Visualization of the regenerating microvasculature and neural fibers in the spinal cord injury of the axolotl
Authors: Svensson Fredrik Karl Erik , Thygesen Mathias Møller , Dittrich Anita , Ding Ming , Pedersen Michael , Lauridsen Henrik 
Published: June 2021

BackgroundSpinal cord injury (SCI) is a severe condition, often resulting in irreversible motoric and sensory deficit. The most common cause of SCI is traffic accidents, inflicting the spinal cord with a contusion trauma. Furthermore, SCI frequently affects young individuals, resulting in a long life with disability. While several pharmaceutical drugs have been tested for possible treatment of SCI, limited success have been found. Consequently, there is today no cure for SCI. Human neural tissue is known to be incapable of neural regeneration. Interestingly; recent studies suggest that angiogenesis may have a more important role than previously thought. In systems where regeneration and improvements in functional recovery are seen, greater densities of blood vessels infiltrating the spinal cord tissue have been observed independently of the therapeutic paradigm used to promote regeneration.To investigate the role of angiogenesis in neural regeneration, we seek to compare neural fiber and microvasculature behavioral after SCI in a vertebrate model capable of complete spinal cord regeneration, the axolotl.Our hypothesis is that it is possible to 3D visualize and compare neural fiber and microvasculature behavioral after SCI in axolotls with state‐of‐the‐art micro‐CT technology and high‐frequency ultrasound imaging. The aim of the study is to introduce a quantifiable measure of the regeneration of vessels and axons following SCI in axolotls using micro‐CT and high‐frequency ultrasound imaging, and compare those findings with conventional histology.Materials and methodsAxolotls (n = 18) were randomized into three groups; sham (n = 6), transection (n =6) SCI and contusion SCI (n = 6). A laminectomy of two vertebrae at the level directly caudal to the hind limbs was performed. Contusion SCI was induced by dropping a 25 g rod in a 30 mm free fall on the exposed spinal cord. Transection SCI was induced by transecting 2 mm of the spinal cord using micro scissors. Sham received identical surgery without SCI.Each axolotl was scanned with high‐frequency ultrasound before injury, directly post‐injury, 3 days post‐injury (dpi), 6 dpi, 12 dpi and 63 dpi. Scans consisted of flow measurements followed by a set of B‐mode images. Flow measurements were used to make flow‐maps of the vessel flow rates around the damaged spinal cord. B‐mode images were used to yield 3D vessel enhancement reconstructions.Micro CT‐scans are yet to be conducted. To visualize the microvascular architecture we will perfuse the axolotls using microfil contrast agent. Subsequently, the tissue samples will be stained with iodine and rescanned. Lastly, the iodine will be washed out, and the tissue will be used for histology.ResultsWith the initial calculations, we found that the number of vessels increase substantially over 63 dpi.Flow‐maps will potentially show the flow rates around the damaged spinal cord.MicroCT will potentially visualize the neural fiber and microvasculature side‐by‐side after SCI.ConclusionsInitial findings demonstrate that neo‐angiogenesis seems to be a crucial part of the neural tissue regeneration in the axolotl after a SCI. Further results might further highlight the relationship between the neural tissue and microvasculature in the regenerative process.These finding might enhance our understanding of how regeneration of the spinal cord is performed and potentially inspire to new ideas of treatment.Support or Funding InformationThis project is supported by Aarhus University, the Else and Mogens Wedell‐Wedellsborgs Fond, the A.P. Møller Fond and Helga and Peter Kornings Fond.