A major barrier to intraspinal regeneration after dorsal root (DR) injury is the DR entry zone (DREZ), the CNS/PNS interface. DR axons stop regenerating at the DREZ, even if regenerative capacity is increased by a nerve conditioning lesion. This potent blockade has long been attributed to myelin-associated inhibitors and CSPGs, but incomplete lesions and conflicting reports have prevented conclusive agreement. Here we evaluated DR regeneration in mice, using novel strategies to facilitate complete lesions and analyses, selective tracing of proprioceptive and mechanoreceptive axons, and the first simultaneous targeting of Nogo/Reticulon-4, MAG, OMgp, CSPGs and GDNF. Co-eliminating myelin inhibitors and CSPGs elicited regeneration of only a few conditioning-lesioned DR axons across the DREZ. Their absence, however, markedly and synergistically enhanced regeneration of GDNF-stimulated axons, highlighting the importance of sufficiently elevating intrinsic growth capacity. We also conclude that myelin inhibitors and CSPGs are not the primary mechanism stopping axons at the DREZ.

Myelomeningocele (MMC) is the most common congenital defect of the central nervous system and results in devastating and lifelong disability. In MMC, the initial failure of neural tube closure early in gestation is followed by a progressive prenatal injury to the exposed spinal cord, which contributes to the deterioration of neurological function in fetuses. Prenatal strategies to control the spinal cord injury offer an appealing therapeutic approach to improve neurological function, although the definitive pathophysiological mechanisms of injury remain to be fully elucidated. A better understanding of these mechanisms at the cellular and molecular level is of paramount importance for the development of targeted prenatal MMC therapies to minimize or eliminate the effects of the injury and improve neurological function. In this review article, we discuss the pathological development of MMC with a focus on in utero injury to the exposed spinal cord. We emphasize the need for a better understanding of the causative factors in MMC spinal cord injury, pathophysiological alterations associated with the injury, and cellular and molecular mechanisms by which these alterations are induced.

Primary sensory axon injury is common after spinal cord and root injuries and causes patients to suffer chronic pain and persistent loss of sensation and motor coordination. The devastating consequences of such injuries are due primarily to the failure of severed axons to regenerate within the damaged CNS. Our understanding of the molecular and cellular events that play key roles in preventing or promoting functional regeneration is far from complete, in part because complex and dynamic changes associated with nerve injury have had to be deduced from comparisons of static images obtained from multiple animals after their death. Revolutionary innovations in optics and mouse transgenics now permit real-time monitoring of regenerating dorsal root axons directly in living animals. Here, we describe detailed procedures for repetitive monitoring of identified axons in a lumbar dorsal root over hours to weeks using both widefield and two-photon microscopes. We also discuss the strengths and limitations of in vivo imaging and provide suggestions based on our own experience for troubleshooting issues associated with repeated anesthetization, an extensive laminectomy, and post-op care. These techniques provide the unprecedented opportunity to obtain novel insights into why sensory axons fail to reenter the spinal cord.

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