Epigenetic processes associated with neonatal spinal transection.

In early development, the spinal cord in healthy or disease states displays remarkable activity-dependent changes in plasticity, which may be in part due to the increased activity of brain derived neurotrophic factor (BDNF). Indeed, BDNF delivery has been efficacious in partially ameliorating many of the neurobiological and behavioral consequences of spinal cord injury (SCI), making elucidating the role of BDNF in the normative developing and injured spinal cord a critical research focus. Recent work in our laboratory provided evidence for aberrant global and locus-specific epigenetic changes in methylation of the Bdnf gene as a consequence of SCI. In the present study, animals underwent thoracic lesions on P1, with cervical and lumbar tissue being later collected on P7, P14, and P21. Levels of Bdnf expression and methylation (exon IX and exon IV), in addition to global methylation levels were quantified at each timepoint. Results indicated locus-specific reductions of Bdnf expression that was accompanied by a parallel increase in methylation caudal to the injury site, with animals displaying increased Bdnf expression at the P14 timepoint. Together, these findings suggest that epigenetic activity of the Bdnf gene may act as biomarker in the etiology and intervention effort efficacy following SCI.

Neonatal Spinal Cord Transection Decreases Hindlimb Weight-Bearing and Affects Formation of Achilles and Tail Tendons.

Mechanical loading may be required for proper tendon formation. However, it is not well understood how tendon formation is impacted by the development of weight-bearing locomotor activity in the neonate. This study assessed tendon mechanical properties, and concomitant changes in weight-bearing locomotion, in neonatal rats subjected to a low thoracic spinal cord transection or a sham surgery at postnatal day (P)1. On P10, spontaneous locomotion was evaluated in spinal cord transected and sham controls to determine impacts on weight-bearing hindlimb movement. The mechanical properties of P10 Achilles tendons (ATs), as representative energy-storing, weight-bearing tendons, and tail tendons (TTs), as representative positional, non-weight-bearing tendons were evaluated. Non- and partial weight-bearing hindlimb activity decreased in spinal cord transected rats compared to sham controls. No spinal cord transected rats showed full weight-bearing locomotion. ATs from spinal cord transected rats had increased elastic modulus, while cross-sectional area trended lower compared to sham rats. TTs from spinal cord transected rats had higher stiffness and cross-sectional area. Collagen structure of ATs and TTs did not appear impacted by surgery condition, and no significant differences were detected in the collagen crimp pattern. Our findings suggest that mechanical loading from weight-bearing locomotor activity during development regulates neonatal AT lateral expansion and maintains tendon compliance, and that TTs may be differentially regulated. The onset and gradual increase of weight-bearing movement in the neonate may provide the mechanical loading needed to direct functional postnatal tendon formation.

Visualization of rat tendon in three dimensions using micro-Computed Tomography.

Micro-computed tomography (CT) is an X-ray-based imaging modality that produces three-dimensional (3D), high-resolution images of whole-mount tissues, but is typically limited to dense tissues, such as bone. The X-rays readily pass-through tendons, rendering them transparent. Contrast-enhancing chemical stains have been explored, but their use to improve contrast in different tendon types and across developmental stages for micro-CT imaging has not been systematically evaluated. Therefore, we investigated how phosphotungstic acid (PTA) staining and tissue hydration impacts tendon contrast for micro-CT imaging. We showed that PTA staining increased X-ray absorption of tendon to enhance tissue contrast and obtain 3D micro-CT images of immature (postnatal day 21) and sexually mature (postnatal day 50) rat tendons within the tail and hindlimb. Further, we demonstrated that tissue hydration state following PTA staining significantly impacts soft tissue contrast. Using this method, we also found that tail tendon fascicles appear to cross between fascicle bundles. Ultimately, contrast-enhanced 3D micro-CT imaging will lead to better understanding of tendon structure, and relationships between the bone and soft tissues.•Simple tissue fixation and staining technique enhances soft tissue contrast for tendon visualization using micro-CT.•3D tendon visualization in situ advances understanding of musculoskeletal tissue structure and organization.

DNA methylation and behavioral changes induced by neonatal spinal transection.

Although the importance of epigenetic mechanisms in behavioral development has been gaining attention in recent years, research has largely focused on the brain. To our knowledge, no studies to date have investigated epigenetic changes in the developing spinal cord to determine the dynamic manner in which the spinal epigenome may respond to environmental input during behavioral development. Animal studies demonstrate that spinal cord plasticity is heightened during early development, is somewhat preserved following neonatal transection, and that spinal injured animals are responsive to sensory feedback. Because epigenetic alterations have been implicated in brain plasticity and are highly responsive to experience, these alterations are promising candidates for molecular substrates of spinal plasticity as well. Thus, the current study investigated behavioral changes in the development of weight-bearing locomotion and epigenetic modifications in the spinal cord of infant rats following a neonatal low-thoracic spinal transection or sham surgery on postnatal day (P)1. Specifically, global levels of methylation and methylation status of the brain-derived neurotrophic factor (Bdnf) gene, a neurotrophin heavily involved in both CNS and behavioral plasticity, particularly in development, were examined in lumbar tissue harvested on P10 from sham and spinal-transected subjects. Behavioral results demonstrate that compared to shams, spinal-transected subjects exhibit significantly reduced partial-weight bearing hindlimb activity. Molecular data demonstrate group differences in global lumbar methylation levels as well as exon-specific group differences in Bdnf methylation. This study represents an initial step toward understanding the relationship between epigenetic mechanisms and plasticity associated with spinal cord and locomotor development.

Onset of neonatal locomotor behavior and the mechanical development of Achilles and tail tendons.

Tendon tissue engineering approaches are challenged by a limited understanding of the role mechanical loading plays in normal tendon development. We propose that the increased loading that developing postnatal tendons experience with the onset of locomotor behavior impacts tendon formation. The objective of this study was to assess the onset of spontaneous weight-bearing locomotion in postnatal day (P) 1, 5, and 10 rats, and characterize the relationship between locomotion and the mechanical development of weight-bearing and non-weight-bearing tendons. Movement was video recorded and scored to determine non-weight-bearing, partial weight-bearing, and full weight-bearing locomotor behavior at P1, P5, and P10. Achilles tendons, as weight-bearing tendons, and tail tendons, as non-weight-bearing tendons, were mechanically evaluated. We observed a significant increase in locomotor behavior in P10 rats, compared to P1 and P5. We also found corresponding significant differences in the maximum force, stiffness, displacement at maximum force, and cross-sectional area in Achilles tendons, as a function of postnatal age. However, the maximum stress, strain at maximum stress, and elastic modulus remained constant. Tail tendons of P10 rats had significantly higher maximum force, maximum stress, elastic modulus, and stiffness compared to P5. Our results suggest that the onset of locomotor behavior may be providing the mechanical cues regulating postnatal tendon growth, and their mechanical development may proceed differently in weight-bearing and non-weight-bearing tendons. Further analysis of how this loading affects developing tendons in vivo may inform future engineering approaches aiming to apply such mechanical cues to regulate engineered tendon formation in vitro.

The Spinal Cord, Not to Be Forgotten: the Final Common Path for Development, Training and Recovery of Motor Function.

Research on learning, memory, and neural plasticity has long focused on the brain. However, the spinal cord also exhibits these phenomena to a remarkable degree. Following a spinal cord injury, the isolated spinal cord in vivo can adapt to the environment and benefit from training. The amount of plasticity or recovery of function following a spinal injury often depends on the age at which the injury occurs. In this overview, we discuss learning in the spinal cord, including associative conditioning, neural mechanisms, development, and applications to clinical populations. We take an integrated approach to the spinal cord, one that combines basic and experimental information about experience-dependent learning in animal models to clinical treatment of spinal cord injuries in humans. From such an approach, an important goal is to better inform therapeutic treatments for individuals with spinal cord injuries, as well as develop a more accurate and complete account of spinal cord and behavioral functioning.