotypic cultures of nervous tissue. Journal of Neuroscience Methods 37, 173182.CrossRefGoogle ScholarPubMed
Stoppini, L., Buchs, P. A., Brun, R., Muller, D., Duport, S., Parisi, L. and Seebeck, T. (2000). Infection of organotypic slice cultures from rat central nervous tissue with Trypanosoma brucei brucei . International Journal of Medical Microbiology 290, 105113.Google Scholar
Strober, W. (2001). Trypan blue exclusion test of cell viability. In Current Protocols in Immunology (ed. Coligan, J. E., Bierer, B. E., Margulies, D. H., Shevach, E. M. and Strober, W.) Appendix 3B.1–3B.2. Greene Publ. Associates and Wiley-Interscience, New York, USA.Google Scholar
Strube, C., Heuer, L. and Janecek, E. (2013). Toxocara spp. infections in paratenic hosts. Veterinary Parasitology 193, 375389.Google Scholar
Summers, B., Cypess, R. H., Dolinsky, Z. S., Burright, R. G. and Donovick, P. J. (1983). Neuropathological studies of experimental toxocariasis in lead exposed mice. Brain Research Bulletin 10, 547550.Google Scholar
Uliasz, T. F. and Hewett, S. J. (2000). A microtiter trypan blue absorbance assay for the quantitative determination of excitotoxic neuronal injury in cell culture. Journal of Neuroscience Methods 100, 157163.Google Scholar
Villano, M., Cerillo, A., Narciso, N., Vizioli, L. and Del Basso De Caro, M. (1992). A rare case of Toxocara canis arachnoidea. Journal of Neurosurgical Sciences 36, 6769.Google Scholar
Vonlaufen, N., Gianinazzi, C., Müller, N., Simon, F., Björkman, C., Jungi, T. W., Leib, S. L. and Hemphill, A. (2002). Infection of organotypic slice cultures from rat central nervous tissue with Neospora caninum: an alternative approach to study host–parasite interactions. International Journal for Parasitology 32, 533542.Google Scholar
Weyermann, J., Lochmann, D. and Zimmer, A. (2005). A practical note on the use of cytotoxicity assays. International Journal of Pharmaceutics 288, 369376.Google Scholar