| Abstract No.: | B-F2179 |
| Country: | Canada |
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| Title: | THE PERIPHERAL-CENTRAL NERVOUS SYSTEM INTERACTION: A NOVEL TECHNIQUE IN ADULT BONE AND NERVOUS TISSUE CRYOSECTIONING. |
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| Authors/Affiliations: | 1 Farhana Begum*; 3 Kim Madec; 2 Mike Namaka; 4 Emma Frost;
1 Dept. Human Anatomy and Cell Science and Manitoba Institute of Child Health, University of Manitoba; 2 Dept. Pharmacy and Manitoba Institute of Child Health, University of Manitoba; 3 Dept. Pharmacy, University of Manitoba; 4 Dept.s Pathology, Human Anatomy and Cell Science, Biochemistry and Medical Genetics, MICH, U of Manitoba;Canada
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| Content: | Objective: To perfect a decalcification technique allowing cryosectioning of the spinal column to maintain the integrity of spinal cord attachments to the dorsal root ganglia (DRG) via the peripheral nerve roots.
Introduction: Studies looking at factors crossing the blood-brain-barrier at the dorsal horn of the spinal cord, encounter obstacles associated with obtaining a cross section of the entire spinal column, including peripheral roots, DRG and spinal cord. Many studies have attempted to compensate by obtaining cryosections of DRG and spinal cord separately, and then matching the sections to provide a single cross-section. Techniques that allow the sectioning of the entire vertebral column are time-consuming and require expensive specialized equipment, as sectioning bone, particularly adult bone, is technically difficult. An alternative approach is to decalcify the vertebral column prior to cryosectioning. However, if the study includes analysis of myelin the decalcification process can disrupt or even destroy the myelin structure. We have devised a novel protocol to decalcify adult rat vertebral column allowing cryosectioning at 10um, which maintains the structure of the DRG and the spinal cord connections, without disrupting myelin structure.
Materials and methods: We compared commercially available decalcification reagents RDO Gold (less than 10% HCl) and Krajian solution (20%-30% formic acid), and a commonly used decalcification solution EDTA-Glycerol, with 6% TCA. The entire spinal column was removed from perfusion-fixed adult rats. Vertebral segments, less than 1cm in length, were placed into decalcification solution for the required period, after which the segments were cryopreserved in 30% sucrose, and embedded in OCT for cryosectioning. Incubation times for the commercial reagents were as per manufacturers instructions, 6% TCA solution and EDTA- G solutions were assessed at several time points using insertion of a sharp needle into the bone to determine the end-point of decalcification. Serial sections, 10μm thickness, were stained for myelin basic protein (MBP) immunoreactivity, performed as previously described1. Images were processed in Image Pro (7.0) via an Olympus IX51 microscope, coupled to a monochrome digital camera. Confocal images were collected via an Olympus FV500 Confocal Microscope.
Results: MBP IHC of RDO GOLD and Krajian solution treated tissue resulted in indistinct myelin structure, with no ring structures visible. MBP IHC showed more distinct myelin structure in EDTA-G treated tissue, however, the rings are not clear. MBP IHC of TCA treated tissue showed clear structure of myelin, with very distinct MBP rings. Further, using confocal microscopy, we are able to visualise the nodes of Ranvier present within the grey matter of the spinal cord.
Conclusion: We conclude that decalcification using 6 % TCA solution results in the maintenance of the myelin structure, allowing accurate visualization of specific structures such as the nodes of Ranvier. Using this decalcification method we can successfully section the entire spinal column, maintaining the integrity of the peripheral root/DRG connections linking the PNS to the CNS. This work was funded by the Manitoba Institute for Child Health
1. Blaschuk, K. et al. Development 2000, 127, 1961
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