Nodes of Ranvier are Incompletely Repaired and Continually Disrupted after Traumatic Spinal Cord Injury
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Date
2018-03
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Abstract
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.
Description
Poster Division: Biological Sciences: 1st Place (The Ohio State University Edward F. Hayes Graduate Research Forum)