CHARACTERIZATION OF AXOLOTL SPINAL FLUID DURING HOMEOSTASIS AND LIMB REGENERATION

Accomplishing regeneration of complex tissues in humans has long been a goal of regenerative medicine. Once lost, humans are incapable of regenerating limbs. The axolotl is a vertebrate salamander capable of regenerating limbs throughout life. This occurs through the formation of the blastema, a mass of proliferating cells that replace missing tissues and is supported by peripheral nerve-derived factors. However, knowledge about how the axolotl brain and associated systems change in response to amputation is still largely understudied. Previous work in mammals has shown that cerebrospinal fluid (CSF) may be a source for bioactive molecules that interact with the central and peripheral nervous systems (CNS/PNS). Here, we describe a novel method for the extraction and characterization of axolotl CSF. Mass spectrometry was used to characterize the proteomic landscape of axolotl CSF during homeostasis and throughout different stages of limb regeneration. We identified 2,784 unique proteins present in axolotl CSF, with the largest class of proteins comprised of proteases and inhibitors. As limb regeneration proceeds, there is an increase in proteins typically associated with limb regeneration and extracellular matrix reorganization. Using tracers, we show that axolotl CSF diffuses into peripheral nerves. To evaluate how peripheral nerves may respond to changes in CSF ligands, we cultured axolotl dorsal root ganglia (DRGs) explants with CSF from different timepoints of limb regeneration. Interestingly, DRGs treated with mid-regeneration CSF exhibited reduced neurite outgrowth. Collectively, this work introduces a novel technique for collecting axolotl CSF while providing evidence for its role in regulating responses to amputation.