Compensatory changes after spinal cord injury in a remyelination deficient mouse model

Abstract

The development of therapeutic strategies to reduce impairments following spinal cord injury (SCI) motivates an active area of research, because there are no effective therapies. One strategy is to address injury‐induced demyelination of spared axons by promoting endogenous or exogenous remyelination. However, previously, we showed that new myelin was not necessary to regain hindlimb stepping following moderate thoracic spinal cord contusion in 3‐month‐old mice. The present analysis investigated two potential mechanisms by which animals can re‐establish locomotion in the absence of remyelination: compensation through intact white matter and conduction through spared axons. We induced a severe contusion injury to reduce the spared white matter rim in the remyelination deficient model, with no differences in recovery between remyelination deficient animals and injured littermate controls. We investigated the nodal properties of the axons at the lesion and found that in the remyelination deficient model, axons express the Nav1.2 voltage‐gated sodium channel, a sub‐type not typically expressed at mature nodes of Ranvier. In a moderate contusion injury, conduction velocities through the lesions of remyelination deficient animals were similar to those in animals with the capacity to remyelinate after injury. Detailed gait analysis and kinematics reveal subtle differences between remyelination deficient animals and remyelination competent controls, but no worse deficits. It is possible that upregulation of Nav1.2 channels may contribute to establishing conduction through the lesion. This conduction could contribute to compensation and regained motor function in mouse models of SCI. Such compensatory mechanism may have implications for interpreting efficacy results for remyelinating interventions in mice and the development of therapies for improving recovery following SCI.

Previous work showed that 3‐month‐old remyelination deficient mice (Myrf ICKOs) regain locomotor function comparably to remyelination competent (Myrf‐Intact) controls after spinal cord injury. This study investigated the mechanisms by which mice compensate for remyelination failure after injury. Spared axons through the lesions of Myrf ICKOs conduct action potentials at comparable velocities to controls. The Myrf ICKOs exhibit subtle differences in gait cycle, without deficits, implying compensatory mechanisms cause subtle locomotor effects. A novel ion channel expression pattern of long sections of Nav1.2 colocalized with Kv1.2 channels was only observed in the Myrf ICKO. These data demonstrate potential compensatory mechanisms for demyelination and may have implications for future therapeutic interventions for spinal cord injury.

Abbreviations

ANOVA

analysis of variance

BMS

Basso Mouse Scale

CAP

compound action potentials

CNS

central nervous system

GFP

green fluorescent protein

ICKO

inducible conditional knockout

LF

lateral funiculus

Myrf

myelin regulatory factor

OPC

oligodendrocyte precursor cell

PBS

phosphate‐buffered saline

PDGFRa

platelet‐derived growth factor receptor alpha

PFA

paraformaldehyde

RRIDs

Research Resource Identifiers (scicrunch.org)

SCI

spinal cord injury

1. INTRODUCTION

Spinal cord injury (SCI) produces deficits in sensory, autonomic, and motor functions. Most injuries are anatomically incomplete, meaning some tissue is preserved (NSCISC, ²⁰¹⁹). This spared tissue represents an opportunity for compensatory and therapeutic strategies for recovery. Myelin, a lamellar membrane structure produced by oligodendrocytes in the central nervous system (CNS), is lost at the lesion site following injury (Powers et al., 2012, 2013). Demyelination leaves axons vulnerable, as myelin provides trophic and metabolic support in addition to increasing action potential conduction speed (Nave, 2010; Waxman, ¹⁹⁸⁰). The oligodendrocyte precursor (OPC) population is in a constant state of proliferation and thus serves as a supply for oligodendrocyte and myelin replacement after injury (Assinck et al., 2017; Blakemore & Keirstead, ¹⁹⁹⁹).

Previously, we disrupted myelin regulatory factor (Myrf), a transcription factor necessary for OPC differentiation and new myelin formation, to explore the role of newly generated myelin after SCI. Animals with lox‐P sites flanking exon 8 of the Myrf gene (Emery et al., 2009) were bred to animals with Cre‐ER^T2 expression under the platelet‐derived growth factor receptor alpha (PDGFRa) promoter (Kang et al., 2010). Extant myelin is intact in this inducible conditional knockout (ICKO), but myelin lost at the injury site is not replaced, and there is no compensatory increase in Schwann cell myelination (Duncan et al., 2018). Animals were injured at 3 months of age and were kept for 6 weeks after injury. Animals kept in a persistent demyelinated state did not differ from injured littermate controls (lacking the PDGFRa Cre driver but homozygous for the floxed Myrf transgene) on any locomotor behaviors assessed in that study (Duncan et al., 2018).

We hypothesized that animals with impaired remyelination relied on compensatory mechanisms to regain motor function after SCI. Knowledge of naturally occurring compensatory mechanisms could reveal their potential as therapeutic targets to improve recovery. Thus, we undertook the current study to explore two potential compensatory mechanisms by which remyelination deficient animals might recover motor function after SCI. First, we investigated the role of the remaining axons in the intact white matter rim that did not undergo demyelination after injury. For this aim, we induced a more severe injury to reduce the amount of spared tissue and consequently the number of myelinated axons. Second, to address strategies by which spared axons in the spared white matter rim re‐establish conduction, we investigated the ability of axons to conduct action potentials by interrogating conduction velocities and ion channel expression in the ICKO model. These data suggest that recovery in remyelination deficient mice may be mediated by adaptations that help maintain conduction through the lesion.

2. METHODS

2.1. Animals

All breeding and experimental procedures were conducted on protocols approved by the University of British Columbia's Animal Care and Use Committee (approval number A21‐0304). As in previous studies (Duncan et al., 2018), inducible conditional knockout (ICKO) of the Myrf gene was accomplished via in‐house breeding of Myrf ^fl/fl mice (Emery et al., 2009) (originally purchased from Jackson Laboratories, Jax strain 010607; RRID:IMSR_JAX:010607) to PDGFRalpha Cre‐ER^T2 (Kang et al., 2010) (originally purchased from Jackson Laboratories, Jax strain 018280; RRID:IMSR_JAX:018280) to produce PDGFRaCreER^T2:Myrf ^fl/fl offspring. Experiments were conducted in ICKOs and littermate controls which were homozygous for the floxed Myrf allele but lacking Cre recombinase (referred to as remyelination competent or Myrf‐Intact). Animals were group housed in open‐top wire lid cages with up to five animals per cage, and maintained on a reverse light–dark cycle with ad libitum access to food and water. 16 Myrf ICKO animals and 16 Myrf‐Intact littermate controls (with approximately equal numbers of males and females in each group) weighing 18–35 g were injured at 3 months of age for severe SCI experiments, and 15 Myrf ICKO animals and 14 Myrf‐Intact controls (with approximately equal numbers of males and females) at a similar age and weight as the severely injured animals were used in the moderate SCI experiments.

2.2. Genotyping

Experimental groups were determined based on animal genotype; homozygous Myrf ^fl/fl animals with one allele of the transgene for the PDGFRa promotor driven Cre‐driver (Myrf ICKOs) were compared against litter‐mate Myrf‐Intact controls (Myrf ^fl/fl and lacking the PDGFRa transgene). To determine genotype, DNA was extracted from ear clippings using the REDExtract‐N‐AMP Tissue Kit (Sigma, St. Louis, MO, cat. no. R4775). Primers for amplification were specific to the transgenes: PDGFRa Forward TCA GCC TTA AGC TGG GAC AT and Reverse ATG TTT AGC TGG CCC AAA TG; Myrf ^fl/fl Forward GGG TAC TAA AGA ATG GCG AAG G and Reverse GCC AGG GGG ATC TTG AAG T.

2.3. Behavior

Animals were handled and acclimatized to personnel and behavior apparatuses. Animals were trained on behavioral tasks and assessed prior to tamoxifen administration and prior to injury to establish baseline scores. All assessments were conducted by personnel blinded to genotype.

The Basso Mouse Scale (BMS) and its subscore are measures of SCI‐induced hindlimb deficits in overground locomotion (Basso et al., 2006). Animals were assessed as they explored in an open plexiglass arena for a maximum of 5 min while being scored by two observers blinded to genotype.

The inclined plane task was used to assess locomotor function in the absence of stepping (Rivlin & Tator, 1977). Three textures were assembled in house: vertical grooves, horizontal grooves, and a homogenized texture.

For kinematics, it was necessary to film animals as they locomoted while wearing reflective dots. To apply these dots, animals were anesthetized initially at 3% isoflurane and then at ~1.2% while maintained on heat support. Under anesthesia, animals were shaved and reflective dots (4 mm diameter) were placed on each hind foot, ankle, knee, hip, and along the spine. Once awake, mice were moved to a variable speed rodent treadmill (Maze Engineers) surrounded by six evenly spaced infrared Vero cameras (Vicon Motion Systems, Oxford, UK) recording at 320 frames/second. Animals completed 10–15 runs each lasting 15 s. Data were recorded using Nexus 2 (Nexus (RRID:SCR_015001)) and ProCalc software (Vicon Motion Systems, Oxford, UK).

2.4. Tamoxifen administration

All animals (Myrf ICKO and Myrf‐Intact) received tamoxifen (Sigma, St. Louis, MO cat. no. C8267) dissolved in corn oil and injected intraperitoneally at 100 mg/kg/day for five consecutive days.

2.5. Injuries

All injuries occurred when the animals were 3 months of age. Mice were initially anesthetized with a mixture of 3% isoflurane in oxygen. After reaching surgical plane, animals were maintained on ~1.2% isoflurane on heat support for the remainder of the procedure. Animals received subcutaneous injections of lactated Ringer's (20 mL/kg), buprenorphine (0.05 mg/kg), and bupivacaine (locally at the incision site, 8 mg/kg). The back was shaved and disinfected with successive chlorhexidine and 70% ethanol washes. An incision was made to reveal the lower thoracic spinal column, and a full laminectomy at thoracic level 9 was performed. All injuries were given using the Infinite Horizon Impactor (Precision Systems). For moderate injuries, the impactor was set to deliver a 70 kDyne injury with no dwell. For severe injuries, the impactor delivered a 70 kDyne impact and was set to dwell for 1 s following displacement. There was no difference between the injuries sustained by Myrf‐Intact controls vs. Myrf ICKOs (Figure S4). Incisions were closed with resorbable suture (6‐0 DemeCAPRONE, DemeTECH, cat. MO176011F13M) and animals awoke in a humidified recovery cage kept at 32°. Animals were returned to their home cage with moistened food and hydration support on the floor of the cage. Supportive fluids and buprenorphine were administered every 8 h for 3 days. Bladders were expressed manually two to three times per day until voluntary micturition returned.

2.6. Inclusion and exclusion

Inclusion criteria included correct genotype (Myrf ICKO or Cre⁻ littermate control), no neurological or physiological abnormalities, and an injury consistent with the rest of the animals in the study based on force and displacement values (Figure S4). An inconsistent injury was defined as a force or displacement values outside of two standard deviations of the mean. No animals were excluded based on these criteria. One animal in each experiment (moderate or severe) required humane euthanasia over the course of the experiment because of complications from bladder expression; these animals were not replaced, and their data were not included in the analysis.

2.7. Immunohistochemistry

Mice were given a lethal dose of 10% chloral hydrate (1 g/kg) via intraperitoneal injection. Once animals lost toe‐pinch and ocular reflexes and went into respiratory arrest, they were transcardially perfused with 20 mL of phosphate‐buffered saline (PBS) followed by 40 mL of freshly hydrolyzed 4% paraformaldehyde (PFA) (Fisher Scientific, Ward Hill, MA cat. A11313) at 6 WPI and 36 WPI. The injury site was identified, then 1 cm of the spinal cord flanking the injury was dissected. Spinal cords were fixed in PFA for 8 h, then incubated in ascending sucrose solutions (12%, 18% and 24% in PBS) for cryoprotection. Tissue was submerged in OCT compound, (Tissue‐Tek, Torrance, CA cat. 4583) frozen on dry ice, and stored at −80°C. Tissue was sectioned using a cryostat (Thermo Scientific, Walldorf, Germany, cat. HM‐525) into 1 cm long and 20 μm thick longitudinal sections, which were mounted in series on 10 slides making each individual section on a slide 200 μm apart. Slides were stored at −80°C.

Slides were thawed then rehydrated in PBS. To effectively stain myelin proteins, the myelin lipids were removed by dipping the slides through ascending, then descending ethanol dilutions (50%, 70%, 90%, 95%, 100%, 95%, 90%, 70%, 50%), followed by three washes of PBS. Tissue was then blocked with 10% normal donkey serum dissolved in PBS with 0.1% Triton X‐100 for 30 min. Primary antibodies were diluted in PBS with 0.1% Triton X‐100 and applied to the slides overnight at room temperature in a humid chamber. The following morning, slides were washed and incubated with donkey Dylight or Alexa Fluor secondary antibodies (Jackson ImmunoResearch Laboratories, Inc.) for 2 h, then washed again before being cover slipped using Fluoromount‐G (Southern Biotech, 0100‐01). Antibodies used were raised against the following antigens: GFP (1:4000, Abcam, ab13970, RRID:AB_300798), PDGFRa (1:200, R and D Systems, AF‐307‐NA, RRID:AB_354459), NF200 (1:1000, Sigma, N0142, RRID:AB_477257), Nav1.2 (1:200, Alamone, ASC‐002, RRID:AB_2040005), and Kv1.2 (1:200, Alamone, APC‐009, RRID:AB_2040144).

2.8. Confocal imaging and ion channel stereology

All analyses were performed blinded to animal genotypes. A Zeiss Axio‐Observer M1 inverted confocal microscope with a Yokogawa spinning disk and Zen 2 software (Zeiss) was used for imaging. Tiled images were taken with a Plan‐Apochromat 63x/1.40 oil objective spanning 1 mm rostro‐caudal, centered around the lesion. Z‐stacks were imaged through the entire depth of the 20 μm thick section with 1 μm spacing between optical sections. Images were taken from the left and right side of the spinal cord starting from the most ventral section and moving up until no spared white matter was observed. The number of axons within the specified volume of the sampling box were counted throughout the Z‐stacks and averaged per section, giving the density of axons per mm³. 300–500 axons were counted per animal.

2.9. Myelin sheath quantification

Spinal cords embedded in resin were sectioned to 1 μm thickness on an ultramicrotome (Ultracut E, Reichert‐Jung). Ultra and semi‐thin sections were collected every 20 μm and semithin sections were viewed under a light microscope to find the injury epicenter, defined by the lowest number of myelinated axons by a rater blinded to genotype. Myelin was visualized in semi‐thin sections by brief staining with 1% toluidine blue and 2% borax solution, then cover slipping with Permount (Fisher Scientific, Fair Lawn, NJ, cat. no. SP15). The imaging of toluidine blue semi‐thin sections was performed on a Zeiss, Axio Imager M2 microscope at 630× magnification (63× objective, NA 1.4). The entire cross section at the lesion epicenter was imaged. A grid with box dimensions of 50 μm × 50 μm was overlaid on top of the merged image. We used systematic arbitrary sampling, counting one in every seven grids. This was done over the extent of the spared ventro‐lateral white matter at the injury epicenter. Spared tissue was indicated by intact cytoarchitecture and the presence of myelin sheaths. Between 1500 and 2500 myelin sheaths were counted per animal.

2.10. Electrophysiology

All electrophysiology experiments were terminal. Mice were initially anesthetized for 3 min with a 3% isofluorane in oxygen. Following induction, anesthesia was maintained at 1.5%–2% isofluorane for the remainder of the procedure. Animals were intubated by inserting a tracheal catheter through an incision between tracheal rings. The catheter was tightly secured with suture. Isoflurane and oxygen were supplied by a ventilator (Inspira Advanced Safety Ventilator by Harvard Apparatus) for the remainder of the experiment. Once ventilation was established, the animal was given a dose of the muscle relaxant gallamine triethiodide (60 mg/kg). The C4‐C6 and the T13‐L2 vertebrae were exposed, and the cord was covered in mineral oil. The animal was then secured with ear bars and a spine clamp and was suspended to reduce ventilation movement. Single tungsten wires were used to stimulate and to record. Compound action potentials (CAPs) were elicited by delivering 1 mAmp biphasic 100 μs pulses at 60 Hz. Averages reflect approximately 180 recordings. Pulses were controlled using ISO‐Flex stimulus isolator (A.M.P.I) and MASTER‐9 stimulator (A.M.P.I). Recordings were amplified and digitized using a Neuro Amp EX front end and Powerlab 16/30 (ADInstruments). LabChart software (ADInstruments, RRID:SCR_023643) was used to monitor heart rate and CAPs. Signals were filtered with a low‐pass filter with a 50 Hz cut‐off. After a high thoracic transection, all signals reported here were lost. Animals were euthanized by a lethal intraperitoneal injection of 10% chloral hydrate (1 g/kg); once the heart rate monitor registered a lack of heart beat, animals were cervically dislocated.

2.11. Kinematics analysis

Kinematics data were analyzed in house using custom Matlab code (The Mathwork Inc., Natick, MA, RRID:SCR_001622). All representations of the “% of step cycles” were calculated by applying cubic spline interpolation to the original data. This obtained exactly 100 data points per step, regardless of small variations between step lengths. Mathematical normalization was done using custom Matlab code. Step cycles were divided into swing phase and plantar phase. The direction of the y‐velocity vector for the animal's toes determined the boundaries between these two phases, as indicated by the transition from negative to positive velocities. Joint angles were calculated in 3D by creating vectors between markers and calculating angles between those vectors.

2.12. Statistics

The number of animals per group was determined by previous studies (Duncan et al., 2018) and a power analysis to detect a meaningful difference in the BMS based on an alpha of 0.05 and a beta of 80%. Statistical analyses were conducted using the Statistical Package for the Social Sciences (SPSS, RRID:SCR_002865) or Graphpad 10.0 (Prism). No outlier tests were conducted on any of the experimental data, and no animals were excluded. Individual data points were displayed when possible and represent a single mouse. All data in graphs portray the mean and the standard error of the mean (SEM). If data met assumptions for normality, tested with the Shapiro–Wilk test, t‐tests were run with or without Welch's correction depending on the homogeneity of variance (tested with Levene's test). Comparisons of the density of recombined oligodendrocytes or lesion area were compared using a two‐way ANOVA with Tukey's post hoc test to detect individual differences. For behavioral analyses, a two‐way repeated measures ANOVA was conducted with comparisons using Tukey's or Šidak post hoc to compare individual groups. Comparisons were two‐tailed and considered statistically significant if p < 0.05.

3. RESULTS

3.1. Remyelination is not necessary for motor recovery in severe injuries

Previously, we showed that 6 weeks after a moderate thoracic contusion injury, regardless of the degree of remyelination, animals regained comparable motor function (Duncan et al., 2018). The moderate injury paradigm entailed a midline 70 kDyne impact with no dwell time. To test whether remyelination accounts for spontaneous recovery of locomotor function, we utilized a more severe injury paradigm to decrease the amount of spared white matter. In this paradigm, the force was maintained at 70 kDyne while the dwell time was increased to 1 s. We compared the spared white matter in remyelination deficient (Myrf ICKOs) and remyelination intact (Myrf‐Intact) animals that sustained these more severe injuries. Comparing the spared white matter in these more severe injuries, we found significantly more myelin sheaths in injured littermate Myrf‐Intact control animals (34 497 sheaths/mm² Figure 1a,c) vs. 25 904 sheaths/mm² in the ICKO (Figure 1b,c; Student's t‐test df = 8, t = 2.475, p = 0.0438). As in our previous studies, tamoxifen administration induced high recombination efficiency (~90%) in the Myrf ICKO mice, disrupting the formation of new myelin (Figure S1).

FIGURE 1.

Spared rim of white matter after severe thoracic spinal cord injury in remyelination incompetent and remyelination competent mice. Examples of toludine blue stained thin sections of injured Myrf‐Intact control (a) and Myrf ICKO (b); scale bar = 24 μm. Quantification of myelinated axons (c) reveals significantly fewer sheaths per mm² in the Myrf ICKO compared to the injured remyelination competent controls. Data are represented as means with standard error of the mean. p = 0.0438. n, number animals.

Having established a more severe injury with less spared white matter, we sought to ascertain if motor recovery would worsen in the absence of remyelination. Myrf ICKO animals and injured Myrf‐Intact littermate controls were pre‐trained on behavioral tasks (Figure 2a). The Basso Mouse Scale (BMS) is a test of hindlimb locomotion sensitive to SCI deficits (Basso et al., 2006). In the 6 weeks after injury, both Myrf ICKO and littermate controls failed to regain stepping (Figure 2b), and thus also could not be assessed on the BMS subscore. Following severe SCI, regular assessments showed both Myrf ICKO and control animals showed limited recovery on the BMS (Figure 2b), with no statistical difference between the two groups (two‐way repeated measures ANOVA (F(1, 29) = 0.9566, p = 0.3361) with Sidak's multiple comparisons test (minimum p > 0.4152)). As the severe injuries limited recovery and precluded weight‐supported plantar stepping, the sensitivity of the BMS in this range is low.

FIGURE 2.

Myrf ICKO and Myrf‐Intact controls demonstrate comparable locomotor recovery from severe thoracic contusion spinal cord injury. Timeline (a) detailing animals' pretraining, tamoxifen administration, a T9/10 contusion injury at 70 kDyne with 1 s of dwell time, and subsequent behavioral testing. 16 animals per experimental condition were injured. One control animal was euthanized at 2 WPI for bladder issues. 12 Myrf ICKO animals and 11 Myrf‐Intact controls were euthanized at experimental endpoint #1, and 4 animals per group were euthanized at endpoint #2. Animals were regularly assessed on the BMS (b) and inclined plane test (c–e). At 6 weeks after injury there are no differences between the minimal recovery observed in both Myrf‐Intact control and Myrf ICKO on the BMS (b). On the inclined plane, both Myrf ICKO and Myrf‐Intact control animals showed a deficit following injury on the vertical grooved surface (c), the homogenous textured surface (d), and the horizontal grooved surface (e). At 4 weeks post injury the Myrf ICKO was able to stand on a more steeply angled platform than the control animals on the vertical grooves (c); all other timepoints were not statistically different between control and Myrf ICKO. Data are represented as means with standard error of the mean. *p = 0.0185. dpi, days post injury; WPI, weeks post injury; n, number animals.

Therefore, animals were also assessed on the inclined plane test as it can detect hindlimb function deficits but does not require stepping (Rivlin & Tator, 1977). In this task, animals rely on hindlimb strength and range of motion to maintain a stance on steadily steeper inclines. The inclined plane allows testing of animals' ability to maintain a grip on a ramp as the angle is increased under three different friction conditions (homogenous texture, horizontal furrows, or vertical furrows). Myrf ICKO mice did not have worse deficits in maintaining grip than injured Myrf‐Intact controls on all three surfaces and at any time point (Figure 2c–e; two‐way repeated measures ANOVA (homogenous F(1, 28) = 0.8512, p = 0.3641; horizontal F(1, 28) = 0.5014, p = 0.4848); Sidak's multiple comparisons test minimum p = 0.7170). When comparing Myrf ICKO mice to controls on the vertical surface, overall there was no difference (Figure 2c; two‐way repeated measures ANOVA F(1, 29) = 3.214, p = 0.0834), but Myrf ICKOs could maintain grip on a greater incline than Myrf‐Intact controls at 4 weeks post‐injury (Sidak's multiple comparisons test p = 0.0185). No other timepoints were statistically significantly different (minimum p = 0.0847). The lack of significant differences in locomotor assessments between Myrf ICKO and Myrf‐Intact control animals persisted out to 36 weeks post‐injury in a small group of animals maintained longitudinally (Figure S2). These data suggest that even when the amount of spared white matter is decreased, animals are still able to partially compensate for persistent demyelination. Thus, we conclude that remyelination of the spared white matter rim is insufficient to account for the spontaneous recovery after SCI.

3.2. Preventing remyelination after SCI yields an altered ion channel expression

To determine whether excitatory domains may be reorganizing to facilitate conduction after remyelination failure following SCI, we examined ion channels typically confined to unmyelinated or demyelinated axons. Mature nodes of Ranvier (referred to hereafter as nodes) express the voltage‐gated sodium channel Nav1.6, while the axon initial segment (AIS), unmyelinated axons, demyelinated axons, and immature nodes express the Nav1.2 variant (Boiko et al., 2003; Craner et al., ²⁰⁰³; Kaplan et al., ¹⁹⁹⁷; Rasband et al., ²⁰⁰³). The potassium channel Kv1.2 is restricted to the juxtaparanode (Rasband & Peles, 2016; Rasband & Trimmer, ²⁰⁰¹). We stained longitudinal sections through the lesion for axons (NF200) and the ion channels Kv1.2 and Nav1.2 (Figure 3). To quantify abnormal nodes and Nav1.2 expression, we counted the number of NF200+ axons with Nav1.2/Kv1.2 expression extending for more than 50 μm from the nodes. A large population of NF200+ axons positive for Kv1.2, but negative for Nav1.2 was found in both Myrf ICKO (1511 axons/mm³) and Myrf‐Intact control mice (1628 axons/mm³; Figure 3a,b, quantified in 3e). This population was comparably sized in both groups (Student's t‐test; df = 6, t = 0.258, p = 0.805), and likely represents the upregulation of potassium channels seen in injured and demyelinated or dysmyelinated axons after SCI (Karimi‐Abdolrezaee et al., 2004). In both Myrf ICKO and Myrf‐Intact control animals we identified “standard” nodes of Ranvier, presumably found on myelinated axons, as determined by distance from one juxtaparanode to the next (Karimi‐Abdolrezaee et al., 2004). The average distance was 31.14 μm in controls and 31.19 μm in Myrf ICKO mice (Figure 3c,d; n.s. Student's t‐test df = 6, t = 0.432, p = 0.6811). A disrupted expression profile, wherein Kv1.2 and Nav1.2 were co‐expressed on NF200+ axons, was observed only in Myrf ICKO (Figure 3a,f). Colocalization of Kv1.2 and Nav1.2 extending over more than 50 μm was frequently observed in Myrf ICKO (2701 axons/mm³) and rarely seen in Myrf‐Intact control mice (111 axons/mm³; Student's t‐test; df = 6, t = 9.72, p < 0.0001; Figure 3f). We concluded that disrupting remyelination after SCI disrupted spatial regulation of channel expression and upregulated the expression of Nav1.2 outside of the node, a phenotype that (to our knowledge) has not been described previously.

FIGURE 3.

Myrf ICKO express Nav1.2 and altered nodal structure after thoracic contusion spinal cord injury. Longitudinal spinal cord sections through the lesion were stained for ion channels. Examples of subsets through z‐stacked images of Myrf ICKO cords (a) co‐stained for Kv1.2, Nav1.2, and NF200 (scale bar = 100 μm; blow out of highlighted regions scale bar = 50 μm) revealing co‐localized sodium and potassium channels on axons at the lesion. Examples from Myrf‐Intact control cords (b) displays NF200+ Kv1.2+ axons lacking signal for Nav1.2. Example of typical nodal structure described by ~30 μm of Kv1.2 signal (c) which was observed in both Myrf ICKO and Myrf‐Intact controls (d). Long sections Kv1.2 was observed in both (e) but colocalized Nav1.2 and Kv1.2 was only observed in Myrf ICKO (f) suggesting chronic demyelination upregulates Nav1.2 channel expression and alters nodal structure. In (d)–(f) bar charts represent mean values from 3 Myrf ICKO and 5 controls and error bars represent the standard error of the mean. ****p < 0.0001.

3.3. Preventing remyelination after moderate SCI does not delay action potential conduction

Thus far, our data have shown that impairing remyelination after SCI alters the ion channel profiles on axons at the lesion. However, the functional, physiological, and behavioral utility of the severe model is limited. Thus, we returned to the moderate injury used in our previous studies (Duncan et al., 2018) to ascertain if we could determine physiological effects in remyelination deficient animals. Myrf ICKO mice and littermate Myrf‐Intact controls were pretrained, treated with tamoxifen, and sustained a moderate 70 kDyne T9/10 contusion injury with no dwell time (Figure 4a). Corroborating our earlier findings, both ICKO mice and controls regained hindlimb stepping (Figure S3).

FIGURE 4.

Conduction velocities through the lesion are comparable between Myrf ICKO and Myrf‐Intact controls in a moderate thoracic contusion. Timeline (a) detailing animals' pretraining, tamoxifen administration, a T9/10 contusion injury at 70 kDyne, and subsequent behavioral testing and electrophysiology. 15 Myrf ICKO animals and 14 littermate Myrf‐Intact controls were injured. One animal was euthanized at 3 wpi for bladder issues. Terminal electrophysiology was performed to measure compound action potentials (CAPs) through the lesion by inserting a stimulating electrode rostral to the injury and a recording electrode caudal to the injury (b). Both Myrf ICKO and Myrf‐Intact demonstrated conduction through the lesion (individual animals in (c); aggregate in (d) representing 5 animals per group). Conduction velocities to the peak did not differ significantly between Myrf‐Intact controls and Myrf ICKOs; n = 4 for uninjured condition (e). Example of an uninjured animal and cessation of signal following transection (f). Bar charts represent means and standard error of the mean. CAP, compound action potential; LF, lateral funiculus; n, number animals.

Upregulated and extended Nav1.2 channels could permit effective conduction in mice unable to remyelinate. We performed terminal electrophysiology in Myrf ICKO and Myrf‐Intact control animals 6 weeks after injury (Figure 4a). While the animals were deeply anesthetized, a stimulating electrode was inserted in the cervical lateral funiculus rostral to the lesion and a recording electrode was inserted into the lateral funiculus caudal to the lesion (Figure 4b). Compound action potentials (CAPs) measured at the recording electrode demonstrate signal transmission through the lesion (individual CAP in Figure 4c; stimulus triggered averages for all animals in Figure 4d). Computed conduction velocities of 28.2 m/s in Myrf ICKO mice were not statistically significantly different than the conduction velocity of 27.2 m/s in Myrf‐Intact controls (one‐way ANOVA with Tukey's multiple comparisons test p = 0.9247; Figure 4e). In all experiments transection at the thoracic level abolished the signal (example of uninjured control and transection in Figure 4f). Based on these conduction velocities, we conclude that Myrf ICKO mice can effectively conduct signal through the lesion.

3.4. Detailed gait analysis reveals only subtle differences in recovery between Myrf ICKO and remyelination competent controls after moderate SCI

Previously we established that remyelination does not contribute to regaining hindlimb stepping and locomotion in animals injured at 3 months of age (Duncan et al., 2018). Having established that persistent demyelination results in altered ion channel expression, but does not disrupt conduction, we wanted to determine if more subtle assessments of gait could detect a difference between Myrf ICKO mice and Myrf‐Intact controls after moderate SCI. To accomplish this, we conducted a detailed analysis of hindlimb movement in injured animals using 3D kinematics. Six weeks after injury reflective markers were placed on the animals' hindlimbs (Figure 5a). Animals were recorded as they walked on a treadmill, permitting detailed analysis of gait parameters, such as joint angles and the durations of step phases, between Myrf ICKO and controls.

FIGURE 5.

Joint angles are comparable between Myrf ICKO and Myrf‐Intact control animals 6 weeks after a moderate thoracic contusion. Reflective markers were placed on the animals' joints (a) to capture movement as they walked on a treadmill. Post‐hoc analysis of the movement of the markers was used to quantify the angles throughout the step cycle made by the hip (b), knee (c), and ankle (d). There are no differences between Myrf ICKO and remyelination competent Myrf‐Intact control animals either before or 6 weeks after injury. The transition from the plantar phase to swing phase is earlier prior to injury (blue dashed lines) reflecting the shorter swing phases after injury (green dashed lines). Traces represent means for 5 Myrf‐Intact and 6 Myrf ICKO animals. WPI, weeks post‐injury.

One possibility is that Myrf ICKOs exhibit a differential strategy to regain locomotor recovery in the absence of remyelination and may subsequently have altered movement in their individual joints. To assess limb and joint movement, the angles of each joint were computed throughout the entirety of the step cycle (Figure 5a). For the hip (Figure 5b) and knee (Figure 5c), there were no statistically significant differences between Myrf‐Intact control animals and Myrf ICKO at any point throughout the cycle (two‐way repeated measures ANOVA; Sidak's multiple comparisons test; p > 0.999). Notably, while the ankle angle also did not differ significantly, the peak angle in the swing phase appeared higher in Myrf‐Intact control animals than Myrf ICKO mice (55° in control vs. 40° in Myrf ICKO), though there was no statistical difference by two‐way repeated measures ANOVA and Sidak's multiple comparisons test; p = 0.583; Figure 5d.

To further characterize gait, we assessed the duration of the animals' step cycles. The step duration is defined by the time elapsed between a foot's successive strikes and consists of the swing phase plus the plantar phase (Figure 6a). There were no differences in the duration of the full step before injury between Myrf ICKO or Myrf‐Intact controls (two‐way repeated measures ANOVA F(3, 18) = 2.068, p = 0.1403; Sidak's multiple comparisons test minimum p > 0.999; Figure 6b). However, 6 weeks after injury both ICKOs and controls exhibited longer step durations as compared to pre‐injury (F(3, 18) = 86.85, p < 0.0001; Figure 6b). Multiple comparisons across genotype after injury showed that the duration of steps for the right and left foot in control animals were significantly larger than the left foot in Myrf ICKO (p = 0.0292 for left ICKO vs. left Myrf‐Intact and p = 0.0192 for left ICKO vs right Myrf‐Intact); no other comparisons between genotypes after injury were significant. We examined the swing phase (the amount of time between when the paw was lifted till it made contact, schematic in Figure 6a, quantification in Figure 6c) and the plantar phase (the amount of time the paw was in contact with the ground, schematic in Figure 6a, quantification in Figure 6d). Six weeks after injury both ICKOs and Myrf‐Intact controls exhibited shorter swing duration (0.08 s at 6 WPI vs 0.15 s in the pre‐injury; Figure 6c) and longer plantar placement (0.36 s at 6 WPI vs 0.18 s in pre‐injury; Figure 6d). The differences before and after injury were statistically significant (two‐way repeated measures ANOVA; Sidak's multiple comparisons test; for swing phase F(3, 18) = 63.93, p < 0.0001; for plantar phase F(3, 18) = 255.2, p < 0.0001) while there were no statistical differences between Myrf ICKO and control animals at either time point for the swing phase or the plantar phase of the step cycle (two‐way repeated measures ANOVA; Sidak's multiple comparisons test p > 0.999). Overall, the kinematics data suggest that inhibition of remyelination after a moderate thoracic contusion injury does not prevent locomotor recovery but may cause small, presumably adaptive gait changes as reflected by ankle angle and step duration.

FIGURE 6.

Myrf ICKO and Myrf‐Intact controls demonstrate subtle differences on step durations after moderate thoracic contusion spinal cord injury. Post‐hoc analysis of the movement of reflective markers was used to quantify the step cycles of each animal, including full step duration, plantar phase, and swing phase (a). There was no difference in the duration of full step before injury in Myrf‐Intact controls and Myrf ICKO, while the left foot of Myrf ICKO exhibited shorter duration than controls after injury (b). After injury both groups had significantly shorter swing phases (c) and significantly longer plantar phases (d), with no differences between groups at any time point on the swing or plantar assessments. Bar charts represent means for 5 Myrf‐Intact animals and 6 Myrf ICKOs, and error bars represent standard error of the mean. *p < 0.05; ***p < 0.0001. WPI, weeks post injury.

4. DISCUSSION

Strategies to promote remyelination after SCI as a means to improve recovery have been extensively studied in rodent models (Papastefanaki & Matsas, 2015; Plemel et al., ²⁰¹⁴). Surprisingly, we demonstrated previously that in a 3‐month‐old mouse model of moderate thoracic contusion injury, remyelination did not contribute to recovery of stepping and hindlimb motor function (Duncan et al., 2018). The current study sought to elucidate how motor recovery might be achieved in the absence of remyelination, utilizing the same Myrf ICKO model to prevent remyelination after SCI.

Reorganization of spared and regenerating tissue is a potential driver of recovery (Fink & Cafferty, 2016; Freund et al., ²⁰¹¹; Siegel et al., ²⁰¹⁵). In rats, sparing of 20% of white matter is sufficient to drive locomotor recovery (Schucht et al., 2002). The injury produced in mice by a thoracic impact of 70 kDyne of force with no dwell is anatomically incomplete, leaving a rim of spared myelinated axons, which might be the source of recovery in the absence of remyelination. We hypothesized that reducing the amount of spared tissue with a more severe injury in the Myrf ICKO would allow us to detect any differences in recovery driven by remyelination. We established a more severe paradigm, wherein the impactor was allowed to dwell for 1 s during a 70 kDyne impact. Analysis of demyelinated axon numbers in the spared rim revealed a 25% decrease in myelinated axons between Myrf ICKO and injured Myrf‐Intact controls. This severe injury produced a more severe behavioral deficit; animals failed to regain stepping even after 36 weeks. Importantly, these sustained deficits in both BMS and the inclined plane task were observed in both the Myrf ICKO and Myrf‐Intact control animals. If the remaining myelinated axons in the spared tissue were able to compensate for any deficits caused by increased numbers of demyelinated axons, reducing their number in the spared rim would permit us to discern these deficits, in particular, when the number of myelinated axons is further reduced by Myrf ICKO. A lack of difference between Myrf ICKO and control animals led us to conclude that the residual myelinated axons in the spared rim are not the only source of compensation after injury in the Myrf ICKO model.

In the intact CNS, regulation of ion channels at nodes of Ranvier by oligodendrocytes dictates their subtypes and spatial organization (Rasband & Peles, 2016). After injury and demyelination this structure breaks down in a phenomenon known as channel spreading (England et al., 1991; Waxman & Ritchie, ¹⁹⁸⁵). Both sodium channels (Hunanyan et al., 2010) and potassium channels (Nashmi et al., 2000) spread outside of the node of Ranvier. We observed the spreading of potassium ion channels in Myrf ICKO and Myrf‐Intact control animals after injury. Interestingly, however, the extended Kv1.2 signal in Myrf ICKO was often associated with Nav1.2 channels, a subtype not typically observed in the mature node (Buttermore et al., 2013). This colocalization was rarely observed in the injured Myrf‐Intact controls. The upregulation of these channels might be one potential mechanism for compensation in remyelination deficient animals. The expression of Nav1.2 channels across long segments of spared axons (>50 μm) could permit conduction through these axons in a manner similar to proposed conduction in unmyelinated axons (Akaishi, 2018).

One limitation of this histological analysis was that we could not determine the status of the Nav1.2+ axons outside of the imaged plane, so it is possible they were severed farther away from the lesion and they are not truly spared. Furthermore, we only utilized antibodies for the 200 kD neurofilament protein, which is not expressed in all axons. Further classification of the axons (including size and myelination status) over greater distances in conjunction with their ion channel expression profiles would establish if demyelinated axons of all sizes express Nav1.2.

We found that conduction velocities through the lesion in Myrf ICKO after a moderate contusion were comparable to those measured in remyelination competent control animals, suggesting axons at the lesion conduct signals comparably. The conduction velocities measured in both Myrf ICKO and Myrf‐Intact control animals are reduced from uninjured animals, which might represent conduction through dysfunctional axons (James et al., 2011) in the vicinity of injury or thinly myelinated axons. In remyelination competent control animals, CAPs evoked in the spinal cords may represent conduction through spared myelinated fibers and fibers that are in the process of remyelination but have not yet established normal myelin thickness and length (Powers et al., 2013). Altogether, these data suggest that spared axons are able to maintain conduction even with increased numbers of demyelinated axonal segments over the relatively short distances of the lesion (1–2 mm), corroborating data showing conduction through long stretches of demyelinated rat axons (Felts et al., 1997). The expression of long stretches of Nav1.2 channels may contribute to this conduction. Future studies which assess conduction in single units would provide more sensitive measurements of conduction velocities. Subsequent marking and imaging of these individual axons may reveal increased expression of Nav1.2.

Finally, we wondered if compensation that was effective at re‐establishing hindlimb stepping would fail to re‐establish more subtle and finer locomotor function. We used a detailed analysis of hind limb movement in 3D 6 weeks after injury in Myrf ICKOs and injured Myrf‐Intact controls. Analyses of gait parameters showed that the Myrf ICKO did not significantly differ from injured Myrf‐Intact control animals in their stride. An analysis of joint angles showed that animals did not exhibit a statistically different range of motion throughout their step cycles. One interesting, though not statistically significant, finding is that the ICKO tended towards a smaller joint angle at the end of the step cycle. Myrf ICKO also showed slightly shorter step durations on one foot as compared to Myrf‐Intact controls. These slight modifications in hindlimb movement may result from small differences in compensation in the absence of remyelination.

A greater understanding of the compensatory mechanisms by which remyelination deficient mice regain locomotion following SCI would have obvious benefits to developing therapeutic strategies. However, further work is necessary to more deeply understand the present data and expand their utility. Performing RNA sequencing of the nuclei of neurons of Myrf ICKO animals following SCI would provide insight into the up or downregulation of other voltage‐gated ion channels. Such an analysis could be conducted in conjunction with a longitudinal histological analysis along the length of the lesion to provide spatial data regarding ion channel expression. Given their widespread expression in the spinal cord and brain, it will be difficult to conduct a loss of function study specific enough to establish the necessity of Nav1.2 channels for conduction in demyelinated axons. If conduction through is permitted by upregulation of Nav1.2 channels, application of a highly specific Nav1.2 channel blocker to the spared axons at the lesion would disrupt the conduction of CAPs observed in this study and differentiate the behavioral recovery of Myrf ICKOs from Myrf‐Intact controls. However, to our knowledge, there are no sodium channel blockers specific to Nav1.2 channels. Genetic manipulations of Nav1.2 expression will be difficult to restrict to demyelinated axons and not the axon initial segment or unmyelinated axons. Finally, while upregulation of Nav1.2 channels might contribute to conduction through a lesion of 2–3 mm in mice (Powers et al., 2013), further research will need to ascertain if altered sodium channel expression could scale up to overcome larger lesions, as are seen in humans.

The impressive compensation following SCI displayed by the mice in this study and our previous work indicates that mice exhibit a robust compensation and change in phenotype when remyelination is inhibited. It remains to be seen if humans sustaining SCI or demyelination exhibit comparable compensation and phenotypic changes. It is necessary to critically assess if studies demonstrating efficacy of OPC transplantation or other remyelination therapies are improving outcomes in young rodents through remyelination or through elevation of other compensatory mechanisms. Our data raise the possibility that compensation may play a role, and thus urge caution when extrapolating efficacy data garnered from rodent models. A mechanistic understanding of the compensation observed in this study may lead to the development of therapies that invoke greater recovery in humans.

AUTHOR CONTRIBUTIONS

S. B. Manesh: Conceptualization; investigation; formal analysis; visualization; writing – original draft; methodology; data curation. B. R. Kondiles: Writing – original draft; validation; formal analysis; data curation; visualization; investigation. S. Wheeler: Investigation; writing – review and editing. J. Liu: Writing – review and editing; methodology; investigation. L. Zhang: Methodology; investigation; writing – review and editing. C. Chernoff: Formal analysis; writing – review and editing; investigation. G. J. Duncan: Conceptualization; investigation; methodology; writing – review and editing. M. S. Ramer: Writing – review and editing; supervision; resources. W. Tetzlaff: Supervision; resources; conceptualization; funding acquisition; writing – review and editing; methodology.

CONFLICT OF INTEREST STATEMENT

The authors have no conflicts of interest to declare.

Supporting information

Figure S1.

Figure S2.

Figure S3.

Figure S4.

Manesh, S. B. , Kondiles, B. R. , Wheeler, S. , Liu, J. , Zhang, L. , Chernoff, C. , Duncan, G. J. , Ramer, M. S. , & Tetzlaff, W. (2025). Compensatory changes after spinal cord injury in a remyelination deficient mouse model. Journal of Neurochemistry, 169, e16220. 10.1111/jnc.16220

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.