Artikel
Investigating cellular processes and molecular cascades secondarily induced in spinal placodes during the prenatal developmental time-course in the rat – a prerequisite for developing innovative therapy strategies for open spinal dysraphism
Untersuchung zu sekundären zellulären Prozessen und molekularen Schadenskaskaden in spinalen Plakoden während des pränatalen Entwicklungszeitverlaufs im Rattenmodell
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Veröffentlicht: | 8. Mai 2019 |
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Objective: Primary therapy for open spinal dysraphism is limited to surgical reconstruction of the spinal defect and symptomatic therapy of long-term sequelae. Future therapies aim for less invasive early fetal repair by tissue engineering to prevent further tissue damage and the development of co-morbidities. The prerequisite for such approaches is a detailed understanding of secondary and tertiary molecular cascades, induced by the “second hit” to the fetal placodes and its microenvironment.
Methods: A retinoic acid mmc model was established early in our lab. Female Sprague-Dawley rat were used. Time dated rats were gavage-fed with all-trans retinoic acid (RA) dissolved in olive oil at day E10. Controls received olive oil only. Fetuses were obtained via caesarean section at E16, E18, E22. Dysraphic lesions and associated findings were photo documented. Spinal cords of both groups were dissected in toto and transferred into PFA. Serial sections were cut and prepared for staining with neuroepithelieal marker expression incl. progenitor cell markers (e.g., GFAP, NeuN, BLBP, Nestin, 2CB2) and inflammatory marker (Vimentin, CD11b, CD68). Pro-inflammatory cytokines like TNFalpha, IL-1beta, and its receptors were analyzed by immunohistochemistry (IHC), real-time RT-PCR, double-labeling. Normal fetal sc of the respective time points served as controls.
Results: Placodes exhibited typical cellular profiles that in general corresponded our previous findings on human post-natal MMC-placodes. Glial and progenitor marker expression changed over the investigated time course and proinflammatory cytokines were induced in the placodes, compared to normal fetal sc tissues. The detailed semiquantitatively evaluation of these markers is still ongoing and will be reported. The time course of the respective cytokines TNFa and IL1a plus their main receptors under investigation will also be presented in detail along with their cellular relationships.
Conclusion: The retinoic mmc model provides a tool to investigate cellular and molecular characteristic of fetal mmc placodes and its microenvironment at different fetal stages. This is a prerequisite for developing strategies to protect cellular scaffolds for tissue engineering and promote endogenous neuroregeneration. All over understanding these processes will offer new targets for innovative adjuvant therapies in open spinal dysraphism.