Meningeal cells and glia establish a permissive environment for axon regeneration after spinal cord injury in newts.

BACKGROUND: Newts have the remarkable ability to regenerate their spinal cords as adults. Their spinal cords regenerate with the regenerating tail after tail amputation, as well as after a gap-inducing spinal cord injury (SCI), such as a complete transection. While most studies on newt spinal cord regeneration have focused on events occurring after tail amputation, less attention has been given to events occurring after an SCI, a context that is more relevant to human SCI. Our goal was to use modern labeling and imaging techniques to observe axons regenerating across a complete transection injury and determine how cells and the extracellular matrix in the injury site might contribute to the regenerative process. RESULTS: We identify stages of axon regeneration following a spinal cord transection and find that axon regrowth across the lesion appears to be enabled, in part, because meningeal cells and glia form a permissive environment for axon regeneration. Meningeal and endothelial cells regenerate into the lesion first and are associated with a loose extracellular matrix that allows axon growth cone migration. This matrix, paradoxically, consists of both permissive and inhibitory proteins. Axons grow into the injury site next and are closely associated with meningeal cells and glial processes extending from cell bodies surrounding the central canal. Later, ependymal tubes lined with glia extend into the lesion as well. Finally, the meningeal cells, axons, and glia move as a unit to close the gap in the spinal cord. After crossing the injury site, axons travel through white matter to reach synaptic targets, and though ascending axons regenerate, sensory axons do not appear to be among them. This entire regenerative process occurs even in the presence of an inflammatory response. CONCLUSIONS: These data reveal, in detail, the cellular and extracellular events that occur during newt spinal cord regeneration after a transection injury and uncover an important role for meningeal and glial cells in facilitating axon regeneration. Given that these cell types interact to form inhibitory barriers in mammals, identifying the mechanisms underlying their permissive behaviors in the newt will provide new insights for improving spinal cord regeneration in mammals.

Fluorescent whole-mount method for visualizing three-dimensional relationships in intact and regenerating adult newt spinal cords.

Adult newts have the remarkable ability to regenerate their spinal cords after a complete transection injury. To understand this process, we have developed a method for visualizing the cellular and molecular events during regeneration in whole-mount preparations using fluorescent probes (streptavidins and antibodies) and confocal microscopy. This method was optimized by varying parameters associated with fixation, tissue trimming, fluorescent probe penetration, and clearing and represents a significant advance in our ability to observe the intact and regenerating newt spinal cord. These methods should also be widely applicable to the study of other newt tissues and adult tissues from other model systems.