gms | German Medical Science

Objective: Myelomeningoceles (MMC) are clinically challenging malformations, improvement in management has been achieved in antenatal diagnosis, prevention and fetal surgery, but cellular mechanisms of damage in the placode are poorly understood. Like in spinal cord injury, the primary lesion induces cellular and molecular cascades promoting ongoing destructive processes, established as "secondary lesion cascades". Mechanical and toxic injuries of the exposed dorsal spinal cord may also induce lesion cascades along the spinal axis. Evaluation of functional aspects and relevance of these findings with regard to fetal development necessitates standardized conditions of an animal model. We established a non-surgical MMC retinoic acid rat model focusing on inflammatory cascades in neural placodes and along the spinal axis in the newborn rat.

Methods: Time-dated Sprague-Dawley rats were gavage fed different doses of retinoic acid (RA, Sigma Aldrich) (50, 60 and 70 mg/kg dissolved in olive oil, applied at E10) (maternal n=15, newborn=163). Control animals received olive oil alone (maternal n=5, newborn n=67). All newborn animals underwent inspection. The neural axis was analyzed by detailed H & E histopathology and immunohistochemical analyses for neuroglial (Neuronal nuclei (NeuN, NF200kD, GFAP, Iba1), neural crest marker expression (Nestin, BLBP), Interleukin-1beta/-receptor (IL-1b/ IL-1R1), TNFalpha/TNF-Receptor (TNFa/TNF-R), Chemokine (C-C motif) ligand 3/ C-C Chemokine receptor type 1 (CCL3/CCR1). Immunoreactivities were analyzed qualitatively and semiquantitatively (ImageJ 1.43u, NIH, USA).

Results: Isolated MMC appeared in 4.3% in the 50mg group, craniofacial anomalies in 7.2%, anencephaly in 1.4% of cases. In the 60mg group MMC appeared in 97.8%, anencephaly in 20%, craniofacial anomalies in 4.4%. Caudal regression was seen in 24.4% of pups. In the 70mg group MMC was observed in 62.1%, anencephaly in 24.1%, craniofacial abnormalities in 37.9%. Investigated placodes exhibited cellular profiles with consistent neuroglial cellular marker expression. Densitometry for investigated cyto- and chemokines confirmed significant induction of pro-inflammatory markers. GFAP and Vimentin were significantly elevated in MMC placodes as well.

Conclusion: Application of 60 mg/kg of RA on E10 reliably induced open spinal dysraphic defects in the offspring. The neural placodes exhibited specific inflammatory reactions, thus establishing a reliable animal model for further investigating fetal dysraphic pathology.