Nodes of Ranvier are Incompletely Repaired and Continually Disrupted after Traumatic Spinal Cord Injury

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Approximately 17,500 new cases of spinal cord injuries (SCI) are reported in the US each year, yet less than 1% of patients experience complete neurological recovery. This is due to the limited ability of injured spinal cord axons to regenerate and reform functional circuits. Novel treatments have emerged that increase sprouting of spared axons through the lesion after SCI; however, many of these axons remain unmyelinated or experience suboptimal remyelination. Thus, strategies that enhance the endogenous ability of the injured cord to remyelinate spared axons are critical to achieving clinically relevant functional recovery after SCI. Oligodendrocytes (OLs), the myelinating cells of the central nervous system (CNS), ensheath axons to ensure timely signal conduction. After myelinating OLs die or retract their processes following injury, oligodendrocyte progenitor cells (OPCs) proliferate and migrate to the lesion where they differentiate into mature OLs. Spontaneous regeneration of OLs persists for months after SCI, suggesting that endogenous OLs continuously attempt to remyelinate spared axons. However, remyelination of spared axons is often incomplete or limited in function. Rapid, saltatory conduction of signals along myelinated axons requires the establishment of discreet domains of structural proteins and ion channels within and around the Node of Ranvier. In healthy, myelinated CNS axons, nodes of Ranvier are highly organized and include the paranodal protein, Caspr, flanked by juxtaparanodal K+ channels (Kv1.2). Nav 1.6 voltage-gated Na+ channels are also clustered in the Node of Ranvier to support regeneration of signals. However, OL death disrupts the organization of these proteins, resulting in compromised signal propagation, aberrant excitability patterns, and neuronal dysfunction. Evidence from a mouse model of multiple sclerosis suggests that in demyelinated lesions, there is a Na+ channel subtype switch from the Nav1.6 isoform to Nav1.2, an embryonic isoform that is expressed on pre-myelinated axons. This suggests that Na+ channel subtype switching occurs after demyelination and may underlie impaired remyelination. Currently, structural abnormalities in the node induced by SCI have not been characterized. Understanding nodal pathology after SCI is essential because suboptimal remyelination of spared axons leaves them vulnerable to degeneration. Such degeneration can enhance pathology and impair functional recovery after SCI. The goal of this work was to characterize structural abnormalities in/around the Node or Ranvier as an index of functional or suboptimal remyelination. Collectively, we tested the theory that functional remyelination after SCI requires specific arrangement of proteins within and around the Node of Ranvier. Accordingly, the following hypotheses were tested: SCI induces diffuse Kv1.2 and Caspr spreading outside of nodes on spared axons: Adult C57BL/6 mice received a partial T9 vertebral laminectomy, followed by a moderate spinal cord contusion (75 kDyn force, Infinite Horizons device). Mice were transcardially perfused with 4% paraformaldehyde at 7 dpi, 18 dpi, 28 dpi, 10 weeks post injury (wpi), and 6 month post injury (mpi). Spinal cords were collected and stained using immunofluorescence for the presence and distribution of voltage gated potassium channel (Kv1.2) and Caspr. Labeling in the proper order (i.e. Kv1.2 in the juxtaparanode and Caspr in the paranode) indicated nodes and was used to generate an index of functional re-myelination in the injured tissues. Diffuse labeling along spared axons indicated demyelination or suboptimal remyelination. Intact nodes were counted, and profile lengths of Caspr and Kv1.2 were normalized to naïves to evaluate aberrant spreading. Data shows that relative to 7dpi, there are increasingly fewer intact nodes at chronic time points, particularly 6 mpi. At 6 mpi, there is also extensive Caspr dysregulation as far distal as 2 mm from the lesion epicenter. This dysregulation is absent 7 dpi, suggesting, for the first time in SCI research, that spared tissue becomes increasingly inhibitory to myelin repair. SCI causes sodium channel subtype switching in demyelinated/remyelinated axons: Mice received the same injury (75 kDyn force, Infinite Horizons device) and tissues were collected as stated above. Spinal cords were immunolabeled for Nav1.2+Caspr and Nav1.6+Caspr. Sections were analyzed for expression of each sodium channel subtype and its association with paranodal Caspr. Positive Nav1.6 labeling in the Node of Ranvier flanked by positive paranodal Caspr labeling indicated an intact node. PCR and Western blots were also used to determine if expression of each Na+ channel is transcriptionally or translationally regulated. Data confirm that myelinated axons in naïve animals have high Nav1.6 expression and low embryonic Nav1.2 expression. Data also show, for the first time, that Na+ channel switching occurs after SCI and may underlie suboptimal endogenous remyelination. Implications: Dysregulation of structural proteins in the nodal area can contribute to neural dysfunction after SCI. My data provides novel evidence that spared tissue is inhibitory to remeylination. The data suggests that nodal proteins whose dysregulation persist chronically after injury may potentially serve as novel therapeutic targets that can restore functional remyelination and facilitate meaningful clinical recovery for patients. We expect that findings from these studies can be applied to other pathologies whose sequelae are driven by aberrant nodal structure such as stroke, MS, and epilepsy – all of which affect large patient populations.


Poster Division: Biological Sciences: 1st Place (The Ohio State University Edward F. Hayes Graduate Research Forum)