Murine spinal cord explants: a model for evaluating axonal growth and myelination in vitro.

In vitro models of myelinating central nervous system axons have mainly been of two types, organotypic or dissociated. In organotypic cultures, the tissue fragment is thick and usually requires sectioning (physically or optically) before visual examination. In dissociated cultures, tissue is dispersed across the culture surface, making it difficult to measure the extent of myelinated fiber growth. We aimed to develop a method of culturing myelinated CNS fibers in defined medium that could be 1) studied by standard immunofluorescence microscopy (i.e., monolayer type culture), 2) used to measure axonal growth, and 3) used to evaluate the effect of substrate and media components on axonal growth and myelination. We used 120-micro m slices of embryonic murine spinal cord as a focal source of CNS tissue from which myelinated axons could extend in a virtual monolayer. Explants were cultured on both poly-L-lysine and astrocytes. The latter were used because they are the scaffold on which axonal growth and myelination occurs during normal development. Outgrowth from the explant and myelination of axons was poor on poly-L-lysine but was promoted by an astrocyte bed layer. The best myelin formation occurred in defined media based on DMEM using N2 mix; it was not promoted by Sato mix or Neurobasal medium with B27 supplement. Neuronal survival was poor in serum-containing medium. This tissue culture model should facilitate the study of factors involved in promoting outgrowth of CNS axons and their myelination. As such it is relevant to studies on myelination and spinal cord repair.

Triphasic MRI of pelvic organ descent: sources of measurement error.

PURPOSE: To identify sources of error when measuring pelvic organ displacement during straining using triphasic dynamic magnetic resonance imaging (MRI). MATERIALS AND METHODS: Ten healthy nulliparous woman underwent triphasic dynamic 1.5 T pelvic MRI twice with 1 week between studies. The bladder was filled with 200 ml of a saline solution, the vagina and rectum were opacified with ultrasound gel. T2 weighted images in the sagittal plane were analysed twice by each of the two observers in a blinded fashion. Horizontal and vertical displacement of the bladder neck, bladder base, introitus vaginae, posterior fornix, cul-de sac, pouch of Douglas, anterior rectal wall, anorectal junction and change of the vaginal axis were measured eight times in each volunteer (two images, each read twice by two observers). Variance components were calculated for subject, observer, week, interactions of these three factors, and pure error. An overall standard error of measurement was calculated for a single observation by one observer on a film from one woman at one visit. RESULTS: For the majority of anatomical reference points, the range of displacements measured was wide and the overall measurement error was large. Intra-observer error and week-to-week variation within a subject were important sources of measurement error. CONCLUSION: Important sources of measurement error when using triphasic dynamic MRI to measure pelvic organ displacement during straining were identified. Recommendations to minimize those errors are made.