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Abstract

Damage to the spinal cord can result in life-long deficits including locomotion. Most spinal cord injury cellular therapies focus on the regeneration of long-distance white matter descending motor tracts. However, locomotion is a complex task involving excitatory and inhibitory circuitry also in the spinal gray matter. The aim of this study was to create a discrete, gray matter lesion in the lumbar spinal cord to investigate the role of spinal interneurons in this region, as well as to establish a model that can evaluate proof-of principle targeted cell replacement therapies. A kainic acid lesion was modified to damage intermediate gray matter (laminae V-VII) in the lumbar spinal enlargement (spinal L2-L4) in 10-week-old female Fischer rats. A thorough, tailored behavioral evaluation revealed deficits in gross hindlimb function, skilled walking, coordination, balance and gait two-weeks post-injury. These deficits strongly correlated with structural deficits in the rostro-caudal axis. Machine-learning quantification confirmed interneuronal damage to laminae V-VII in spinal L2-L4 correlates with hindlimb dysfunction. White matter area and lower motoneuron numbers at lesion epicenters did not correlate with behavioral deficits. Animals do not regain lost sensorimotor function three months after injury, indicating that natural recovery mechanisms of the spinal cord cannot compensate for loss of laminae V-VII neurons. Together, this established model is ideal to evaluate cellular transplantation therapies that replace these lost neuronal pools vital to sensorimotor function.

Rat spinal cells taken from embryos age 14 contain a mix of neural stem cells and neural precursor cells and have shown potential to aid functional recovery. Studies have shown that they survive and differentiate into gray matter interneurons both in vitro and in vivo, despite being transplanted into white matter lesions. Using the newly established KA-model, E14 spinal graft survival in a lumbar gray matter lesion could be evaluated for future proof-of-principle, targeted cell replacement experiments. Histological analysis revealed that grafted cells survive well both in the intact and KA-lesioned lumbar spinal cord without additional growth factors. Furthermore, grafts differentiate into neuronal NeuN+ cells both in the white and the gray matter. While future experiments will need to adjust injection parameters as well as evaluate graft differentiation and functional benefits, this work lays the groundwork to assess the potential cellular replacement has to restore lost function after a lumbar spinal cord injury.