Experimental Neurology

Spinal cord injury (SCI) can result in temporary or permanent alterations in sensory, motor, and autonomic functions as a result of primary mechanical damage to the spinal cord. Functional recovery is often limited due to persistent secondary injury mechanisms like inflammation, vascular breakdown, and cellular damage. Mitochondrial dysfunction is a key driver of secondary injury pathology, and while mitochondrial-targeted therapies have shown promise in rodent models of injury, functional improvements fail to translate to humans. Pigs are excellent models for understanding both the behavioral and molecular consequences of SCI because of their physiological similarity to humans, which could bridge the translational gap between rodent research and clinical implementation. To develop effective, mechanistic-based therapies, we must understand the molecular underpinnings of SCI using both male and female animal models with high translational fidelity at multiple time points after injury. To date, research on mitochondrial dysfunction following SCI has been limited to female rodent models measured acutely (6h-7d) after injury. Here, we studied mitochondrial dysfunction at three different time points in male pigs to establish a relative time course of mitochondrial impairment following SCI that may be therapeutically targeted to treat secondary complications of injury. We measured mitochondrial bioenergetic function and electron transport chain (ETC) complex activities, as well as qualified mitochondrial dynamics and oxidative damage acutely (2h), sub-acutely (24h), and chronically (9wk) after SCI in adult male pigs. The results show distinct patterns of mitochondrial dysfunction between time points with functional deficits occurring 2h post-SCI, increased mitochondrial fragmentation at 24h post-SCI, and mitochondrial recovery by 9wks post-SCI. These studies offer insight into mitochondrial changes across time in a clinically relevant animal model of SCI in hopes of bridging the translational research gap.

Spinal cord injury (SCI) often impairs motor functions such as voluntary movement and fine motor control, with the corticospinal tract (CST) being a crucial pathway affected. While CST-targeted rehabilitation, such as treadmill training, supports motor recovery, gaps remain in understanding the topographical changes within the CST and how they correlate with behavioral outcomes. In this study, we utilized a custom Emx1Cre;LSL-SynGFP mouse line to quantify CST plasticity following moderate contusion SCI, both with and without exercise (treadmill) training. Fluorescent labeling of cortical synapses allowed for detailed visualization of descending CST rewiring, and we assessed its relationship to behavioral outcomes, including kinematics analysis and motivational state. Mice were stratified by motivational state using the Progressive Ratio Assay, and locomotor recovery was evaluated through the Basso Mouse Scale (BMS), joint/limb kinematics, and Motion Sequencing (MoSeq) analysis. Our findings indicate that treadmill training enhances CST rewiring, especially in highly motivated animals, leading to increased synaptic density in the ventral horn and improved BMS subscores. Motivation further influenced specific kinematic parameters, such as toe clearance, while treadmill training significantly improved speed by reducing the stance phase. Results suggest that while treadmill training induces broad beneficial outcomes, motivation may fine-tune recovery, influencing neural circuit and behavioral changes. This suggests multiple mechanisms converge to promote recovery--those we cannot control and those we can. These results underscore the combined role of task-specific training and also perhaps motivation in driving CST plasticity and functional recovery after SCI.

Spinal cord injury (SCI) pathology is highly difficult to treat due to substantial heterogeneity in injury presentation and spread, along with unclear mechanisms linking damage to pathology. Damages from injury forces (primary injury) are exacerbated by a series of biochemical events that follow the initial damage and injure additional tissue, known as secondary injury. Reactive aldehydes, such as acrolein, play a key role in propagating secondary injury cascades following SCI. Targeting acrolein after SCI has demonstrated therapeutic potential in limiting injury spread and pathology. However, injury mechanisms linking reactive aldehydes to SCI outcome have not been fully characterized. To gain a more comprehensive understanding of the cellular and molecular mechanisms underlying SCI, we generated proteomic profiles of rat spinal cords 24 h (acute phase) after subjection to SCI, sham injury, saline injection, or acrolein injection. We performed gene set enrichment analysis (GSEA) to characterize proteins and pathways significantly enriched after SCI and acrolein-injection. We then used Translatable Components Regression (TransComp-R), a framework for translating biological signatures across systems, to assess whether acrolein-associated spinal cord signatures can stratify SCI from sham outcomes. Our proteomics analysis revealed 467 differentially expressed proteins (DEPs) between the sham and SCI groups and 7 DEPs between saline and acrolein injection groups. Notably, the complement and coagulation cascades were upregulated in spinal cords subjected to SCI and acrolein injection. Our TransComp-R analysis further demonstrated that acrolein-associated signatures could distinguish SCI from sham conditions. Taken together, our findings suggest that acrolein induces proteomic alterations during the acute phase of SCI and is associated with complement and coagulation cascade activation, among other pathways. Therefore, this study reinforces the notion that understanding the role of acrolein in the acute phase of secondary SCI may be beneficial.

Modulation of neural activity is a promising strategy to influence the growth of axons and improve behavioral recovery after damage to the central nervous system. The benefits of neuromodulation likely depend on optimization across multiple input parameters. Here we used a chemogenetic approach to achieve continuous, long-term elevation of neural activity in murine corticospinal tract (CST) neurons. To specifically target CST neurons, AAV2-retro-DIO-hM3Dq-mCherry or matched mCherry control was injected to the cervical spinal cord of adult Emx1-Cre transgenic mice. Pilot studies verified efficient transgene expression in CST neurons and effective elevation of neural activity as assessed by cFos immunohistochemistry. In subsequent experiments mice were administered either DIO-hM3Dq-mCherry or control DIO-mCherry, were pre-trained on a pellet retrieval task, and then received unilateral pyramidotomy injury to selectively ablate the right CST. Mice then received continual clozapine via drinking water and weekly testing on the pellet retrieval task, followed by cortical injection of a viral tracer to assess cross-midline sprouting by the spared CST. After sacrifice at eight weeks post-injury immunohistochemistry for cFos verified elevated CST activity in hM3Dq-treated animals and immunohistochemistry for PKC-gamma verified unilateral ablation of the CST in all animals. Despite the chronic elevation of CST activity, however, both groups showed similar levels of cross-midline CST sprouting and similar success in the pellet retrieval task. These data indicate that continuous, long-term elevation of activity that is targeted specifically to CST neurons does not affect compensatory sprouting or directed forelimb movements.

Cognitive deficits frequently arise after traumatic brain injury. The murine closed head injury (CHI) models these deficits since injured mice cannot acquire Barnes maze. Dosing of minocycline plus N-acetylcysteine beginning 12 hours post-CHI (MN12) restores Barnes maze acquisition by an unknown mechanism. Increased hippocampal synaptic efficacy is needed to acquire Barnes maze, synaptic long-term potentiation (LTP) models this increased synaptic efficacy in vitro. LTP has an early phase (E-LTP) lasting up to one hour that is mediated by second messengers that is followed by a late phase (L-LTP) that needs new synthesis of protein kinase M zeta (PKM{zeta}). PKM{zeta} has constitutive kinase activity because it lacks the autoinhibitory regulatory domain found in other PKCs. Due to its constitutive activity, the amount of PKM{zeta} kinase activity is determined by PKM{zeta} protein levels. We report that CHI bilaterally decreases PKM{zeta} levels in the CA3 and CA1 hippocampus. MN12 increases CA1 PKM{zeta} expression. CHI inhibits E-LTP in slices from the ipsilesional hippocampus and inhibits L-LTP in slices from both hippocamppi. MN12 treatment reestablishes both E-LTP and L-LTP in slices from the injured MN12-treated hippocampus. The restoration of L-LTP from injured MN12-treated hippocampus is mediated by PKM{zeta} because L-LTP is blocked by the specific PKM{zeta} inhibitor, {zeta}-stat. Hippocampal {zeta}-stat infusions also prevents Barnes maze acquisition in injured, MN12-treated mice. These data suggest that post-injury minocycline plus N-acetylcysteine targets PKM{zeta} to improve synaptic plasticity and cognition in mice with closed-head injury.

Acute spinal cord injury triggers a complex secondary injury cascade characterized by lesion expansion, neuroinflammation, glial reactivity, and oligodendrocyte degeneration, which together limit endogenous repair. Identifying neuroprotective interventions capable of targeting distinct components of this cascade remains a major challenge. In this study, we compared the neuroprotective profiles of minocycline, a tetracycline derivative with anti-inflammatory and antioxidant properties, and bone marrow mononuclear cells (BMMCs), which exert paracrine immunomodulatory and trophic effects, using a model of complete thoracic spinal cord transection in adult rats. Animals received either BMMCs (5 x 106 cells, intravenously, 24 h post-injury) or minocycline (50 mg/kg twice daily for 48 h, followed by 25 mg/kg for five days). Histological and immunohistochemical analyses revealed that both treatments attenuated secondary damage, reducing lesion area, microglial/macrophage activation (ED1+ cells), and oligodendrocyte pathology (Tau-1+ cells). However, the magnitude and pattern of protection differed between interventions: minocycline produced a stronger reduction in lesion area, whereas BMMCs exerted greater suppression of microglial/macrophage activation and superior preservation of oligodendrocytes. Astrocyte counts (GFAP+ cells) did not differ quantitatively among groups, despite qualitative differences in astrocytic morphology. Integrated effect size analysis further highlighted these complementary neuroprotective profiles across outcomes. Collectively, these findings indicate that minocycline and BMMCs target distinct components of secondary injury after severe spinal cord injury, providing a mechanistic rationale for future studies exploring multi-targeted or combinatorial therapeutic strategies.

Although the behavioral outcome of Constraint-Induced Movement Therapy (CIMT) is well known, and that a combination of CIMT and arm use training potentiates the effect, there has been limited study of the brain circuits involved that respond to therapy. An understanding of CIMT from a brain network level would be useful for guiding the duration of effective therapy, the type of training regime to potentiate the outcome, as well as brain regional targets that might be amenable for direct neuromodulation. Here we investigated the effect of CIMT therapy alone unconfounded by additional rehabilitation training in order to determine the impact of intervention at the circuit level. Adult rats were injured by controlled cortical impact injury and studied before and then after 2wks of CIMT or noCIMT at 1-3wks post-injury using a combination of forelimb behavioral tasks and task-based and resting state functional magnetic resonance imaging at 3 and 7wks post-injury and compared to sham rats. There was no difference in behavior or functional imaging between CIMT and noCIMT after injury before intervention so that data are unlikely to be confounded by differences in injury severity. CIMT produced only a transient reduction in limb deficits compared to noCIMT immediately after the intervention, but no difference thereafter. However, CIMT resulted in a persistent reduction in contralesional limb-evoked activation and a corresponding ipsilesional cortical plasticity compared to noCIMT that endured 4wks after intervention. This was associated with a significant amelioration of intra and inter-hemispheric connectivity present in the noCIMT group at 7wks post-injury.

Spinal cord injury (SCI) triggers secondary pathophysiological cascades, including glutamate excitotoxicity, that result in neuronal loss and impair functional recovery. We have previously shown that lysophosphatidylethanolamine (LPE), a lysophospholipid, promotes neurite outgrowth and protects against glutamate excitotoxicity in cultured cortical neurons. However, whether these effects extend to spinal cord neurons and occur in vivo has remained unclear. In this study, we compared the effects of different LPE species: myristoyl-LPE (14:0 LPE), palmitoyl-LPE (16:0 LPE), stearoyl-LPE (18:0 LPE), and oleoyl-LPE (18:1 LPE) in cultured spinal commissural neurons, and evaluated their effects in vivo using a mouse model of SCI. In cultured neurons, all LPE species promoted neurite outgrowth. Although several species demonstrated a tendency toward neuroprotection, only 16:0 LPE exhibited a statistically significant protective effect against glutamate-induced excitotoxic cell death. Intrathecal administration of 16:0 LPE after SCI reduced TUNEL-positive cells in the acute phase and attenuated lesion expansion at 8 weeks post-injury. Moreover, 5-HT fluorescence intensity was increased in 16:0 LPE-treated mice, suggesting enhanced serotonergic innervation. Furthermore, administration of 16:0 LPE after SCI significantly improved hind-limb motor performance compared with vehicle controls, as assessed by the Basso Mouse Scale. Collectively, these findings suggest that intrathecal administration of 16:0 LPE reduces secondary injury and promotes functional recovery following SCI. Our findings highlight its potential as a therapeutic candidate for SCI.

Preclinical traumatic brain injury (TBI) research relies on experimental models that vary by mechanism, parameters, surgical procedures, species, strains, and ages, to name a few. While these models are crucial for understanding injury mechanisms and testing therapies, the progress in translating this knowledge to the clinic has been limited. This is in part due to fragmented resources and inconsistent reporting of critical variables. Here, we introduce the PRECISE-TBI model catalog, a centralized, queryable resource that consolidates metadata from published studies. The catalog integrates curated annotations from more than 450 papers, including details such as age, sex, strain, model type, device, and injury parameters. Where available, entries are also linked to protocols and datasets to enhance transparency and reproducibility. The Model Catalog serves as a living resource that enables cross-study comparison, identifies gaps in reporting, and connects the literature to datasets, protocols, device information, and other relevant resources. Analysis of the initial catalog entries revealed gaps in the reporting of device, age, and weight. In contrast, the reporting of sex improved over time, with over 90% of recent studies within the catalog papers reporting sex. Strain was also reported in most studies, with consistent reporting of specificity, especially for the C57 mice substrain. We expect the Model Catalog to serve as a valuable tool to enhance study design and reproducibility in preclinical TBI research while advancing FAIR data principles in the TBI field.

During development, cadherins Celsr2 and Celsr3 control axon navigation. Unlike Celsr3, Celsr2 remains expressed in the adult, suggesting unexplored roles in maintenance and repair. Here we show that Celsr2 knockdown promotes motor axon regeneration in mouse and human spinal cord explants and cultured motor neurons. Celsr2 downregulation is accompanied by increased levels of GTP-bound Rac1 and Cdc42, and of JNK and c-Jun proteins. Using a branchial plexus injury model, we show that forelimb functional recovery is improved in Celsr2 mutant versus control mice. Compared to controls, in mutant mice, reinnervated biceps muscles are less atrophic, contain more newly formed neuromuscular junctions, and generate larger electromyographic potentials, while motor neuron survival and axon regeneration are improved. GTP-bound Rac1 and Cdc42, JNK and c-Jun are upregulated in injured mutant versus control spinal cord. In conclusion, Celsr2 negatively regulates motor axon regeneration via Cdc42/Rac1/JNK/c-Jun signaling and is a target for neural repair.

Cervical spinal cord injury (SCI) frequently leads to life-threatening respiratory insufficiency by disrupting descending phrenic pathways. There is growing interest in non-invasive neuromodulatory approaches to enhance plasticity of spared respiratory circuits. We investigated whether cervical repetitive magnetic stimulation (rMS) applied to the injured cervical spinal cord promotes ventilatory recovery in a preclinical mouse model. Adult mice received a unilateral C3 hemicontusion followed by either rMS or sham stimulation. We found that rMS-treated mice significantly improved recovery of tidal volume and minute ventilation at 21 days post injury(dpi) compared to sham controls under various breathing conditions (isoflurane anesthesia, poikilocapnic phase and hypercapnic challenge). Correspondingly, diaphragm EMG enhanced ipsilateral hemidiaphragm activity in ventral and medial regions, and even contralateral hemidiaphragm activity in its ventral part. This was associated with a marked attenuation of the inflammatory response at the cervical spinal cord level. Indeed, rMS lowered astroglial, fibrotic scarring, pro-inflammatory CD68-, Iba1- microglial/macrophage markers. Moreover, perineuronal net expression (WFA positive staining) is globally reduced in the ventral spinal horn, whereas at the lesion site it is markedly increased and tightly wrapped around motoneurons. Together, these findings demonstrate that rMS promotes functional respiratory recovery after cervical SCI through combined enhancement of diaphragmatic motor output and modulation of the inflammatory and extracellular environment. Together, these functional and cellular findings indicate that spinal rMS promotes a permissive, pro-regenerative environment supporting respiratory circuit plasticity. We conclude that rMS significantly enhances ventilatory recovery via reduced inflammatory response and improved intraspinal rewiring after high cervical SCI, suggesting it is a promising non-invasive strategy. The ability of rMS to engage spared respiratory networks and support neuroplasticity highlights its promise as a safe, non-invasive therapeutic strategy with translational potential for rehabilitation of breathing function after SCI. One Sentence SummaryNoninvasive cervical magnetic stimulation improves breathing after spinal cord injury by boosting diaphragm activity and reducing inflammation.

Functional recovery after spinal cord injury (SCI) is hindered by the limited ability of axons to regenerate in the adult mammalian central nervous system (CNS). Overcoming this barrier is critical for achieving effective recovery. Axonal regeneration depends on the activation of intracellular processes like transcription factor induction, protein and lipid trafficking, and cytoskeletal remodelling. Targeting these pathways offers a promising approach for promoting neuronal repair. This study examined the combined therapeutic effects of dibutyryl-cAMP (db-cAMP), which primes neurons for growth, and integrin 9 overexpression, which supports axonal extension. Using in vitro models with dorsal root ganglion (DRG) neurons and astrocytes, as well as an in vivo SCI model, we evaluated the potential of this approach. In vitro, the combination of db-cAMP and integrin 9 significantly enhanced neuronal growth. However, in vivo results were less consistent, with db-cAMP affecting AAV-mediated transcription and the expression of tenascin C (TnC) in neurons and astrocytes. These findings highlight the potential of modulating intracellular signalling and integrin activation but underscore the challenges posed by the complexity of the in vivo environment. Further studies are necessary to unravel these mechanisms and refine therapeutic strategies for effective SCI recovery. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/653846v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1473630org.highwire.dtl.DTLVardef@36a8beorg.highwire.dtl.DTLVardef@80593corg.highwire.dtl.DTLVardef@6285e3_HPS_FORMAT_FIGEXP M_FIG C_FIG

Traumatic spinal cord injury (SCI) in humans occurs most frequently in the cervical spine where it can cause substantial sensorimotor impairments to upper limb function. The altered input to spinal circuits below the lesion leads to maladaptive reorganisation which often leads to hyperreflexia in proprioceptive circuits. Neurotrophin 3 (NT3) is growth factor essential for the development of proprioceptive neurons. We have previously shown that following bilateral corticospinal tract axotomy, intramuscular delivery of an Adeno-Associated Viral vector encoding NT3 (AAV-NT3) induces proprioceptive circuit reorganisation linked to functional recovery. To assess its therapeutic effects following a clinically relevant bilateral C5-C6 contusion in rats, AAV-NT3 was injected intramuscularly into the dominant limb 24 hours after injury and forelimb function was assessed over 13 weeks. The injury generated hyperreflexia of a distal forelimb proprioceptive circuit. There was also loss of fine motor skills during reach-and-grasp and walking on a horizontal ladder. Ex vivo magnetic resonance imaging (MRI) revealed atrophy of the spinal cord and white matter disruption throughout the lesion site together with extensive loss of grey matter. Unexpectedly, animals treated with AAV-NT3 had a slightly smaller lesion in the regions close to the epicentre compared to PBS treated animals. Rats treated with AAV-NT3 showed subtly better performance on the horizontal ladder and transient benefits on reach-and-grasp. AAV-NT3 did not normalise hyperreflexia in a treated muscle. The treatment increased the amount of NT3 in treated muscles but, unexpectedly, serum levels were only elevated in a small subset of animals. These results show that this dose and delivery of AAV-NT3 may generate subtle improvements in locomotion but additional treatments will be required to overcome the widespread sensorimotor deficits caused by contusion injury.

Remote secondary injury in the thalamus has been observed following cortical infarct, however the mechanisms are not well understood. We used the distal MCAO stroke model (pdMCAO) to explore the cellular and temporal gliosis response in secondary thalamic injury in mice. At 3 days post-stroke (PSD3), primary infarct was limited to the cortex, with no infarct in the thalamus. However, at 2 weeks after stroke (PSD14), the ipsilateral thalamus demonstrated degenerating and severely damaged neurons. Staining for GFAP (astrogliosis) or IBA-1 (microgliosis) was first apparent in the ipsilateral thalamus by PSD3, and showed a progressive increase through PSD14. The number of activated microglia was increased within the thalamus at PSD14, reflecting proliferation of resident microglia as well as infiltration of peripheral monocytes. Interestingly, astrogliosis within the thalamus was enduring, as it was still evident at two years post-stroke. Furthermore, the astrogliosis at two years (but not at 6 weeks) demonstrated glial scar-like characteristics. Lastly, we demonstrated that post-stroke treatment with an NMDA receptor antagonist (memantine) reduces gliosis in the thalamus at PSD14. These findings highlight the development of lasting secondary injury in the thalamus following cortical stroke and support the value of memantine treatment in the mitigation of this injury.

Traumatic brain injuries (TBI) are diverse with heterogeneous injury pathologies, which creates challenges for the clinical treatment and prevention of secondary pathologies such as post-traumatic epilepsy and subsequent dementias. To develop pharmacological strategies that treat TBI and prevent complications, animal models must capture the spectrum of TBI severity to better understand pathophysiological events that occur during and after injury. To address such issues, we improved upon our recent larval zebrafish TBI paradigm emphasizing titrating to different injury levels. We observed coordination between an increase in injury level and clinically relevant injury phenotypes including post-traumatic seizures (PTS) and tau aggregation. This preclinical TBI model is simple to implement, allows dosing of injury levels to model diverse pathologies, and can be scaled to medium- or high-throughput screening.

The circadian rhythms of gene expression drive diurnal oscillations of physiological processes that determine the acute injury response including immunity, inflammation and hemostasis. While outcomes of various acute injuries are affected by the time of day at which the original insult occurred, such diurnal influences on recovery after spinal cord injury (SCI) are unknown. We report that several key regulators of circadian gene expression are differentially expressed in uninjured spinal cord tissue of naive mice at Zeitgeber time 1 (ZT1) or ZT12, where ZT0 or ZT12 are times when lights are turned on or off, respectively. However, mice that received moderate, T9 contusive SCI at ZT0 or ZT12 showed similar recovery of locomotion as determined using the ladder walking test and the Basso mouse scale (BMS) over a 6 week post-injury period. Consistent with those findings, terminal histological analysis revealed no significant differences in white matter sparing at the injury epicenter. Therefore, locomotor recovery after thoracic contusive SCI is not affected by the time of day at which the neurotrauma occurred at least when comparing the beginning to the end of the mouse active period.

Disruption of neural tracts descending from the brain to the spinal cord after brain trauma and stroke causes postural and sensorimotor deficits. We previously showed that unilateral lesion to the sensorimotor cortex in rats with completely transected thoracic spinal cord produced asymmetry in hindlimb posture and withdrawal reflexes. Supraspinal signals to hindlimb muscles may be transmitted through the paravertebral chain of sympathetic ganglia that remain intact after the transection. We here demonstrated that prior transection of the spinal cord at the cervical level that was rostrally to segments with preganglionic sympathetic neurons, did not abolish formation of asymmetry in hindlimb posture and musculo-articular resistance to stretch after unilateral brain injury. Thus not the sympathetic system but humoral signals may mediate the effects of brain injury on the lumbar spinal circuits. The asymmetric responses in rats with transected spinal cords were eliminated by bilateral lumbar dorsal rhizotomy after the left-side brain injury, but resistant to deafferentation after the right-side brain lesion. Two mechanisms, one dependent on and one independent of afferent input may account for asymmetric hindlimb motor responses. Resistance to deafferentation may be due to sustained stretch- and effort-unrelated muscle contractions that is often observed in patients with central lesions. Left-right asymmetry is unusual feature of these mechanisms that both are activated by humoral signals. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/488460v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@14770e6org.highwire.dtl.DTLVardef@1452343org.highwire.dtl.DTLVardef@e1aedorg.highwire.dtl.DTLVardef@9c8ea_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundThe entopeduncular nucleus (EP), corresponding to the human globus pallidus internal segment, is a basal ganglia output nucleus, and plays a critical role in motor control. However, the impact of EP damage on skilled motor function and the relationship between its damage in stroke, such as internal capsule hemorrhage (ICH), and motor dysfunction remains unclear. This study aimed to clarify whether EP damage causes motor dysfunction in two disease models. MethodsEP-related motor dysfunction was investigated by inducing localized unilateral EP damage in Long-Evans rats using a stereotactic kainic acid (KA) injection. Motor function was assessed using a single-pellet reaching task pre-injection and on postoperative days 2, 7, 14, 21, and 28. Immunohistochemical staining for NeuN, somatostatin (SST), and parvalbumin was conducted to quantify damage and its correlation with motor outcomes. In addition, unilateral ICH was induced via stereotactic injection of collagenase type IV, which dissolves the vascular basement membrane, into the internal capsule (IC) of Long-Evans rats. Injury sites were classified into the IC, dorsomedial region from the IC, ventral lateral region from the IC, and EP, and their volumes were measured. Measured volumes were analyzed for correlations with motor function assessments. ResultsKA-induced EP damage significantly reduced reaching success rates on postoperative day 2 compared to those in the control group (p<0.05). Immunohistochemical analysis showed that reaching success rates on day 28 positively correlated with the numbers of remaining NeuN-positive and SST-positive neurons (p<0.05). In the ICH experiment, all rats significantly reduced the success rate of the reaching task to 0% on day 2, and the success rate on day 28 correlated positively with the remaining EP volume, but not with total lesion volume. ConclusionsEP damage was strongly associated with motor impairments, highlighting its critical role in motor control and recovery.

During the acute phase of spinal cord injury (SCI), neutrophils infiltrate in large numbers and can exacerbate inflammation, secondary tissue damage, and neurological deficits. L-selectin is a signaling and adhesion receptor that has been shown to facilitate neutrophil recruitment and secondary injury after SCI. During neutrophil activation, L-selectin is typically cleaved or shed from the cell surface and augmenting L-selectin shedding can improve hindlimb recovery and tissue sparing following SCI in male mice. However, it is unclear how endogenous L-selectin shedding regulates neutrophil responses and functional recovery after SCI, particularly when also considering sex as a biological variable. In this study, we investigated the sex-dependent role of endogenous L-selectin shedding in neutrophil function and long-term outcomes in a murine thoracic contusion model of SCI. We found that endogenous L-selectin shedding improves long-term functional recovery and white matter sparing in female, but not male, mice. In addition, we demonstrate that L-selectin shedding alters neutrophil accumulation in a sex-dependent manner. While L-selectin shedding does not mediate neutrophil activation or effector functions, we found that neutrophil clearance is facilitated by L-selectin shedding in female mice alone. These results demonstrate that endogenous L-selectin shedding is a critical and sex-dependent mediator of neutrophil accumulation and clearance, as well as long-term functional outcomes, after SCI.

Spinal cord injury (SCI) results in significant neurological deficits, with no currently available curative therapies. Neural progenitor cell (NPC) transplantation has emerged as a promising approach for neural repair, as graft-derived neurons (GDNs) can integrate into the host spinal cord and support axon regeneration. However, the mechanisms underlying functional recovery remain poorly understood. In this study, we investigate the synaptic integration of NPC-derived neurons into locomotor circuits, the projection patterns of distinct neuronal subtypes, and their potential to modulate motor circuit activity. Using transsynaptic tracing in a mouse thoracic contusion SCI model, we found that NPC-derived neurons form synaptic connections with host locomotor circuits, albeit at low frequencies. Furthermore, we mapped the axon projections of V0C and V2a interneurons, revealing distinct termination patterns within host spinal cord laminae. To assess functional integration, we employed chemogenetic activation of GDNs, which induced muscle activity in a subset of transplanted animals. However, NPC transplantation alone did not significantly improve locomotor recovery, highlighting a key challenge in the field. Our findings suggest that while GDNs can integrate into host circuits and modulate motor activity, synaptic connectivity remains a limiting factor in functional recovery. Future studies should focus on enhancing graft-host connectivity and optimizing transplantation strategies to maximize therapeutic benefits for SCI.