Netrin-1 Therapy Restores Partial Hindlimb Movement in a Rat Model of High-Severity Chronic Spinal Cord Injury

Abstract

Spinal trauma caused by contusion or compression is the leading cause of spinal cord injury (SCI) worldwide. These injuries frequently progress to a chronic phase, especially in cases of severe damage. This process results in permanent impairment, affecting both physiological functions and voluntary motor control below the lesion level. At cellular level, the formation of a glial scar, which delineates the cystic cavity, interrupts the connectivity between the central nervous system (CNS) and muscles, as well as the neural communication between the peripheral and CNSs. This process, combined with the CNS inability to promote its self-repair to prevent the progression to a chronic phase, contributes to the exacerbation of spinal cord damage, resulting in a devastating pathology. Currently, there is no effective medical treatment to address the consequences of this condition, apart from physiotherapy, which has variable success depending on the type of injury and the degree of neural tissue preservation in the affected spinal cord. Considering this last, the development of new strategies to promote neuronal repair is essential for reversing this pathology in the future. Therefore, we propose Netrin-1—a developmental guidance molecule known to direct corticospinal tract (CST) growth during CNS development—as a potential therapeutic approach for enhancing neuronal repair in severe chronic SCI. Previously, we demonstrated in an acute phase model of transected SCI that this protein effectively promotes axonal regrowth, axonal reconnection, and recovery of locomotor activity. Based on these findings, we hypothesize that Netrin-1 may additionally act as a neuroreparative molecule in chronic SCI, promoting the recovery of hindlimb movement impaired by injury. To test the therapeutic potential of this molecule, we performed a rat model of chronic SCI with a high-severity lesion at Th10–Th11 thoracic level. We demonstrate that the delivery of Netrin-1 to the epicenter of the lesion promotes significant recovery of extensive movement in the three hindlimb joints, including full flexion and extension, previously impaired by chronic injury. Additionally, it restores functional abilities such as climbing and grasping to some extent. These functional findings correlate with anatomical and cellular observations, including regrowth, sprouting and remyelination of the CST; regrowth–reconnection of extrapyramidal tracts; regrowth–reconnection of serotonergic and dopaminergic axons; prevention of transsynaptic degeneration of lower motoneurons; and neuroprotection of both myelinated sciatic nerve fibers and ascending sensory pathways. In conclusion, this study extends the neuroreparative properties of Netrin-1 to chronic conditions. These findings support its use as potential therapeutic strategy in future human clinical trials.

Keywords

axonal regeneration axonal reconnection BBB score corticospinal tract motoneurons Netrin-1 spinal cord injury

Introduction

Spinal cord injury (SCI) is a debilitating condition, primarily caused by traumatic events, that disrupts spinal circuits and axonal pathways, severing communication between the brain and the spinal cord. This damage often results in profound motor, sensory, and autonomic impairments, with limited prospects for recovery, particularly in severe and chronic cases.^1,2

From an epidemiological standpoint, the SCI is one of the leading causes of disability in young adults worldwide.³ The World Health Organization estimates that between 250,000 and 500,000 new injuries occur each year.⁴ Remarkably, many patients with SCI remain disabled during the chronic phase.⁵ The functional deficits in SCI are multifaceted, being motor function loss notably impairing independence in daily activities and substantially diminishing the overall quality of life.

Currently, treatment options for patients with chronic SCI are largely limited to rehabilitation,⁶ where the success of therapies varies depending on the type of injury and the degree of both axonal tracts and spinal neuronal preservation after injury. Emerging trial therapies aim to enhance neurological function, emphasizing the critical need for the development of innovative therapeutic strategies.

From a functional standpoint, voluntary motor control relies on a complex wiring connectome comprising descending axons originating from motor cortex neurons (pyramidal pathway) and brainstem control centers (extrapyramidal pathways). These pathways converge to interact with spinal circuits responsible for coordinating movement. Nevertheless, the corticospinal tract (CST) is the key pathway responsible for voluntary motor control. It originates from pyramidal neurons located in layer 5 of the primary motor cortex, as well as from other regions such as the premotor and sensory cortices. The CST is unique in being the only direct pathway linking the motor cortex to the spinal cord, enabling precise control of fine motor movements.⁷ Furthermore, the decussation of the CST at the medullary pyramids enables the transmission of motor signals from both cerebral hemispheres to coordinate movement in both forelimbs and hindlimbs during locomotion.⁸

It is well established that after SCI, the limited intrinsic regenerative capacity of neurons in the adult mammalian central nervous system (CNS), along with the lack of sustained neurotrophic support in the injured environment, significantly hinders spinal cord tissue regeneration and rebuilding of cellular architecture.^9,10 Despite that, in human SCI, even in the most severe chronic cases, the lesion is mainly anatomically incomplete, with preservation of white matter regions containing spared axons. These preserved white matter axons surround the lesion and attempt to partially maintain synaptic interactions with spinal circuits above and below the injury site.¹¹ Nevertheless, once the pathology progresses to the chronic phase, characterized by the formation of a fluid-filled cavity and the maturation of the glial scar, the environment becomes highly inhibitory to neuronal regrowth and contributes to the enhanced die back of residual axons.¹² This process alters synaptic conduction at the edges of the lesion, reducing the efficiency of spontaneous reconnection between residual axons and target neurons and hindering spontaneous motor recovery.¹³ Therefore, the disruption of most descending axonal tracts after injury suppresses the sources of modulation and excitation that are essential for the proper functioning of spinal circuits.¹⁴ In line with this, although the preservation of local circuits after injury may be sufficient to generate basic muscular activity, voluntary movement is impossible without supraspinal input, specifically when the integrity of descending axonal tracts is compromised. Thus, promoting the regrowth and synaptic reconnection of pyramidal and extrapyramidal tracts with preserved circuits below the injury site is essential for the recovery of locomotor function.¹⁵

In our previous studies, we demonstrated the regenerative potential of Netrin-1, a developmental axon guidance cue critically involved in CST navigation during CNS development, primarily through its receptors UNC5h and DCC, which mediate repulsive or attractive signaling, respectively.¹⁶ We showed that Netrin-1 promotes CST regrowth and synaptic reconnection in a transection model of acute SCI, resulting in significant improvements in voluntary locomotor function.¹⁷ This underscores its therapeutic potential as a candidate for promoting neuronal repair and motor function recovery in models of spinal lesions that more accurately replicate the complexity of severe human SCI in chronic phase.

Therefore, in this study, we investigated the therapeutic potential of Netrin-1 as a treatment to enhance axonal regrowth across the lesion site and facilitate its synaptic reconnection in a chronic SCI. Our aim was to restore essential hindlimb movements that were impaired by the injury.

To test this molecule, we performed a chronic SCI contusion model using a severe compression injury at the thoracic level (Th10–Th11), which produced a high-severity lesion. Once the chronic phase was reached, we administered Netrin-1 intramedullary at the epicenter of the lesion (2000 and 4000 ng at 100 ng/μL).

We observed a significant effect in the recovery of extensive movement in the three hindlimb joints, including full flexion and extension as well as some recovery of functional abilities such as climbing and grasping. This response was the same for both doses of Netrin. We correlated this functional recovery with the follow observation at cellular level of (1) CST regrowth, reconnection, remyelination, and die back prevention; (2) avoiding transsynaptic degeneration of lower motoneurons; (3) synaptic preservation of extrapyramidal pathways; (4) regrowth and reconnection of serotonergic and dopaminergic axons; (5) preservation of sensory axons; and (6) preservation of myelination of sciatic nerve fibers. Furthermore, using magnetic resonance imaging (MRI), we observed spinal tissue sparing at the epicenter of the lesion.

Our results reveal that the therapeutic potential of Netrin-1 extends beyond acute SCI, showing efficacy in a chronic and severe model as well and supporting its future development as a viable biological therapy for patients with SCI.

Materials and Methods

Animals

Healthy adult male Wistar Kyoto rats from Charles River Laboratories were used in this study. The rats were 8–11 weeks old, and their weight was 250–290 g. All animal experiments were conducted in the “Laboratorio de Medicina Experimental,” Hospital Alemán, Buenos Aires, Argentina. The animal experimentation was approved by the Ethics Committee From “Sociedad Argentina de Investigación Clínica—SAIC” (Register code: 03-22) and was conducted according to the Guide for the Care and Use of Laboratory Animals.¹⁸

Throughout the study, the rats were housed in a temperature-controlled room at 22°C ± 2°C, with 50–60% humidity and a 12-h light/dark cycle, with access to food and water ad libitum.¹⁷

Lateral compression–contusion SCI

The animals were anesthetized using ketamine (75 mg per kg body weight), xylazine (10 mg per kg body weight), and acepromazine (2 mg per kg body weight) and placed over a heated platform to maintain body temperature during the surgery (Kent Scientific Corporation).

Animals underwent contusion–compression SCI at the Th10–Th11 laminar level under aseptic conditions. Briefly, after Th10–Th11 laminectomy, the dura was opened and a lateral contusion–compression was applied using an aneurysm clip with a closing force of 70 g. The clip was applied for two consecutive 60-sec intervals, resulting in a total compression time of 120 sec, which is sufficient to induce a high-severity SCI (the technique used in the present study is a modification of the methods previously described by Triplet et al. and Poon et al.^19,20). Lesion was inspected using a stereoscopic microscope (Stereo Zoom ST-6D) before closing in layers the muscles and skin. Sham-operated rats received only a Th10–Th11 laminectomy, without spinal cord compression, and as expected no motor deficits were observed.

After surgery, the rats were individually housed and received subcutaneous injection of tramadol hydrochloride (5 mg per kg body weight) as analgesia, gentamicin (5 mg per kg body weight) to prevent infections, silver sulfadiazine (Platsul-A) as topical ointment to prevent pressure sores, and 6 mL lactated Ringer’s solution to prevent dehydration. Then, rats were kept warm (29°C) for 4 h, placed on beds of sawdust, and given manual bladder evacuation three times per day until recovery of the micturition reflex. Moreover, hindlimb muscle stretch was carried out in each rat.

Food was provided on the cage floor, and water bottles were placed to have no difficulty reaching after injury. Supplemental oral feedings, topical ointments, Ringer’s solution, and/or analgesia were given as necessary.

Re-lesion surgeries were conducted 15 weeks after the initial spinal contusion–compression injury, targeting the same thoracic region (Th10–Th11) as the original SCI.

Netrin-1 therapeutical administration

The animals were divided into four groups: sham, vehicle control, Netrin-1 (2000 ng), and Netrin-1 (4000 ng) and housed individually for the entire duration of the experiment, which was 15 weeks.

Five weeks after injury, when the rats had reached the chronic phase,²¹ we performed a second surgery to deliver Netrin-1 to the epicenter of the lesion.

Recombinant Netrin-1 (Human Netrin-1 Cat. No. 6419-N1, R&D Systems) was reconstituted at 100 ng/μL in phosphate-buffered saline (PBS). At the time of application, Netrin-1 or vehicle (PBS) was delivered to the lesion epicenter, filling the entire injured area.

Netrin-1 was administered using a neuro-syringe (Hamilton Model 75 RN, 33-gauge), with one injection delivered perpendicularly into the lesion epicenter and two additional injections angled at 45° cranially and caudally, respectively, following previously described protocols.^17,22 A total of 2000 or 4000 ng of Netrin-1 was delivered. The whole procedure was performed under a stereoscopic microscope.

Neurobehavioral testing

To evaluate the effects of Netrin-1 treatment on the recovery of motor function following chronic SCI, we conducted a series of neurobehavioral tests. Prior to inducing the injury, all animals underwent a habituation protocol, initiated 1 week before the SCI. The habituation consisted of two 10-min cycles per session test, ensuring consistent acclimatization to the experimental conditions.²²

The neurobehavioral tests were carried out in a sound-attenuated room and with a blind experimental design. −

The Basso, Beattie, and Bresnahan locomotor rating scale (BBB score) was assessed in rats with SCI to identify, define, and rank patterns of locomotor recovery. This test was performed twice a week on an open field platform (1 m² diameter) and in a straight platform (1 m of length).²³

−

The negative geotaxis test was evaluated in rats 15 weeks after SCI. This test is used to assess motor coordination and vestibular sensitivity.²⁴ The rats were positioned at the top of a sloping grid, oriented with their heads facing downward. Since the response to stimulus, or taxis, is an innate behavior, we evaluated the time it takes the rats to reorient themselves toward an upwards position.²⁵ The score reflects either the animal’s ability to turn upward and climb or its failure to do so—manifested as an inability to turn or a fall to the ground—within 60 sec.

−

Kinematic analysis of climbing was evaluated in sham and Netrin-1-treated rats after SCI. Using Kinovea video analysis software, we determined the movement of the hindlimb joints during the execution of platform climbing.²⁶ We quantify the angle of displacement of the three joints during the pushing movement to climb a step height of 10 cm.

Magnetic resonance imaging

In order to assess the extent of tissue damage in the spinal cord in vivo, we carried out MRI analyses on rats with chronic SCI as well as on injured rats treated with Netrin-1. The imaging was performed using an Achieva 1.5 Tesla Philips MRI scanner. T2-weighted three-dimensional (3D) Fast Spin Echo sequence images were acquired with a “neurovascular” solenoid coil under the following parameters: field of view of 130 mm (FH), 130 mm (AP), and 35 mm (RL); voxel size of 0.508 mm (FH), 0.63 mm (AP), and 0.7 mm (RL); reconstruction matrix of 256 × 256; one stack consisting of 50 slices; fold-over direction in the anterior–posterior (AP) axis; slice gap of 0.3 mm; slice thickness of 2 mm; and a total scan time of 6 min.

Tissue isolation and preparation

Rats were transcardially perfused with PBS containing 0.05% (v/v) heparin, followed by fixation with 4% paraformaldehyde in PBS. The spinal cord, brain, and sciatic nerve were then dissected and post-fixed in the same fixative solution for 48 h at 4°C. The tissues were then incubated in a sucrose gradient (15–30%) for 48 h to prevent ice crystal formation during freezing.^22,27 Following sucrose treatment, the dura mater (meninges) was removed.

Immunohistochemistry

Spinal cord tissue was frozen and sectioned into 30-μm longitudinal cryostat slices (cranial-to-caudal and dorsal-to-ventral) using a Leica CM1850 cryotome. For analysis, 12 serial dorsal-to-ventral spinal cord sections were collected from each specimen (sham, vehicle control, and Netrin-1). Similarly, transverse spinal cord sections were obtained at a thickness of 15 μm as previously described.¹⁷

Free-floating slices were first incubated in a high-ionic-force blocking buffer (HIFBB) for 50 min, followed by permeabilization with 0.15% Triton X-100 in PBS for 20 min. Primary and secondary antibody solutions were prepared in HIFBB. Slices were incubated with primary antibodies for 3 h at room temperature, whereas secondary antibodies were applied overnight at 4°C. Nuclear labeling was performed using 4′,6-diamidino-2-phenylindole (DAPI) for 1 h at room temperature. To assess nonspecific binding, control slices were treated only with secondary antibodies corresponding to different IgG isotypes. All analyses were conducted by an observer blinded to experimental conditions.

CST labeling

Retrograde tracing of CST axons in the spinal cord was performed in rats treated with vehicle control or Netrin-1 15 weeks after chronic SCI. Fluoro-Ruby (FR) Dextran tracer (15% in dH2O; Molecular Probes, D1817, Eugene, OR, USA) was injected into the right dorsal CST, whereas Wheat Germ Agglutinin Fluorescent conjugate (WGA, 2 mg/mL, W11261, Alexa488, Molecular Probes) was loaded into the left dorsal CST. The delivery of tracers was carried out using a calibrated electric pump (Ap&S, CABA, Argentina) with a microcapillary tube (Sigma-Aldrich) at the tip. A total of 800 nL of tracers was injected on each side at a flow rate of 10 nL/sec, following a modification of a previously described method.²⁷ The injections were performed under a stereoscopic microscope (Stereo Zoom ST-6D) using a stereotaxic apparatus (Harvard Apparatus Compact Stereotaxic Instrument), following the coordinates: Th13-L1 spinal cord level; + 0.12 mm lateral from the midline and−0.87 mm depth for FR; and −0.12 mm lateral from the midline and−0.87 mm depth for WGA. Five days post-injection, rats were euthanized, and brain, spinal cord, and sciatic nerve tissues were collected through a perfusion–fixation procedure.

Lumbar motoneurons tracing

Retrograde tracing of lumbar motoneurons was performed as previously described.²² Briefly, 15 weeks after chronic SCI, rats treated with vehicle control or Netrin-1 received injections of cholera toxin subunit B (CTB) conjugated to Alexa Fluor 488 (1 mg/mL; Molecular Probes Inc., Eugene, OR, USA) into the right and left sciatic nerves to trace motor circuitry in the spinal cord. A total of 1 μL of CTB was injected into each sciatic nerve using a microcapillary tube and a calibrated electric pump, with an average flow rate of 1–2 μL/min. The microcapillary tube remained in place for 2 min post-injection to ensure proper tracer diffusion.

Four days after injection, rats were euthanized by perfusion–fixation, and lumbar spinal cord segments were dissected and sectioned longitudinally (cranial-to-caudal) at 45-μm thickness using a cryostat. Quantification of CTB-positive cell bodies was performed on three serial sections corresponding to ventral horn segments from each animal, in triplicate. The total number of CTB-positive cell bodies per sample was determined by summing the counts from each serial section. Automated counting was performed using the “Analyze Particles” plugin in Fiji image analysis software (NIH, USA).

All surgical procedures were performed under aseptic conditions.

Myelin labeling

Myelin sheath labeling in axons was performed on sectioned spinal cord tissues. Perfusion-fixed tissues were washed with PBS and incubated with FluoroMyelin Green Fluorescent Stain (1:150 dilution; F34651, Molecular Probes) for 50 min at room temperature. Following incubation, tissues were washed three times with PBS to remove excess stain. Immunohistochemistry (IHC) was then performed as described above.

Clearing technique

To render tissues transparent, whole rat spinal cords were incubated in tetrahydrofuran (THF) (Biopack, Prod. Química., Buenos Aires, Argentina)–distilled water [50% (vol/vol)] for 1 h, followed by incubation in THF-distilled water [80% (vol/vol)] for 2 h, and then in 100% THF for 1.5 h. This was followed by a fresh incubation in 100% THF for 24 h and subsequently in 100% dichloromethane (Biopack) for 2–24 h. Finally, the spinal cords were incubated in a BABB solution (a mixture of benzyl alcohol [Biopack] and benzyl benzoate [Biopack] at a 1:2 ratio) until the samples became transparent, as previously described.²⁷ The same protocol was applied to brain tissues, with extended THF incubation times. All incubation steps were conducted in glass tubes under continuous rotation (0.022 g) in light-shielded conditions.

Image acquisition

Epifluorescence microscopy

Images were acquired using a Nikon Eclipse E400 epifluorescence microscope (Nikon Instrument Group, Melville, NY, USA) equipped with CFI Plan Apo Lambda objectives (4× NA 0.1, 10× NA 0.45, 20× NA 0.75, and 60× NA 1.4, all suitable for confocal microscopy) and a Nikon DS-Fi1 camera (Nikon Instrument Group, Melville, NY, USA). Illumination source was provided by a mercury arc lamp, and the beam was passed through bandpass filters: a DAPI cube (375/28), an Alexa 488 cube (480/30), and a Cy3 cube (Chroma Technology Corp.). Images were acquired at 8-bit depth in TIFF format.

One-photon confocal microscopy

Images were acquired using an Olympus Fluoview 1000 confocal microscope (Olympus Headquarters Corporate, Philadelphia, PA, USA) equipped with UPLSAPO 10× NA 0.40, UPLSAPO 20× NA 0.75, and PLANAPO 60× NA 1.42 (oil immersion) objectives. The 3D images were obtained by acquiring z-series of 350–1000 optical slices with an Airy unit of 1 airy disk, using interval spacing optimized according to Nyquist theory to ensure optimal overlap and minimize photobleaching.^17,27 Confocal images were acquired at 12-bit per pixel. Scan speed (time per pixel in arbitrary Olympus units) was set to 8.0 for 10× magnification and 4.0 for 20× and 60× magnifications. Excitation wavelengths used were 473 nm for Alexa Fluor 488 or Cy2 and 559 nm for Cy3 or FR. The photomultiplier tube settings were, on average, 400 for voltage, 3125 for gain, and 1000 for offset (all in arbitrary Olympus units).

Image processing and analysis

Assessments of the lesioned gap

Gap quantification was performed using the length measurement function in Fiji Image Software. MRI images were calibrated upon import, and the region of interest (ROI) was delineated using the freehand selection tool. Gap measurements were taken five times per image, and the obtained values were averaged.

Assessment of CST myelinated area

Quantification of CST myelinated area was performed using the area measurement function in Fiji Image Software. Tissue images were calibrated using the set-scale plugin, and the ROI was delineated using the freehand selection tool. ROI area analysis was performed five times per image, and the obtained values were averaged.

Deconvolution

Image deconvolution was applied when necessary. Images were imported into Huygens Core 3.5.2p3 (64-bit) software, where the Tikhonov method and Laplacian stencil were applied. Theoretical point spread functions (PSFs) were generated using the Diffraction PSF 3D plugin in Fiji software.

Axonal quantification

The z-axis image stacks of the spinal cord, acquired by confocal microscopy, were imported into IMARIS 3D software v6.3.1 (Bitplane Sci Software, Zurich, Switzerland). Quantification of the number of axonal tracts in three dimensions was performed using the Measurement Point function in IMARIS. The trajectory of each axonal tract was manually traced using measurement points from the beginning to the end of each tract, generating a 3D filament rendering that replicated the shape of the axons (X/Z). This approach enabled precise measurement of axonal tract length, as previously described.²⁸ Since most axons at the lesion epicenter originate from the gray matter, their quantification was considered to reflect individual axons.

Axonal branching

Quantification of axonal branching was performed using z-stack images rendered at epicenter of the lesion, as well as upstream and downstream lesion segments. Branching was quantified using the Filament function in IMARIS software and the Volume Viewer plugin in Fiji software, when necessary.

Axonal mask quantification

Z-stack images in TIFF format were imported into the Axon Tracer macro for automated quantification of axon regeneration in spinal cord tissue.²⁹ A ROI was applied to define the lesion area. Axons were displayed using a multi-color mask in order to facilitate the quantification.

CST axons quantification

Quantification of dorsal CST axons was carried out using the Analyze Particle function from Fiji software. Circularity was defined between 0.00 and 1.00 as was previously described.¹⁷

Clustering quantification

Quantification of different types of clusters (pre- and postsynaptic, as well as Netrin-1) was performed using the manual multi-point counting function on 60× images. The same confocal images were used to generate 3D reconstructions for colocalization analysis when necessary, as previously described.³⁰

3D video animation

A motion video based on a series of z-stack images from one-photon confocal microscopy was generated using the Animation plugin in IMARIS 3D.

Antibodies

The following primary antibodies were used for IHC: mouse monoclonal IgG anti-Bassoon (1:200; Millipore), rabbit polyclonal IgG anti-Homer1 (1:200; Invitrogen), rabbit polyclonal IgG anti-Netrin-1 (1:200; Invitrogen), rabbit polyclonal IgG anti-Parvalbumin (1:50; Invitrogen), mouse monoclonal IgG2a anti-PKCγ (C4)-Alexa 488 (1:50; Santa Cruz), mouse polyclonal IgG anti-5HT (1:400; Immunostar), rabbit polyclonal IgG anti-Tyrosine Hydroxylase (1:200; Invitrogen), mouse monoclonal IgG anti-S100β (1:200; Sigma), mouse monoclonal IgG anti-Neurofilament Medium Molecular Mass (NFM) (1:500; Invitrogen), and mouse monoclonal IgG anti-βIII-Tubulin (neuronal) (1:500; Sigma).

Secondary antibodies for IHC included DyLight™ 488, Cy2™, and Cy3™, all from Jackson ImmunoResearch. DAPI was obtained from Molecular Probes (Eugene, OR, USA).

The specificity of the secondary antibodies was validated by omitting the primary antibodies; no detectable immunoreactivity was observed in control tissue samples.

Statistical analysis

Data analysis was performed using GraphPad Prism software (version 5.0, GraphPad Software, Inc., La Jolla, CA, USA). Results are expressed as mean ± standard deviation (SD).

A priori power analysis was conducted using G*Power (version 3.1.9.4, University of Kiel, Germany) to determine the minimum required sample size, ensuring adequate statistical power while minimizing the number of animals in accordance with ethical guidelines for laboratory animal use. For in vivo behavioral studies, sample size estimation was based on a power analysis with α = 0.04; effect size |ρ| = 0.85, and power (1 − β) = 0.75, resulting in a minimum required sample size of 6 per group. Accordingly, the experimental groups consisted of 6 animals in the sham group, 6 in the vehicle control group, 7 in the Netrin-1 4000 ng group, and 6 in the Netrin-1 2000 ng group, yielding a total of 25 animals.

For the imaging analyses, group sizes ranged from three to five animals per condition due to the ethical and logistical constraints inherent to chronic SCI models. However, multiple anatomically matched spinal cord sections—or whole-cleared tissue samples—were analyzed per animal. Consistent patterns were observed across samples, ensuring that the data obtained were robust and representative.

The statistical comparisons were performed using an unpaired two-tailed Student’s t test or one-way analysis of variance (ANOVA), followed by Tukey’s or Bonferroni’s post hoc test, as appropriate.

The significance threshold was set at p < 0.05, unless otherwise specified in the corresponding figure legends.

Results

Targeted application of Netrin-1 induces partial hindlimb recovery in a rat model of severe chronic SCI

To investigate the therapeutic potential of Netrin-1 to restore hindlimb movement after chronic SCI, we used a contusion–compression model. As detailed in the Materials and Methods section, compression was applied at the Th10–Th11 level, resulting in immediate hindlimb paralysis. Five weeks post-injury, at the onset of the chronic phase, we administered 2000/4000 ng of Netrin-1 or vehicle control directly into the lesion epicenter (Fig. 1A).

FIG. 1.

Quantification of locomotor recovery following chronic spinal cord injury (SCI). (A) Experimental timeline illustrating SCI induction and treatment protocol. Inset shows the lesion induced by clip compression. (B) Representative image sequence from a vehicle-treated rat. Yellow arrowheads indicate sustained hindlimb hyperextension with no voluntary movement, observed consistently during the chronic phase and following vehicle administration. (C) Representative image sequence of a Netrin-1-treated rat. Yellow arrowheads indicate persistent hindlimb hyperextension with no voluntary movement during the established chronic phase. After Netrin-1 treatment, yellow arrowheads highlight the full range of motion of the hip, knee, and ankle joints during stepping. (D) Basso, Beattie, and Bresnahan (BBB) score quantification. Rats were treated with vehicle; 2000 ng of Netrin-1; and 4000 ng of Netrin-1. Values represent the mean ± standard deviation (SD); n = 6 rats per group (sham, vehicle, and Netrin-1 [2000 ng]), and n = 7 to Netrin-1 (4000 ng) group. ***p < 0.001 using one-way analysis of variance and Tukey multiple comparison test. (E) Column graph showing no significant (ns) differences between the Netrin-1 treatment groups. Values represent the mean ± SD (ns, p > 0.05) using unpaired two-tailed Student t test.

To assess recovery of hindlimb function, we performed the BBB locomotor rating scale, evaluating animals prior to injury, during the chronic phase, and following treatment (Fig. 1B, C).

No significant functional recovery was observed in vehicle-treated animals, which exhibited a BBB score of 1.375 ± 1.13, indicative of limited movement (slight) in one or two hindlimb joints (typically the hip and/or knee), with an inability to bear weight (Fig. 1B, D and Supplementary Video S1). In contrast, Netrin-1-treated rats demonstrated substantial recovery of hindlimb movement, with extensive movement of all three joints and the ability to perform some degree of plantar placement (Fig. 1C and Supplementary Video S2). This functional recovery was reflected in significantly increased BBB scores in the Netrin-1-treated groups (Netrin-1 2000 ng: 8 ± 1.01; Netrin-1 4000 ng: 8.52 ± 0.32) compared with control animals (Fig. 1D). Notably, at the study end-point, no statistically significant differences were found between the 4000 ng and 2000 ng treatment groups (Fig. 1E). However, the 4000 ng dose exhibited a lower SD, suggesting a more consistent response within this group.

To further evaluate the recovery of vestibular function and certain aspects of motor coordination, we conducted the negative geotaxis test. Fifteen weeks post-injury, vehicle-treated rats were unable to grasp, descend, reorient upwards, or climb to the top of the inclined grid, instead falling directly to the floor. In contrast, Netrin-1-treated rats exhibited grasping ability, upward reorientation, and climbing behavior, comparable to sham rats (Fig. 2A and Supplementary Videos S3, S4, and S5), demonstrating a significant improvement compared with controls (Fig. 2B). Since no statistically significant differences were observed between the two Netrin-1 doses (2000 and 4000 ng), we selected the 4000 ng dose for subsequent analyses due to its more consistent response. Additionally, as expected, vehicle-treated rats exhibited a characteristic tail-dangling posture, in contrast to the tail positioning observed in sham-operated animals. Interestingly, Netrin-1-treated rats showed a partial correction in tail positioning, resembling, and, to some extent, the posture observed in the sham group (Fig. 2C).

FIG. 2.

Quantification of motor coordination and vestibular function. (A) Negative geotaxis test on an inclined grid. Right, schematic representation of the setup. Left, representative image sequences from sham, vehicle-treated, and Netrin-1-treated rats. Only sham and Netrin-1-treated animals were able to reorient and climb the grid, as indicated by yellow arrowheads showing paw placement in each sequence. In contrast, vehicle-treated rats were unable to perform the task and fell from the grid. (B) Bar graph quantifying the time required for rats to turn upward and climb the grid. No significant difference (ns) was observed between sham and Netrin-1-treated groups. Data are presented as mean ± standard deviation. Statistical analysis was performed using one-way analysis of variance followed by Bonferroni’s multiple comparisons test (ns, p > 0.05). (C) Representative images highlighting the tail position in sham, vehicle-treated, and Netrin-1-treated rats (yellow lines highlight the angle of the proximal tail relative to the paw). (D) Kinematic analysis during the climbing task. Insets in each image magnify the hindlimb joint angles. Graphs on the right highlight the kinematic analysis. Red arrowheads indicate the two steps required by Netrin-1-treated rats to climb the platform.

Finally, we assessed the extent of joint movement in rats that regained hindlimb mobility after chronic SCI. To this end, we conducted a kinematic analysis during the climbing task. Sham-operated rats were able to climb the platform with full joint extension, completing the movement in a single step. In contrast, although Netrin-1-treated rats climbed the platform with more extended joint angles and were able to support their body weight, they required two steps to complete the ascent (Fig. 2D and Supplementary Videos S6–S7). Vehicle-treated rats, however, failed to perform the climbing task altogether (Supplementary Video S8).

Netrin-1 preserves spinal cord tissue continuity by reducing the cystic cavity following chronic injury

Upon reaching the chronic phase of the lesion, rats’ spinal cord exhibited a prominent cystic cavity, disrupting the transmission of information above and below the injury site.

Treatment with Netrin-1 significantly reduced the size of the cavity, preserving tissue continuity at the lesion site. MRI analysis (1.5 Tesla, STIR-3D) revealed a distinct gap flanked by two inflamed segments in untreated rats (chronic lesion prior to treatment) and vehicle-treated rats. In contrast, Netrin-1-treated rats exhibited continuous tissue, although quite thinned at the lesion site, yet structurally preserved, with no detectable gap (yellow arrowheads; Fig. 3A, Supplementary Videos S9, S10, and S11). Quantification confirmed the absence of a measurable gap in Netrin-1-treated rats (Fig. 3B).

FIG. 3.

Magnetic resonance imaging (MRI). (A) Representative MRI images from sham rats and animals with chronic spinal cord injury before treatment and after vehicle or Netrin-1 administration. Yellow arrowheads indicate the lesion level. Insets on the right show magnified views of intact spinal cord tissue in sham rats and lesion sites under each experimental condition. (B) Measurement of lesion gap across experimental conditions, data are presented as mean ± standard deviation.

Axonal regrowth at the epicenter of the lesion

We focused our analysis on the epicenter of the lesion, specifically examining the spinal cord gray matter-intermediate area. In vehicle-treated rats, we observed an absence of axonal regrowth, with only a few NFM-positive signals that correspond to fragmented axons and retraction bulbs (white arrowheads; Fig. 4A). Additionally, no Netrin-1 signal was detected in this region. In contrast, Netrin-1-treated rats exhibited a marked increase in the number of NFM-positive axons in the core of the lesion. Notably, Netrin-1 signal was localized along the re-regrown axonal shafts as discrete clusters, exclusively in the Netrin-1-treated group (white frame, yellow arrowhead; Fig. 4A, Supplementary Video S12). Quantification revealed a significant increase in axonal regrowth, branching, and Netrin-1 clusters in Netrin-1-treated rats compared with vehicle-treated controls (***p < 0.0001; **p = 0.0037; Fig. 4B). These results indicate that Netrin-1 promotes axonal regrowth, with the newly growing axons in this region originating primarily from spinal neurons.

FIG. 4.

(A) Representative immunohistochemical analysis of regrown axons using antibodies against neurofilament medium molecular mass (NFM, red) and Netrin-1 (green). Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole. The middle panel shows the absence of both axons and Netrin-1 signal in the lesion core, highlighted in the boxed area, whereas white arrowheads mark retraction bulbs, which are likewise devoid of Netrin-1. In contrast, the boxed area in the right panel highlights axonal regrowth, with the corresponding magnification (below) showing Netrin-1 clusters along the axonal shaft of regrowing axons (yellow arrowhead). Scale bar, 30 μm. (B) Quantification of axonal profiles at the lesion epicenter. Bar graphs represent the number of regrowing axons; axonal branches; and Netrin-1 clusters over the vicinity of focal plane of regrown axons. Data are expressed as mean ± standard deviation; n = 3 rats per group, with multiple serial sections evaluated per animal. Statistical significance was determined using unpaired two-tailed Student’s t test; **p = 0.0037; ***p < 0.0001, where p < 0.05 was considered statistically significant.

Restoration of dorsal CST continuity across the lesion epicenter in chronic SCI

After reaching the chronic phase, we evaluated the effect of Netrin-1 treatment on the regrowth of the dorsal CST (dCST), the primary axonal pathway involved in precise and dexterous voluntary movements. To label both the right and left dCST, FR and WGA were injected, respectively, as shown in Figure 5A. Tissue analysis was performed using whole-cleared spinal cord samples under one-photon microscopy.

FIG. 5.

Regrowth, branching, and reconnection of the dorsal corticospinal tract (dCST) at the epicenter of the lesion. (A) Schematic illustration of Fluoro-Ruby (FR) and wheat germ agglutinin (WGA) tracers injections and subsequent analysis in a chronic spinal cord injury (SCI) model induced by severe compression. (B) Representative z-projected image from a transparent spinal cord of a sham rat. Bilateral dCSTs (red and green) are clearly visible both at the lumbar site where the tracers were injected and at the thoracic level. White boxes (1–4), magnified on the right, highlight axons from each dCST. (C) Representative z-projected image from a transparent spinal cord from a vehicle-treated rat. The yellow arrowhead in the left panel (cleared) marks the cystic cavity and glial scar at the lesion site. In the lumbar segment (panel 1), a few FR-labeled axons are visible (inset). However, no labeled fibers are detected at the lesion epicenter (panel 2), as shown in the corresponding boxes regions (1–2) and their magnified insets. Similarly, the thoracic segment (panel 3) displays a complete absence of FR- and WGA-labeled fibers, as indicated in the boxed areas (1–2) and corresponding magnifications. (D) Representative z-projected image from a transparent spinal cord from a Netrin-1-treated rat. In the lumbar segment (panel 1), both tracers distinctly label the bilateral dCST; green and red fibers are highlighted in the insets. dCST fibers are also present at the lesion epicenter (panel 2). Boxes 1 and 2 show regrown dCST axons traversing the lesion core, while box 3 marks lateral branching of red-labeled fibers in close proximity to the focal plane of green-labeled neuronal somas. The magnified view of box 3 illustrates this interaction, as schematized in cartoon (D′). At the thoracic level, bilateral dCST axons (green and red) are visible; boxes 1–3 and their magnified views reveal strong fluorescence signals from both sides. Boxes 4 and 5 show magnified views detailing fiber morphology. Scale bar, 200 μm. (E–H) Bar graphs showing: (E) the number of axonal branches; (F) WGA-labeled transsynaptic contacts at the lesion epicenter; (G) dCST axonal branches interacting with WGA-positive neurons; and (H) the number and percentage of regrown dCST axons traversing the lesion center. Data are presented as mean ± standard deviation, with n = 3 rats per group. For each animal, the entire spinal cord was cleared and analyzed. Statistical significance was assessed using unpaired two-tailed Student’s t test in (E–G): p = 0.0048, p = 0.0025, and p = 0.0096, respectively. In (H), one-way analysis of variance followed by Tukey’s post hoc test was used; p < 0.05 was considered statistically significant.

After tracer injection at the lumbar level, vehicle-treated rats exhibited fragmentation of dCST axons, likely due to Wallerian degeneration. This axonal pattern was in stark contrast to that observed in sham rats, where continuous fluorescence signals were detected along both dCST segments ascending through the spinal cord (Fig. 5B and insets 1–2; Fig. 5C(1)). Notably, in Netrin-1-treated rats, preservation of both lumbar dCST axons was observed, as evidenced by the continuous red and green fluorescence signals, which were conserved compared with the fragmentation observed in vehicle-treated rats (Fig. 5D (1) and insets in the bottom and top right corners).

At the epicenter of the lesion, with a transverse length of 2.716 mm ±0.39 generated by lateral compression, Netrin-1-treated rats exhibited FR-positive fibers, in contrast to vehicle-treated rats, where no FR-positive fibers were observed due to the presence of a marked cystic cavity that interrupted the retrograde transport of the tracers (Fig. 5C(2)−D(2) and insets 1–2). Furthermore, the number of FR-positive axonal branches in Netrin-1-treated rats showed a significant increase compared with vehicle-treated controls (**p = 0.0048; Fig. 5E).

Regarding WGA, which possesses transsynaptic properties, we observed that in Netrin-1-treated rats, WGA-positive axons interacted with propriospinal neurons at the epicenter of the lesion, transferring their fluorescence (Fig. 5D(2) inset 3, white frame). This interaction was significantly greater than in vehicle-treated animals (**p = 0.0025; Fig. 5F). Additionally, WGA-positive propriospinal neurons appeared to make contact with the ramifications of FR-positive axons in the same focal plane (Fig. 5D). This close interaction was significantly greater than that observed in vehicle-treated rats (p = 0.0096; Fig. 5G). In addition, we demonstrated that the regrown axons in Netrin-1-treated rats were able to pass through the lesion center, as evidenced by the presence of the upstream of lesion of both FR-positive and WGA-positive dCST axons (Fig. 5D(3) and insets 1–5). In contrast, no such axons were observed in vehicle-treated rats (Fig. 5C(3) and insets 1–2). This significant difference is quantified in Figure 5H. Furthermore, the regrowth of dCST axons was supported by a significant recovery of PKC-γ fluorescence signal in the spinal cord of Netrin-1-treated rats compared with vehicle-treated controls, below the lesion site (Supplementary Fig. S1).

Finally, we validate the regrowth of CST axons by demonstrating the restoration of intra-axonal retrograde cargo traffic across the lesion after chronic SCI. To assess this, FR was injected into the lumbar spinal cord (Fig. 6A), and its presence was detected in the soma of upper motoneurons in the primary layer 5 of the motor cortex but not in control rats (Fig. 6B). This suggests that injured axons not only regenerated across the lesion site but also reestablished functional retrograde transport, allowing the tracer to reach the neuronal soma.

FIG. 6.

Retrograde tracing of upper motoneurons. (A) Schematic illustration of the experimental methodology and single-photon confocal imaging used to analyze brain samples in the chronic spinal cord injury compression model. (B) Detection of Fluoro-Ruby (FR)-labeled upper motoneurons in optically cleared whole brains. FR-positive cell bodies were observed in layer V of the motor cortex in both sham and Netrin-1-treated rats. Representative white arrowheads indicate the position of labeled neuronal somata. Scale bar, 50 μm.

Remyelination of Netrin-1-induced regrowing axons prevents CST dieback

As described above, Netrin-1 promotes the regrowth of damaged axons after SCI. Under normal conditions, axons are uniformly covered by myelin along their entire shaft (Fig. 7A). However, after SCI, we observed extensive myelin debris accumulation at the lesion epicenter in vehicle-treated rats, with only a few regrown axons exhibiting partial remyelination (Fig. 7B). In contrast, treatment with Netrin-1 in rats with chronic SCI significantly enhanced axonal remyelination, which was accompanied by the formation of nodes of Ranvier (***p = 0.0009; Fig. 7C–D and Supplementary Video S13).

FIG. 7.

Remyelination of spinal axons at the lesion epicenter. (A) Representative image of a healthy (sham) spinal cord section (4× magnification). The dotted box indicates the area shown at higher magnification (60×), where axons are labeled in red (βIII-tubulin), nuclei in blue (DAPI), and myelin in green (FluoroMyelin). The asterisk in the middle panel marks the region magnified on the right, illustrating a representative axonal shaft and its myelin sheath. A three-dimensional (3D) rendering further depicts how myelin ensheathes the axons in this region. (B) Representative image of a vehicle-treated rat. The dotted box at the lesion epicenter highlights the accumulation of cellular debris and fragmented myelin (green). The white arrowhead in the right panel magnification indicates myelin debris, which is also visualized in the corresponding 3D rendering. (C) Representative image of a Netrin-1-treated rat. The dotted box at the lesion epicenter highlights remyelination (green) and regrowth of spinal axons (red). The right panel magnification shows axonal shafts ensheathed by myelin, which is further visualized in the corresponding 3D rendering. The inset in this rendering displays myelin (green) wrapping around the axon (red), indicated by a blue arrowhead. Additionally, the formation of nodes of Ranvier is visualized by white arrowheads. Scale bars, 600 μm for 4×, 40 μm for 60×. (Voxel size of x, y, and z was 0.331,·0.331,·and 0.55 μm) (D) Bar graph shows the quantification of remyelinated axons at the epicenter of the lesion. Data are expressed as mean ± standard deviation; n = 4 rats per group, with multiple serial sections evaluated per animal. Statistical significance was determined using unpaired two-tailed Student’s t test; ***p < 0.0009 where p < 0.05 was considered statistically significant.

Given that chronic SCI induces CST axonal dieback upstream of the lesion, a process associated with cytoskeletal collapse and progressive myelin degradation along the axonal shaft, we next examined whether Netrin-1 could also prevent the retrograde axonal degeneration proximal to the injury site. We found a significant preservation of FuoroMyelin-positive CST axons upstream of the lesion in spinal cords from Netrin-1-treated rats compared with vehicle-treated controls (p < 0.05; Fig. 8A, B).

FIG. 8.

Dorsal corticospinal tract (dCST) remyelination after chronic spinal cord injury. (A) Representative coronal images of spinal cord sections cranial to the lesion site, showing myelinated dCST fibers (green). The dotted lines delineate the dCST region. The upper panel shows a sham rat, the middle panel a vehicle-treated rat, and the lower panel a Netrin-1-treated rat. Magnifications of the dotted areas highlight the myelinated dCST section in each condition. The right panels display corresponding pseudocolor spectrum maps generated using ICA-Lut in Fiji software, where high-intensity signals are shown in yellow/white and low-intensity signals in cyan/blue. Scale bars: 500 μm for 4×, 50 μm for 60×. (B) Quantification of the myelinated dCST section across groups. Data are presented as mean ± standard deviation; n = 3 rats per group, with multiple serial sections analyzed per animal. Statistical analysis was performed using one-way analysis of variance followed by Bonferroni’s multiple comparisons test, p = 0.0006. Differences were considered significant at p < 0.05.

Regeneration of serotonergic fibers through the lesion site and preservation of dopaminergic projections surrounding the spinal injury

We comprehensively characterized the axonal repair process, dissecting individual descending pathways. In this context, we focused on serotonergic axons. Under normal conditions (Sham), descending serotonergic fibers exhibited a variable caliber and a highly branched distribution (Fig. 9A, left panel). In contrast, following SCI, vehicle-treated rats showed a marked reduction in 5-HT-positive signal at the lesion epicenter, accompanied by axonal bulb-derived debris accumulation (Fig. 9A, middle panel).

FIG. 9.

Regrowth of serotonergic fibers after Netrin-1 treatment. (A) Representative longitudinal spinal cord sections showing 5-hydroxytryptamine (5-HT, green) immunolabeling. Left panel: Sham rats exhibit continuous 5-HT fibers (dotted boxes 1–2 and corresponding magnifications). Middle panel: Vehicle-treated rats show scarce 5-HT signal. Magnified images from dotted boxes 1–3 (caudal to cranial) highlight the lesion core, showing fragmented axons (asterisk) and bulb-derived debris (white arrowheads). Right panel: Netrin-1-treated rats display evident 5-HT-positive axons at the lesion epicenter, both within the white matter (dotted boxes 1 and 3) and crossing the lesion core (dotted box 2), as shown in the magnifications. Binary mask images were generated to enhance axon detection and enable quantitative analysis. Scale bars: 1000 μm for 4×, 50 μm for 60×. (B) Quantification of 5-HT-positive axons across experimental groups. Data are presented as mean ± standard deviation; n = 3 rats per group, with multiple serial sections analyzed per animal. Statistical significance was assessed using one-way analysis of variance followed by Tukey’s post hoc test; p = 0.0092. Differences were considered significant at p < 0.05.

Remarkably, Netrin-1-treated rats displayed a substantial increase in 5-HT-positive fibers throughout the lesion site. These fibers not only recovered the typical 5-HT labeling along the axonal shaft but also exhibited the classical periodic punctate-like accumulations, indicative of presynaptic specializations in the regenerating axons (Fig. 9A, right panel). Quantification of serotonergic axons revealed a significant increase in 5-HT-positive axons at the lesion site in Netrin-1-treated animals compared with vehicle-treated controls (**p = 0.0016; *p = 0.0407; Fig. 9B). Furthermore, upstream of the lesion, we observed preservation of serotonergic fibers in Netrin-1-treated rats. These 5-HT fibers entered the gray matter and established synaptic contacts with propriospinal neurons, exhibiting punctate accumulations surrounding their surface within the same focal plane. In contrast, vehicle control rats also showed such contacts but at significantly reduced numbers (Supplementary Fig. S2). Besides, we also found a significant regrowth of 5-HT fibers below the lesion in the white matter of lumbar segments only in Netrin-1-treated rats (p = 0.0016; Supplementary Fig. S3).

Moreover, we observed decussation of tyrosine hydroxylase (TH)-positive fibers through the central canal of the spinal cord both upstream and downstream of the lesion, exclusively in Netrin-1-treated rats. In contrast, vehicle control rats exhibited only cellular debris at these levels (Supplementary Fig. S4A, B).

Additionally, we found that TH-positive fibers upstream of the lesion established synaptic contacts with the soma and dendrites of propriospinal neurons within the spinal cord gray matter, exclusively in Netrin-1-treated rats. Conversely, in vehicle-treated animals, only sparse TH punctate signals were detected, with no clear evidence of synaptic interaction (Supplementary Fig. S4A′, arrowheads indicate synaptic contacts). A similar pattern was observed downstream of the lesion, where synaptic interactions between TH-positive fibers and propriospinal neurons were detected only in Netrin-1-treated rats (Supplementary Fig. S4B′, yellow arrowheads).

Axonal regrowth prevents transsynaptic degeneration and supports preservation of lumbar motor circuits

To further evaluate the integrity of motor circuits, we performed retrograde tracing with the CTB to assess the connectivity between lumbar motoneurons and their peripheral targets (Fig. 10A). In Netrin-1-treated rats, we observed robust CTB labeling of lumbar motoneurons projecting to muscle, indicating preservation of axonal connections. By contrast, vehicle-treated rats showed a marked reduction in labeled motoneurons, reflecting compromised connectivity (Fig. 10B, C). Consistent with these findings, axonal and myelin preservation was observed in the sciatic nerve of Netrin-1-treated rats compared with controls (Supplementary Fig. S5).

FIG. 10.

Retrograde cholera toxin subunit B (CTB) tracing of motoneurons. (A) Schematic illustration of the experimental workflow for retrograde CTB tracing in a chronic spinal cord injury (SCI) model. (B) Representative images of lumbar spinal cord sections showing CTB-labeled motoneurons (green). The upper panel corresponds to sham rats, with magnifications of the dotted box highlighting CTB-positive motoneurons. The middle and lower panels show vehicle- and Netrin-1-treated rats, respectively, analyzed at the chronic stage (15 weeks post-injury). Magnified views of dotted boxes highlight CTB-labeled motoneurons in each condition. Scale bars: 400 μm for 4×, 20 μm for 20×. (C) Quantification of CTB-positive motoneurons across experimental groups. Data are presented as mean ± standard deviation; n = 3 rats per group, with multiple serial sections analyzed per animal. Statistical significance was assessed using one-way analysis of variance followed by Bonferroni’s post hoc test, p < 0.0001. Differences were considered significant at p < 0.01. (D) Representative high-magnification confocal images showing CTB-labeled motoneurons (green), postsynaptic Homer1 clusters (red), and DAPI (cyan) in the lumbar spinal cord. Upper, middle, and lower panels correspond to sham, vehicle-treated, and Netrin-1-treated rats, respectively. Scale bars: 20 μm for 60×. (E) Quantification of Homer1 clusters in the lumbar spinal cord. Data are expressed as mean ± standard deviation (SD); n = 3 rats per group, with multiple serial sections analyzed per animal. Statistical significance was determined by one-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test, p = 0.0006. Differences were considered significant at p < 0.05. (F) Quantification of Homer1 clusters per CTB-positive motor neuron. Data are expressed as mean ± SD; n = 3 rats per group, with multiple serial sections analyzed per animal. Statistical significance was determined by one-way ANOVA followed by Bonferroni’s post hoc test, p < 0.0001. Differences were considered significant at p < 0.001.

The motoneurons preservation may be attributed to the regrowth of axonal tracts through the lesion site, extending to the lumbar segments (Supplementary Fig. S6), thereby restoring synaptic contact with lumbar motoneurons and preventing their transsynaptic degeneration. The preserved structural integrity of these motoneurons was supported by the finding that CTB-labeled neurons—those with functional projections to muscle—exhibited a significantly higher number of Homer1-positive postsynaptic clusters in Netrin-1-treated rats compared with vehicle-treated controls (Fig. 10D, E, F). These findings indicate that regenerating axons form stable synaptic connections that protect target neurons from secondary degeneration.

Bassoon–Homer1 colocalization as evidence of synaptic restoration below the lesion following Netrin-1-induced rubrospinal tract regrowth

Given the specific anatomical localization of the rubrospinal tract (RST) within the spinal cord (Supplementary Fig. S7A), we assessed the degree of colocalization between the presynaptic marker Bassoon (originating from RST terminals) and the postsynaptic marker Homer1 (expressed in dendrites of gray matter neurons).

In sham rats, we observed the characteristic distribution pattern and spatial colocalization of Bassoon and Homer1 (Supplementary Fig. S7B, left panel; magnified views and colocalization mask in white). This organization was preserved only in Netrin-1-treated rats, which displayed a similar synaptic pattern to that of sham animals (Supplementary Fig. S7B, right panel). In contrast, vehicle-treated rats exhibited a marked reduction in Bassoon signal and a loss of Bassoon–Homer1 spatial colocalization (Supplementary Fig. S7B, middle panel).

Quantitative colocalization analysis using Mander’s overlap coefficient revealed that approximately 60% of Bassoon-positive clusters overlapped with Homer1 in Netrin-1-treated rats, compared with only 20% in vehicle-treated controls and 80% in Sham animals (Supplementary Fig. S7C). This intermediate value suggests that Netrin-1 promotes the partial restoration of synaptic architecture, bringing it closer to physiological levels. These findings support the notion that Netrin-1-induced RST regeneration enables the reestablishment of functional synaptic contacts below the lesion.

Regeneration of ascending and descending axons across the lesion restores neural connectivity and supports hindlimbs movement recovery

To rule out the possibility that the observed recovery of extensive hindlimb movements was due to intrinsic spinal circuit plasticity below the lesion—such as from central pattern generator activity—we performed a re-lesion in Netrin-1-treated rats 15 weeks after the initial SCI, targeting the same thoracic segment. Following this second injury, the previously recovered hindlimb movements were abolished, with rats reverting to complete hindlimb dragging and a BBB score that dropped to 1 and remained unchanged for 15 days (Supplementary Fig. S8). These results strongly indicate that the locomotor recovery in Netrin-1-treated rats depended on long-distance axonal regeneration and reconnection across the original lesion site. Notably, this includes both descending motor tracts and ascending sensory projections, such as those from the gracile fasciculus as well as from spinothalamic tracts (Supplementary Fig. S9), ruling out a solely sublesional plasticity-driven mechanism.

Discussion

In this study, we demonstrate that the targeted application of Netrin-1 significantly enhances the recovery of full hindlimb mobility—including climbing and weight-supported propulsion—through spinal tissue preservation and axonal regeneration/reconnection following severe chronic phase SCI.

Netrin-1, a guidance cue recognized for its involvement in axon growth, CST decussation, and its synaptic connection in the spinal cord,^31,32 has shown promising therapeutic effects in SCI models, where regenerative capacity is often limited. Our previous findings¹⁷ and the results presented in this study further underscore the clinical potential of Netrin-1 in treating severe SCI. While its ability to significantly enhance previously lost hindlimb mobility is crucial, what is equal remarkable is its capacity to promote axonal regrowth and facilitate synaptic reconnection, offering a broader therapeutic benefit for long-term recovery.

Locomotion was most pronounced in Netrin-1-treated animals, where we observed substantial improvements in hindlimb movement, reflected in a significantly increase in the BBB scores reaching an “Intermediate Stage of Locomotor Recovery.” This improvement was accompanied by the ability to perform more complex motor tasks, such as climbing on the inclined geotaxis grid, behaviors that were absent in control animals and directly associated with the reestablishment of the vestibular pathway. The ability of Netrin-1 to induce movement in all three joints of the hindlimb suggests that it not only promotes general movement but also facilitates more coordinated motor actions, a crucial aspect for the restoration of hypothetical voluntary movement following SCI.

One of the most striking findings in our study was the preservation of spinal cord tissue continuity, evidenced by reduced cystic cavitation in Netrin-1-treated rats. This result is particularly significant, as Netrin-1 has been shown to reduce macrophage infiltration at the core of lesion.^17,33,34 The formation of glial scar and cystic cavitation at the site of the lesion is a major barrier to both axonal regeneration and functional recovery following SCI.³⁵ Our MRI and histological analyses revealed that Netrin-1-treated rats exhibited a continuous, albeit thinned, tissue structure at the lesion site, while vehicle-treated rats showed extensive tissue disruption. This preservation likely contributes to the observed improvements in hindlimbs movement, as continuous spinal cord tissue is essential for relaying signals between the brain and peripheral targets.³⁶ It is noteworthy that the chronic SCI model employed in this study represents an exceptionally severe form of injury. Specifically, we used an aneurysm clip with a closing force of 70 g applied to the spinal cord for 2 min, a condition that substantially exceeds the severity thresholds commonly employed in rodent models of SCI. For instance, Poon et al.²⁰ used a clip force of 35 g for only 60 sec to produce a moderate to severe lesion. More severe injuries, such as those induced by a 50 g clip, have been shown to replicate the clinical and histological characteristics of a complete SCI, corresponding to ASIA A classification in humans.³⁷ Therefore, the extent of damage in our model is considerably greater. This may help explain why control animals in our study exhibited a mean BBB score of only 1.3, indicating near-complete loss of hindlimb function and minimal, if any, spontaneous recovery. From this perspective, the model used here provides a highly stringent platform for evaluating the therapeutic potential of biological interventions such as Netrin-1.

At cellular level, Netrin-1 treatment promoted marked axonal regeneration at the lesion epicenter, as evidenced by a significant increase in neurofilament medium (NFM)-positive axons—a marker of active axonal growth. NFM, an intermediate filament protein, is predominantly expressed in axons undergoing regrowth and plays a critical role in regulating axonal caliber and, consequently, nerve conduction velocity, while preserving cytoskeletal architecture.³⁸ Furthermore, Netrin-1 was found spotted in clusters along the surface of regrowing axonal shafts, suggesting that it may contribute to both the stimulation and directional guidance of axonal regeneration through attractive signaling mechanisms.^39,40

The ability of Netrin-1 to restore the continuity of the CST was also demonstrated in this study. The CST is crucial for voluntary and precise motor control, and its preservation is a major challenge in spinal cord repair.⁴¹ In Netrin-1-treated rats, the dorsal CST tract showed significant preservation and regrowth across the lesion epicenter, a finding that contrasts sharply with the fragmentation observed in control animals. Moreover, using a transsynaptic neuronal tracing technique, we demonstrated that regenerated CST axons not only traversed the lesion site but also formed collateral branches capable of reaching and potentially interacting with propriospinal neurons. This result highlights the potential of Netrin-1 to support the regeneration of descending motor pathways, which are often severely disrupted following SCI.^42,43 Interestingly, the typically muted regenerative response of CST neurons to SCI—attributed to the distal localization of axonal damage⁴⁴—may be at least partially overcome following Netrin-1 treatment.

Our study also revealed that Netrin-1-induced regrowing axons undergo remyelination, a critical prerequisite for meaningful functional recovery. Remyelination is essential for restoring axonal conduction, preserving axonal integrity, stabilizing the plasma membrane, and preventing secondary degeneration following SCI.^45,46 In Netrin-1-treated animals, we observed substantial remyelination, particularly at the lesion epicenter, along with a marked reduction in myelin debris relative to controls. The newly formed nodes of Ranvier further indicate that these remyelinated axons are functionally competent, as these structures are required for efficient saltatory conduction and to neuron–glia interaction.⁴⁷ Additionally, this therapeutic intervention prevented CST dieback and demyelination upstream of the lesion, thereby potentially limiting the spread of secondary injury.

Additionally, we evaluated the regrowth of serotonergic and dopaminergic fibers, which are critical for the initiation, modulation, and fine-tuning of locomotor patterns following SCI.⁴⁸ Netrin-1 treatment promoted the regeneration of both fiber systems across the lesion and preserved their synaptic contacts with spinal neurons. This preservation likely contributes to the functional recovery observed, as these monoaminergic systems are essential modulators of spinal motor networks.⁴⁹ Supporting our findings, Jasmin et al. demonstrated that Netrin-1 plays a neuroprotective and neurorestorative role in dopaminergic neurons, highlighting its broader potential in CNS repair.⁵⁰

Targeting the regeneration of descending extrapyramidal tracts is particularly relevant, since the restoration of serotonergic and dopaminergic fibers enhances intrinsic spinal circuits plasticity and facilitates endogenous repair mechanisms after SCI.⁵¹

Furthermore, our findings indicate that Netrin-1 not only promotes the regrowth of long axonal tracts but also preserves the connectivity of lumbar motor circuits, which is critical for the recovery of locomotor function. CTB retrograde tracing experiments revealed that Netrin-1-treated rats maintained functional connections between lumbar motoneurons and their peripheral nerves, while vehicle-treated animals exhibited a significant loss of this connectivity. The preservation of these motor relay circuits supports the idea that regenerated descending fibers can reestablish connections with spared or newly formed intraspinal interneuronal networks, thereby preventing the concomitant transsynaptic degeneration of lumbar motoneurons and allowing them to remain functional.⁵² This assumption was supported by the restoration of synaptic contacts below the lesion, since—as previously mentioned—axonal regeneration without reconnection is not sufficient to achieve locomotor recovery after SCI.⁴³ Evidence presented here shows that the presynaptic cytomatrix protein Bassoon, which is localized in both excitatory and inhibitory synapses,⁵³ co-localizes with Homer1, a dendritic protein that regulates glutamatergic synapses. This further underscores the potential of Netrin-1 to facilitate the formation of functional synapses in regenerating spinal cord circuits. This restoration of synaptic connectivity appears particularly in areas consistent with the anatomical location of the RST, providing further evidence of Netrin-1’s role in supporting the complex neuroplasticity required for functional recovery after SCI. Notably, a complete retransection at the original injury site abolished locomotor function, confirming that the regenerated axons were functionally integrated and essential for the observed recovery.

In conclusion, our study provides compelling evidence that Netrin-1 facilitates locomotor recovery following a severe SCI in the chronic phase. This recovery is mediated by several mechanisms, including the preservation of spinal cord tissue, axonal regeneration, restoration of both descending and ascending motor pathways, prevention of transsynaptic degeneration, maintenance of peripheral nerve myelination, and the formation and reconnection of new synaptic contacts. These findings highlight the potential of Netrin-1 as a promising therapeutic strategy for improving outcomes in patients with chronic SCI, especially those with severe lesions where other therapeutic approaches have proven ineffective. Our findings are consistent with studies from other groups demonstrating the neuroprotective effects of Netrin-1^54,55 supporting its role not only in promoting locomotor recovery after SCI but also in other neurological conditions, such as Parkinson’s disease.⁵⁰

While our findings provide strong support for the therapeutic efficacy of Netrin-1 in a chronic severe SCI model, several important limitations must be acknowledged. First, the intervention was based on a single intramedullary administration, and the potential benefits—or risks—of repeated dosing remain unexplored. Second, the follow-up period was limited to 15 weeks, restricting our ability to assess the long-term stability of the observed effects or the emergence of delayed complications. Third, although rodent models offer valuable mechanistic insights, the translation of these findings to human patients remains a significant challenge, in part due to species-specific differences in CST organization that influence plasticity and recovery.⁴¹ Addressing these limitations in future studies—including prolonged follow-up and testing in primates models—will be essential to advance the clinical relevance of Netrin-1-based therapies. Nevertheless, the current data position Netrin-1 as a compelling candidate for promoting functional recovery in individuals with SCI.

Transparency, rigor, and reproducibility statement ethics approval and consent to participate

This article is classified as basic research with a strong translational focus, as it involves nonhuman animal models relevant to human SCI. All animal procedures were thoroughly reviewed and approved by the Ethics Committee of the Sociedad Argentina de Investigación Clínica—SAIC (Register code: 03-22) and conducted in full accordance with the Guide for the Care and Use of Laboratory Animals (U.S. National Research Council).

Sample size estimation for behavioral studies was performed using G*Power analysis (α = 0.04, effect size |ρ| = 0.85, power = 0.75), resulting in a minimum of six animals per group. The final group sizes were sham (n = 6), vehicle control (n = 6), Netrin-1 2000 ng (n = 6), and Netrin-1 4000 ng (n = 7), totaling 25 animals. These numbers were specifically determined to ensure sufficient statistical power while minimizing animal use, in strict adherence to ethical guidelines.

Statistical comparisons were performed using unpaired two-tailed Student’s t tests or one-way ANOVA, followed by Tukey’s or Bonferroni’s post hoc tests, as appropriate. The significance threshold was set at p < 0.05, unless otherwise specified in the corresponding figure legends.

The analysis plan was preestablished prior to study initiation. Investigators were blinded to group allocation during all data analyses. All materials used are commercially available.

This research was primarily funded by Fundación Florencio Fiorini (Argentina), with additional support from PICT–Argentina and a Minnesota grant.

Authors’ Contributions

H.R.Q. conceived and coordinated the study, and designed, performed, and analyzed the experiments. J.S. assisted with microscopy analysis and surgical procedures. A.U. and A.S. provided support with animal care and surgeries. H.R.Q. and R.B. supported the study. H.R.Q. wrote the article. All authors reviewed the results and approved the final version of the article.

Footnotes

Acknowledgments

The authors would like to thank Horacio Romeo (BIOMED-UCA-CONICET) for providing the stereotaxic surgery equipment and Pablo Lopez (INIMEC-CONICET-UNC) for supplying the WGA tracer. The authors also grateful to technicians Lic. Claudia Calo and Lic. Matias Arce (Hospital Alemán) for their assistance with MRI image acquisition and to Eduardo Eyheremendy, Head of the Imaging Service at Hospital Alemán. The authors sincerely thank the Board of Directors of Hospital Alemán for their financial support. The authors are also thankful to Bruno Buchholz (School of Medicine, University of Buenos Aires) for his valuable insights, Osvaldo Ponzo (School of Medicine, University of Buenos Aires) for his assistance, and Leslie Morse, Chair of the Department of Physical Medicine and Rehabilitation at the Miller School of Medicine, University of Miami, for her support throughout this process. The authors also acknowledge the continued support of the National Scientific and Technical Research Council (CONICET), Argentina.

Author Disclosure Statement

The authors declare no conflicts of interest.

Funding Information

This work was supported by a grant from the Florencio Fiorini Foundation Argentina and PICT-2019 Argentina awarded both to Héctor Ramiro Quintá, and the Minnesota grant awarded to Ricardo Battaglino and Héctor Ramiro Quintá.

Supplemental Material

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