Label-free Imaging of Tissue Architecture during Axolotl Peripheral Nerve Regeneration in Comparison to Functional Recovery

Abstract

Human peripheral nerves hold the potential to regenerate after injuries; however, whether a successful axonal regrowth was achieved can be elucidated only months after injury by assessing function. The axolotl salamander is a regenerative model where nerves always regenerate quickly and fully after all types of injury. Here, de- and regeneration of the axolotl sciatic nerve were investigated in a single and double injury model by label-free multiphoton imaging in comparison to functional recovery. We used coherent anti-Stokes Raman scattering to visualize myelin fragmentation and axonal regeneration. The presence of axons at the lesion site corresponded to onset of functional recovery in both lesion models. In addition, we detected axonal regrowth later in the double injury model in agreement with a higher severity of injury. Moreover, endogenous two-photon excited fluorescence visualized macrophages and revealed a similar timecourse of inflammation in both injury models, which did not correlate with functional recovery. Finally, using the same techniques, axonal structure and status of myelin were visualized in vivo after sciatic nerve injury. Label-free imaging is a new experimental approach that provides mechanistic insights in animal models, with the potential to be used in the future for investigation of regeneration after nerve injuries in humans.

Imaging Arm Regeneration: Label-Free Multiphoton Microscopy to Dissect the Process in Octopus vulgaris

“…Hemocytes invade the stump, changing their appearances from circulating, roundshaped cells to amebocyte-like cells (Figure 3). These latter cells resemble vertebrate macrophages (Aurora and Olson, 2014;Uckermann et al, 2019), thus suggesting their involvement in debris removal.…”

“…One of the potentially most useful applications of this technique is in the evaluation of mammalian disease states, including the production of high-resolution imaging of myelin sheets in physiological and pathological conditions (Huff and Cheng, 2007), and the identification of vessel tissue components to monitor the onset and progression of arterial diseases, such as atherosclerosis or aneurysms (Wang et al, 2008;Sehm et al, 2020). This approach has proved to be exceptionally versatile; it has been applied to the study of axon regeneration after spinal cord or peripheral nerve lesions in mammals (Morisaki et al, 2013), amphibians (Uckermann et al, 2019) and even invertebrates , facilitating comparison among animal species because it does not rely on species-related epitopes.…”

Effect of the neurodynamic mobilization technique combined with electroacupuncture on functional recovery and GAP-43 expression after sciatic nerve injury in rabbits

“…Peripheral nerve injury (PNI) is mainly caused by accidents, degenerative diseases, repetitive compression, or surgical interventions (Uckermann et al 2019;Li et al 2017), with an incidence of approximately 5% among all trauma cases (Gallaher and Steward 2018), and it results in restricted activity or life-long disability (Li et al 2017). Injury to peripheral nerves results in the destruction of severed axons distal to the injury site via Wallerian degeneration (Davies et al 2019;Conforti, Gilley, and Coleman 2014), thereby triggering Schwann cells (SCs) to rapidly dedifferentiate and proliferate (Mao et al 2019;Scheib and Höke 2013) and to elongate along their basal lamina in bands of Bungner to provide scaffolding for new axonal growth (Jones, Eisenberg, and Jia 2016; Gaudet, Popovich, and Ramer 2011).…”

In Vivo Cellular‐Level 3D Imaging of Peripheral Nerves Using a Dual‐Focusing Technique for Intra‐Neural Interface Implantation

“…Magnetic resonance imaging and ultrasonography, used in clinical practice, can help visualize the peripheral nerve structure in vivo; however, their spatial resolution of hundreds of micrometers is insufficient to visualize cellular structures such as myelinated axons with a diameter of 12–20 µm. [ 11 , 12 , 13 ] Recently, spectral confocal reflectance microscopy [ 14 ] and coherent anti‐Stokes Raman spectroscopy [ 15 ] were successfully applied to visualize the myelinated axons of rodent and rabbit sciatic nerves, respectively, in vivo. However, their narrow field of view (FOV) and nonvolumetric imaging properties provide information limited only to the nerve surface.…”