K. A. Walker1, G. Jiang1, J. Di Bernardo1, K. O’Shea3, L. D. Shea4, S. M. Kunisaki1 4University Of Michigan,Biomedical Engineering,Ann Arbor, MI, USA 1University Of Michigan (Michigan Medicine),Surgery, Pediatric Surgery Section,Ann Arbor, MI, USA 3University Of Michigan (Michigan Medicine),Cell And Developmental Biology,Ann Arbor, MI, USA
Introduction: Fetoscopic repair is an emerging surgical option to help preserve neural function in prenatally diagnosed myelomeningocele (MMC). However, the exact in utero repair strategy that would facilitate maximal regeneration of damaged neural tissue remains unknown. Our laboratory has recently developed a hydrogel-based surgical patch to facilitate tissue coverage during fetoscopic MMC repair. The purpose of this study was to determine whether an organotypic slice culture (OSC) model of fetal MMC could be established to study the effects of patch repair on underlying spinal cord development.
Methods: Sprague-Dawley pregnant dams were gavaged with retinoic acid (60 mg/kg) at E10 gestation to induce fetal MMC. E21 rat pup spinal cords were longitudinally sectioned (400 microns) and cultured ex vivo in the presence of fibrin hydrogels (40 mg/mL) loaded with 6×105 rat dorsal root ganglion cells (group 1, n=10). Sections exposed to acellular fibrin hydrogels were used as controls (group 2, n=10). Neurotrophin expression, cell proliferation, apoptosis, and neural phenotype were subsequently analyzed at E21+7 and E21+14 by phase microscopy, qPCR, ELISA, and immunohistochemistry (IHC). The optical density (OD) of IHC sections was quantified using ImageJ. Statistical comparisons were by the unpaired t-test with significance set at p<0.05.
Results: Fetal OSCs were successfully cultured for up to 14 days while maintaining morphological integrity and demonstrating progressive neurite extension. qPCR showed upregulation of several neurotrophic genes, including neurotrophin-4/5 (NT4/5), ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 compared to native control tissues. ELISA demonstrated an increase in NT4/5 over time whereas BDNF and CNTF decreased over time. IHC (Figure) showed no significant differences between group 1 and group 2 in terms of proliferation and apoptosis based on Ki67 and Cas3 expression, respectively. The neural progenitor marker, Nestin, was significantly increased in group 1 compared to group 2 at E21+7 (mean OD: 0.60±0.14 vs. 0.33±0.16, p<0.03), but these levels were similar at E21+14 (mean OD: 0.50±0.06 vs. 0.53±0.07, p=0.38). At both time points, there were no significant differences with respect to astrocyte (GFAP) and neuronal (beta-III tubulin, NeuN, and MAP2) differentiation with the damaged spinal cord.
Conclusion: For the first time, this study demonstrates a novel ex vivo OSC model that can be sustained for a sufficient period of time to study the impact of patch repair on MMC neural tissue. Further work utilizing this organotypic approach to evaluate the effects of biomaterials, cells, and growth factors on MMC spinal cord development is warranted.
