Method for quantification of the myelin content using luxol fast blue staining in injured spinal cord of adult rats and whole body vibration as a possible method for enhancing (re)myelination

Abstract

Introduction: Luxol fast blue (LFB) is a dye that binds stoichiometrically to myelin and allows quantifica­tion of the myelination in preparations from the CNS. Therefore, LFB`s can be used to evaluate the dam­age after spinal cord injury (SCI) and the effect of diverse treatment methods on remyelination in the in­jured spinal cord. Materials and methods: 37 female Wistar rats were subjected to a compressive SCI at a low-thoracic lev­el (vertebra Th8). The animals were randomized to four groups treated daily with whole body vibra­tion (WBV) starting 1, 7, 14 or 28 days after injury (WBV1, WBV7, WBV14, WBV28) that continued for 12-weeks. Four groups served as controls - rats euthanized in the first 2 weeks post SCI (subacute), un­treated (Sham therapy), treated with passive flexion-extension (PFE) and intact animals. We measured the optical density (OD) of longitudinal spinal cord sections stained with LFB. The entire slice area and a re­gion of interest spanning 2.5 mm on both sides of the compression site were evaluated. In a second step we applied an image mask to exclude the `empty` fields from the section (due to vessels, tears or damage vacuoles). Thus, the OD is evaluated once in the entire sectional plane and a second time only in the pre­served tissues (correspondingly the preserved neural tissue bridging the compression (PNTB)). The two measurement modes allowed the calculation of different quantitative features of the LFB staining. Results: WBV groups showed different degree of remyelination compared to sham therapy. A trend for better (re)myelination in the PNTB was observed in the WBV14 group. Conclusion: Using LFB staining for analysis in SCI animal models is suitable for quantification of the my­elination. The method allows an evaluation and comparison between the different groups and treatment options. WBV could enhance remyelination depending on the day it was initiated.