Spinal Cord Transection In Rats Research Articles

Effect of robotic gait training on muscle and bone characteristics in spinal cord transected rats.

Osteoporosis and loss of muscle mass are secondary issues with spinal cord injury. Robotic gait training has provided evidence of increasing bone density and muscle mass, but its effect on bone strength is undetermined. The purpose of this study was to determine the effect of a 6-week robotic locomotion training program on skeletal muscle mass and bone characteristics. Twelve female Sprague-Dawley rats received a mid-thoracic spinal cord transection at 5 days old and at 3 weeks old were assigned to a Control or Trained Group. The Trained Group performed 5-min sessions on the Rat Stepper 5 days a week for 6 weeks with 90% of body weight supported. At the end of the 6 weeks, body mass was obtained and right femurs and four lower extremity muscles were harvested. Femur bone mineral density was measured with DXA and mechanical characteristics of the femur were determined via 3-point bending testing. Independent t-tests, effects sizes and percent differences were computed between the two groups (p < 0.05). The Trained Group had significantly larger normalized femur mass (p = 0.007) and normalized soleus muscle mass (p = 0.033) when compared to the Control Group. There was a medium or large effect size with the Trained Groups' femurs having larger mass, bone mineral density, rupture loads, cortical wall thickness, shaft cross sectional area, soleus mass, normalized gastrocnemius mass, and smaller shaft inner diameters compared to the Control Group. These changes may contribute to decreasing osteoporosis and fracture risk in those with spinal cord injuries.

Vof16-miR-185-5p-GAP43 network improves the outcomes following spinal cord injury via enhancing self-repair and promoting axonal growth.

Self-repair of spinal cord injury (SCI) has been found in humans and experimental animals with partial recovery of neurological functions. However, the regulatory mechanisms underlying the spontaneous locomotion recovery after SCI are elusive. This study was aimed at evaluating the pathological changes in injured spinal cord and exploring the possible mechanism related to the spontaneous recovery. Immunofluorescence staining was performed to detect GAP43 expression in lesion site after spinal cord transection (SCT) in rats. Then RNA sequencing and gene ontology (GO) analysis were employed to predict lncRNA that correlates with GAP43. LncRNA smart-silencing was applied to verify the function of lncRNA vof16 invitro, and knockout rats were used to evaluate its role in neurobehavioral functions after SCT. MicroRNA sequencing, target scan, and RNA22 prediction were performed to further explore the underlying regulatory mechanisms, and miR-185-5p stands out. A miR-185-5p site-regulated relationship with GAP43 and vof16 was determined by luciferase activity analysis. GAP43-silencing, miR-185-5p-mimic/inhibitor, and miR-185-5p knockout rats were also applied to elucidate their effects on spinal cord neurite growth and neurobehavioral function after SCT. We found that a time-dependent increase of GAP43 corresponded with the limited neurological recovery in rats with SCT. CRNA chip and GO analysis revealed lncRNA vof16 was the most functional in targeting GAP43 in SCT rats. Additionally, silencing vof16 suppressed neurite growth and attenuated the motor dysfunction in SCT rats. Luciferase reporter assay showed that miR-185-5p competitively bound the same regulatory region of vof16 and GAP43. Our data indicated miR-185-5p could be a detrimental factor in SCT, and vof16 may function as a ceRNA by competitively binding miR-185-5p to modulate GAP43 in the process of self-recovery after SCT. Our study revealed a novel vof16-miR-185-5p-GAP43 regulatory network in neurological self-repair after SCT and may underlie the potential treatment target for SCI.

Sprouting of afferent and efferent inputs to pelvic organs after spinal cord injury.

Neural plasticity occurs within the central and peripheral nervous systems after spinal cord injury (SCI). Although central alterations have extensively been studied, it is largely unknown whether afferent and efferent fibers in pelvic viscera undergo similar morphological changes. Using a rat spinal cord transection model, we conducted immunohistochemistry to investigate afferent and efferent innervations to the kidney, colon, and bladder. Approximately 3-4 weeks after injury, immunostaining demonstrated that tyrosine hydroxylase (TH)-labeled postganglionic sympathetic fibers and calcitonin gene-related peptide (CGRP)-immunoreactive sensory terminals sprout in the renal pelvis and colon. Morphologically, sprouted afferent or efferent projections showed a disorganized structure. In the bladder, however, denser CGRP-positive primary sensory fibers emerged in rats with SCI, whereas TH-positive sympathetic efferent fibers did not change. Numerous CGRP-positive afferents were observed in the muscle layer and the lamina propria of the bladder following SCI. TH-positive efferent inputs displayed hypertrophy with large diameters, but their innervation patterns were sustained. Collectively, afferent or efferent inputs sprout widely in the pelvic organs after SCI, which may be one of the morphological bases underlying functional adaptation or maladaptation.

Effects of tail nerve electrical stimulation on the activation and plasticity of the lumbar locomotor circuits and the prevention of skeletal muscle atrophy after spinal cord transection in rats.

Severe spinal cord injury results in the loss of neurons in the relatively intact spinal cord below the injury area and skeletal muscle atrophy in the paralyzed limbs. These pathological processes are significant obstacles for motor function reconstruction. We performed tail nerve electrical stimulation (TNES) to activate the motor neural circuits below the injury site of the spinal cord to elucidate the regulatory mechanisms of the excitatory afferent neurons in promoting the reconstruction of locomotor function. Eight days after T10 spinal cord transection in rats, TNES was performed for 7 weeks. Behavioral scores were assessed weekly. Electrophysiological tests and double retrograde tracings were performed at week 8. After 7 weeks of TNES treatment, there was restoration in innervation, the number of stem cells, and mitochondrial metabolism in the rats' hindlimb muscles. Double retrograde tracings of the tail nerve and sciatic nerve further confirmed the presence of synaptic connections between the tail nerve and central pattern generator (CPG) neurons in the lumbar spinal cord, as well as motor neurons innervating the hindlimb muscles. The mechanisms of TNES induced by the stimulation of primary afferent nerve fibers involves efficient activation of the motor neural circuits in the lumbosacral segment, alterations of synaptic plasticity, and the improvement of muscle and nerve regeneration, which provides the structural and functional foundation for the future use of cutting-edge biological treatment strategies to restore voluntary movement of paralyzed hindlimbs.

Multimodal therapy strategy based on a bioactive hydrogel for repair of spinal cord injury

Traumatic spinal cord injury results in permanent and serious neurological impairment, but there is no effective treatment yet. Tissue engineering approaches offer great potential for the treatment of SCI, but spinal cord complexity poses great challenges. In this study, the composite scaffold consists of a hyaluronic acid-based hydrogel, decellularized brain matrix (DBM), and bioactive compounds such as polydeoxyribonucleotide (PDRN), tumor necrosis factor-α/interferon-γ primed mesenchymal stem cell-derived extracellular vesicles (TI-EVs), and human embryonic stem cell-derived neural progenitor cells (NPC). The composite scaffold showed significant effects on regenerative prosses including angiogenesis, anti-inflammation, anti-apoptosis, and neural differentiation. In addition, the composite scaffold (DBM/PDRN/TI-EV/NPC@Gel) induced an effective spinal cord regeneration in a rat spinal cord transection model. Therefore, this multimodal approach using an integrated bioactive scaffold coupled with biochemical cues from PDRN and TI-EVs could be used as an advanced tissue engineering platform for spinal cord regeneration.

Adult spinal cord tissue transplantation combined with local tacrolimus sustained‐release collagen hydrogel promotes complete spinal cord injury repair

The strategy of replacing a completely damaged spinal cord with allogenic adult spinal cord tissues (aSCs) can potentially repair complete spinal cord injury (SCI) in combination with immunosuppressive drugs, such as tacrolimus (Tac), which suppress transplant rejection and improve graft survival. However, daily systemic administration of immunosuppressive agents may cause harsh side effects. Herein, a localized, sustained Tac-release collagen hydrogel (Col/Tac) was developed to maximize the immune regulatory efficacy but minimize the side effects of Tac after aSC transplantation in complete SCI recipients. Thoracic aSCs of rat donors were transplanted into the complete thoracic spinal cord transection rat recipients, after which Col/Tac hydrogel was implanted. The Tac-encapsulated collagen hydrogel exhibited suitable mechanical properties and long-term sustained Tac release behaviour. After Col/Tac hydrogel implantation in SCI rats with aSC transplantation, the recipients' survival rate significantly improved and the side effects on tissues were reduced compared with those with conventional Tac medication. Moreover, treatment with the Col/Tac hydrogel exhibited similarly reduced immune rejection levels by regulating immune responses and promoted neurogenesis compared to daily Tac injections, and thus improved functional restoration. Localized delivery of immunosuppressive agents by the Col/Tac hydrogel may be a promising strategy for overcoming immune rejection of transplants, with significant potential for clinical application in the future.

Forced Remyelination Promotes Axon Regeneration in a Rat Model of Spinal Cord Injury

Spinal cord injuries result in the loss of motor and sensory functions controlled by neurons located at the site of the lesion and below. We hypothesized that experimentally enhanced remyelination supports axon preservation and/or growth in the total spinal cord transection in rats. Multifocal demyelination was induced by injection of ethidium bromide (EB), either at the time of transection or twice during transection and at 5 days post-injury. We demonstrated that the number of oligodendrocyte progenitor cells (OPCs) significantly increased 14 days after demyelination. Most OPCs differentiated into mature oligodendrocytes by 60-90 dpi in double-EB-injected rats; however, most axons were remyelinated by Schwann cells. A significant number of axons passed the injury epicenter and entered the distant segments of the spinal cord in the double-EB-injected rats. Moreover, some serotoninergic fibers, not detected in control animals, grew caudally through the injury site. Behavioral tests performed at 60-90 dpi revealed significant improvement in locomotor function recovery in double-EB-injected rats, which was impaired by the blockade of serotonin receptors, confirming the important role of restored serotonergic fibers in functional recovery. Our findings indicate that enhanced remyelination per se, without substantial inhibition of glial scar formation, is an important component of spinal cord injury regeneration.

Molecular Identification of Pro-Excitogenic Receptor and Channel Phenotypes of the Deafferented Lumbar Motoneurons in the Early Phase after SCT in Rats.

Spasticity impacts the quality of life of patients suffering spinal cord injury and impedes the recovery of locomotion. At the cellular level, spasticity is considered to be primarily caused by the hyperexcitability of spinal α-motoneurons (MNs) within the spinal stretch reflex circuit. Here, we hypothesized that after a complete spinal cord transection in rats, fast adaptive molecular responses of lumbar MNs develop in return for the loss of inputs. We assumed that early loss of glutamatergic afferents changes the expression of glutamatergic AMPA and NMDA receptor subunits, which may be the forerunners of the developing spasticity of hindlimb muscles. To better understand its molecular underpinnings, concomitant expression of GABA and Glycinergic receptors and serotoninergic and noradrenergic receptors, which regulate the persistent inward currents crucial for sustained discharges in MNs, were examined together with voltage-gated ion channels and cation-chloride cotransporters. Using quantitative real-time PCR, we showed in the tracer-identified MNs innervating extensor and flexor muscles of the ankle joint multiple increases in transcripts coding for AMPAR and 5-HTR subunits, along with a profound decrease in GABAAR, GlyR subunits, and KCC2. Our study demonstrated that both MNs groups similarly adapt to a more excitable state, which may increase the occurrence of extensor and flexor muscle spasms.

Stimulation by Exosomes from Hypoxia Preconditioned Human Umbilical Vein Endothelial Cells Facilitates Mesenchymal Stem Cells Angiogenic Function for Spinal Cord Repair.

Revascularization treatment is a critical measure for tissue engineering therapies like spinal cord repair. As multipotent stem cells, mesenchymal stem cells (MSCs) have proven to regulate the lesion microenvironment through feedback to the microenvironment signals. The angiogenic capacities of MSCs have been reported to be facilitated by vein endothelial cells in the niche. As emerging evidence demonstrated the roles of exosomes in cell-cell and cell-microenvironment communications, to cope with the ischemia complication for treatment of traumatic spinal cord injury, the study extracts the microenvironment factors to stimulate angiogenic MSCs through using exosomes (EX) derived from hypoxic preconditioned (HPC) human umbilical vein endothelial cells (HUVEC). The HPC treatment with a hypoxia time segment of only 15 min efficiently enhanced the function of EX in facilitating MSCs angiogenesis activity. MSCs stimulated by HPC-EX showed significant tube formation within 2 h, and the in vivo transplantation of the stimulated MSCs elicited effective nerve tissue repair after rat spinal cord transection, which could be attributed to the pro-angiogenic and anti-inflammatory impacts of the MSCs. Through the simulation of MSCs using HPC-tailored HUVEC exosomes, the results proposed an efficient angiogenic nerve tissue repair strategy for spinal cord injury treatment and could provide inspiration for therapies based on stem cells and exosomes.

Expression profiles of protein markers regulating the mitochondrial lifecycle in skeletal muscle of acute spinal cord transected rats

Spinal cord transection (ST) inactivates the neuromuscular complex and triggers progressive muscular atrophy of the affected skeletal muscles. Disruption in neural input also leads to a reduction in mitochondrial volume. The mitochondria lifecycle can be generally divided into processes of biogenesis, expression of OXPHOS proteins, fusion, fission, and mito/autophagy; specifically, the balance between biogenesis vs. mitophagy dictates the rate of formation and destruction, respectively. Here, we examined the expression profiles of the markers associated with the mitochondrial lifecycle up to one month following ST in phenotypically slow (soleus; SOL) and mixed (plantaris; PLT) skeletal muscle. We hypothesized that ST would induce a reduction in mitochondrial DNA (mtDNA) copy number, decrease expression in markers associated with biogenesis, and increased proteins that regulate mito/autophagy. Adult female Sprague Dawley rats were randomly divided into control (CON; n=6), 1 day (1dST; n=5), 8 day (8dST; n=8), and 28 day post‐ST (28dST; n=8). Compared to CON relative SOL and PLT muscle masses (absolute mass / body mass) were 98, 65, and 62% and 85, 60, and 73% at 1, 8 and 28d post‐ST, respectively (p&lt;0.05). Compared to CON, no significant changes were observed in mtDNA copy number in ST muscles following amplification using standard end‐point PCR protocols for mtDNA (ND1, COX1, or ATP6) and nuclear (β2M) genes (p&gt;0.05). Total protein was isolated from SOL and PLT, separated using standard SDS‐PAGE protocol, and probed for markers associated with various stages of the mitochondrial lifecycle using standard western blot protocols. Upstream markers known to influence mitochondrial lifecycle signaling (Rev‐Erbα, AMPK) showed a varied temporal and muscle‐specific response: in 1dST SOL muscles, Rev‐Erbα increased 74% from CON then decreased 43% at 28dST, whereas 8dST PLT muscle exhibited a 44% increase (p&lt;0.05). In 8dST SOL and PLT muscles, the pAMPK:AMPK ratio was elevated 366 and 85% from CON, respectively (p&lt;0.05). Markers for mitochondrial biogenesis (PGC‐1α, NRF1, NRF2) in SOL and PLT muscles were largely unchanged, although a ~50% decrease in Tfam protein expression was observed at each ST time point compared to CON (p&lt;0.05). Mitofusion2, a marker for mitochondrial fusion, was unchanged (p&gt;0.05). However, for markers detecting mitochondrial fission (MFF, Drp1, Fis1), we observed a ~60% increase in MFF expression in 1dST SOL and PLT muscles (p&lt;0.05). As expected markers for mitophagy (Parkin, PINK, p62, LC3 I/II) were significantly elevated: Parkin increased ~70% at 28dST (p&lt;0.05), whereas LC3 I/II increased 65% and 112% at 1dST in in SOL and PLT muscles, respectively (p&lt;0.05), and remained significantly elevated in PLT muscles at 8dST (27%) and 28d (37%) (p&lt;0.05). Taken together, these data suggest that mitochondrial volume and expression of markers regulating mitochondrial biogenesis and fusion are not influenced by acute ST despite a decrease in muscle mass. However, selective markers for fission and mitophagy are significantly elevated in both SOL and PLT muscles, suggesting that mitochondrial destruction mechanism are activated acutely following ST injury. Lastly, selective proteins of the OXPHOS protein complex significantly increased in 1dST SOL and PLT muscles suggesting possible activity‐dependent regulation of mitochondrial gene expression.

Three-dimensional bioprinting sodium alginate/gelatin scaffold combined with neural stem cells and oligodendrocytes markedly promoting nerve regeneration after spinal cord injury.

Accumulating research has indicated that the transplantation of combined stem cells and scaffolds is an effective method for spinal cord injury (SCI). The development of three-dimensional (3D) bioprinting technology can make the 3D scaffolds combined with cells more accurate and effective for SCI treatment. However, unmyelinated newborn nerve fibers have no nerve signaling conduction, hampering recovery of motor function. In this study, we designed and printed a type of sodium alginate/gelatin scaffold loaded with neural stem cells and oligodendrocytes, which were involved in the formation of the myelin sheaths of neural cell axons. In order to observe the effectiveness of this 3D bioprinting scaffold, we transplanted it into the completely transected rat spinal cord, and then immunofluorescence staining, hematoxylin–eosin staining and behavioral assessment were performed. The results showed that this 3D bioprinting scaffold markedly improved the hindlimb motor function and promoted nerve regeneration. These findings suggested that this novel 3D bioprinting scaffold was a good carrier for cells transplantation, thereby enhancing spinal cord repair following injury.

Cervical Spinal Cord Transection (SCT) Increases Gastric Compliance in Anesthetized Rats

Abstract Spinal cord injury (SCI) can markedly alter the autonomic nervous system’s functions. It immediately causes autonomic and somatic hypo- or arreflexia, a state known as spinal shock. SCI determines the enhancement of two gastric reflexes that appear in normal conditions: the receptive and adaptative reflexes. Furthermore, this study also tried to evaluate the causes that led to such modifications: either vagal control, nitric oxide (NO)-pathways or intestine-intestinal reflexes. Male Wistar rats (N=25) were subjected to laminectomy (Sham group) or laminectomy + complete Spinal Cord Transection (SCT), between C7 and T1 vertebrae under anesthesia (SCT group). Before the surgery, the rats had water and food ad libitum; after the surgery, rats were fasted for 24 hours, still having access to water. The next day, all animals were subjected to a catheterism of their right carotid artery, a tracheostomy and a flexible balloon introduction down to the stomach’s fundix region, also under anesthesia. Rats were connected to a Power-Lab® system, via a pressure transducer (in order to measure the arterial pressure – MAP - and the heart rate - HR), and to a modified Plethysmometer (in order to measure the fundical gastric volume - GV variation). Vagal control was studied via cervical vagotomy; NO-mediation - via NO synthesis inhibitors (L-NAME); intestinointestinal reflexes - by using laxatives (Lactulone). Statistical analysis revealed, in some cases, that GV is significantly (p&lt;0.05) augmented in SCT rats (2,4±0,09; 3,1±0,05; 3,7±0,06 ml) than in Sham (2,2±0,12; 2,9±0,12; 3,3±0,15 ml). Nevertheless, MAP and HR, were subsequently lower (p&lt;0.05) in SCT (63,5±21,1mm Hg; 227,1±25,2 BPM) than in the control group (88,2±14,4mm Hg; 427,0±19,2 BPM). In summary, the increase of GV in SCT, seems to involve vagal pathways and/or NO-mediation, but still not neglecting the colic-gastric reflexes.

Pyrrole Plasma Polymer-Coated Fibrillar Scaffold Implant: Pilot Study in Rat Spinal Cord Transection with MRI.

Despite extensive research on spinal cord injury (SCI) therapies for the recovery of motor, sensory and autonomic function, currently there are no effective treatments to completely restore tissue structure and function. In this work, a polylactic acid (PLA) fibrillar scaffold coated with pyrrole plasma polymer doped with iodine (pPPy/I), was studied as therapeutic strategy in a SCI transection model. Magnetic resonance imaging (MRI) was used to evaluate tissue response to the implant. Behavioral analysis using the BBB open-field testing was conducted to evaluate functional response. MRI analysis showed the SCI model completely disrupted tissue continuity, and diffusion indices were altered at the injury site. The animals had completely paralyzed hindlimbs and bladder control loss after injury. After 8 weeks of treatment, in contrast to control and PLA-implanted animals, PLA+pPPy/I-implanted animal had regained bladder control autonomy and frequent to consistent weight supported plantar steps and occasional coordination between forelimbs and hindlimbs. These results suggest fibrillar scaffolds coated with pPPy/I constitute a promising therapy for SCI.

Rapid and effective treatment of traumatic spinal cord injury using stem cell derived exosomes

Traumatic spinal cord injury is a fatal acute event without effective clinical therapies. Following the trauma, immediate neural protection and microenvironment mitigation are vitally important for nerve tissue repair, where stem cell-based therapies could be eclipsed by the deficiency of cells due to the hostile microenvironment as well as the transport and preservation processes. Effective emergency strategies are required to be convenient, biocompatible, and stable. Herein, we assess an emergency cell-free treatment using mesenchymal stem cell-derived exosomes, which have proven capable of comprehensive mitigation of the inhibitory lesion microenvironment. The clinically validated fibrin glue is utilized to encapsulate the exosomes and in-situ gelates in transected rat spinal cords to provide a substrate for exosome delivery as well as nerve tissue growth. The emergency treatment alleviates the inflammatory and oxidative microenvironment, inducing effective nerve tissue repair and functional recovery. The therapy presents a promising strategy for effective emergency treatment of central nervous system trauma.

Cell-adaptable dynamic hydrogel reinforced with stem cells improves the functional repair of spinal cord injury by alleviating neuroinflammation

Spinal cord injury (SCI) is one of the most challenging clinical issues. It is characterized by the disruption of neural circuitry and connectivity, resulting in neurological disability. Adipose-derived stem cells (ADSCs) serve as a promising source of therapeutic cells for SCI treatment. However, the therapeutic outcomes of direct ADSCs transplantation are limited in the presence of an inflammatory microenvironment. Herein, a cell-adaptable neurogenic (CaNeu) hydrogel was developed as a delivery vehicle for ADSCs to promote neuronal regeneration after SCI. The dynamic network of CaNeu hydrogel loaded with ADSCs provides a cell-infiltratable matrix that enhances axonal growth and eventually leads to improved motor evoked potential, hindlimb strength, and coordination of complete spinal cord transection in rats. Furthermore, the CaNeu hydrogel also establishes an anti-inflammatory microenvironment by inducing a shift in the polarization of the recruited macrophages toward the pro-regeneration (M2) phenotype. Our study showed that the CaNeu-hydrogel‒mediated ADSCs delivery resulted in significantly suppressed neuroinflammation and apoptosis, and that this phenomenon involved the PI3K/Akt signaling pathway. Our findings indicate that the CaNeu hydrogel is a valuable delivery vehicle to assist stem cell therapy for SCI, providing a promising strategy for central nervous system diseases.

The corticospinal tract structure of collagen/silk fibroin scaffold implants using 3D printing promotes functional recovery after complete spinal cord transection in rats

No effective treatment has been established for nerve dysfunction caused by spinal cord injury (SCI). Orderly axonal growth at the site of spinal cord transection and creation of an appropriate biological microenvironment are important for functional recovery. To axially guiding axonal growth, designing a collagen/silk fibroin scaffold fabricated with 3D printing technology (3D-C/SF) emulated the corticospinal tract. The normal collagen/silk fibroin scaffold with freeze-drying technology (C/SF) or 3D-C/SF scaffold were implanted into rats with completely transected SCI to evaluate its effect on nerve repair during an 8-week observation period. Electrophysiological analysis and locomotor performance showed that the 3D-C/SF implants contributed to significant improvements in the neurogolical function of rats compared to C/SF group. By magnetic resonance imaging, 3D-C/SF implants promoted a striking degree of axonal regeneration and connection between the proximal and distal SCI sites. Compared with C/SF group, rats with 3D-C/SF scaffold exhibited fewer lesions and disordered structures in histological analysis and more GAP43-positive profiles at the lesion site. The above results indicated that the corticospinal tract structure of 3D printing collagen/silk fibroin scaffold improved axonal regeneration and promoted orderly connections within the neural network, which could provided a promising and innovative approach for tissue repair after SCI.

Intraspinal Administration of Netrin-1 Promotes Locomotor Recovery after Complete Spinal Cord Transection.

Complete spinal cord lesions interrupt the connection of all axonal projections with their neuronal targets below and above the lesion site. In particular, the interruption of connections with the neurons at lumbar segments after thoracic injuries impairs voluntary body control below the injury. The failure of spontaneous regrowth of transected axons across the lesion prevents the reconnection and reinnervation of the neuronal targets. At present, the only treatment in humans that has proven to promote some degree of locomotor recovery is physical therapy. The success of these strategies, however, depends greatly on the type of lesion and the level of preservation of neural tissue in the spinal cord after injury. That is the reason it is key to design strategies to promote axonal regrowth and neuronal reconnection. Here, we test the use of a developmental axon guidance molecule as a biological agent to promote axonal regrowth, axonal reconnection, and recovery of locomotor activity after spinal cord injury (SCI). This molecule, netrin-1, guides the growth of the corticospinal tract (CST) during the development of the central nervous system. To assess the potential of this molecule, we used a model of complete spinal cord transection in rats, at thoracic level 10-11. We show that in situ delivery of netrin-1 at the epicenter of the lesion: (1) promotes regrowth of CST through the lesion and prevents CST dieback, (2) promotes synaptic reconnection of regenerated motor and sensory axons, and (3) preserves the polymerization of the neurofilaments in the sciatic nerve axons. These anatomical findings correlate with a significant recovery of locomotor function. Our work identifies netrin-1 as a biological agent with the capacity to promote the functional repair and recovery of locomotor function after SCI. These findings support the use of netrin-1 as a therapeutic intervention to be tested in humans.

Neonatal Spinal Cord Transection Decreases Hindlimb Weight-Bearing and Affects Formation of Achilles and Tail Tendons.

Mechanical loading may be required for proper tendon formation. However, it is not well understood how tendon formation is impacted by the development of weight-bearing locomotor activity in the neonate. This study assessed tendon mechanical properties, and concomitant changes in weight-bearing locomotion, in neonatal rats subjected to a low thoracic spinal cord transection or a sham surgery at postnatal day (P)1. On P10, spontaneous locomotion was evaluated in spinal cord transected and sham controls to determine impacts on weight-bearing hindlimb movement. The mechanical properties of P10 Achilles tendons (ATs), as representative energy-storing, weight-bearing tendons, and tail tendons (TTs), as representative positional, non-weight-bearing tendons were evaluated. Non- and partial weight-bearing hindlimb activity decreased in spinal cord transected rats compared to sham controls. No spinal cord transected rats showed full weight-bearing locomotion. ATs from spinal cord transected rats had increased elastic modulus, while cross-sectional area trended lower compared to sham rats. TTs from spinal cord transected rats had higher stiffness and cross-sectional area. Collagen structure of ATs and TTs did not appear impacted by surgery condition, and no significant differences were detected in the collagen crimp pattern. Our findings suggest that mechanical loading from weight-bearing locomotor activity during development regulates neonatal AT lateral expansion and maintains tendon compliance, and that TTs may be differentially regulated. The onset and gradual increase of weight-bearing movement in the neonate may provide the mechanical loading needed to direct functional postnatal tendon formation.

MicroRNA339 Targeting PDXK Improves Motor Dysfunction and Promotes Neurite Growth in the Remote Cortex Subjected to Spinal Cord Transection.

Spinal cord injury (SCI) is a fatal disease that can cause severe disability. Cortical reorganization subserved the recovery of spontaneous function after SCI, although the potential molecular mechanism in this remote control is largely unknown. Therefore, using proteomics analysis, RNA interference/overexpression, and CRISPR/Cas9 in vivo and in vitro, we analyzed how the molecular network functions in neurological improvement, especially in the recovery of motor function after spinal cord transection (SCT) via the remote regulation of cerebral cortex. We discovered that the overexpression of pyridoxal kinase (PDXK) in the motor cortex enhanced neuronal growth and survival and improved locomotor function in the hindlimb. In addition, PDXK was confirmed as a target of miR-339 but not miR-124. MiR-339 knockout (KO) significantly increased the neurite outgrowth and decreased cell apoptosis in cortical neurons. Moreover, miR-339 KO rats exhibited functional recovery indicated by improved Basso, Beattie, and Bresnehan (BBB) score. Furthermore, bioinformatics prediction showed that PDXK was associated with GAP43, a crucial molecule related to neurite growth and functional improvement. The current research therefore confirmed that miR-339 targeting PDXK facilitated neurological recovery in the motor cortex of SCT rats, and the underlying mechanism was associated with regulating GAP43 in the remote cortex of rats subjected to SCT. These findings may uncover a new understanding of remoting cortex control following SCI and provide a new therapeutic strategy for the recovery of SCI in future clinical trials.

CacyBP/SIP in the rat spinal cord in norm and after transection – Influence on the phosphorylation state of ERK1/2 and p38 kinases

IntroductionCacyBP/SIP is a multifunctional protein present in various mammalian tissues, among them in brain. Recently, it has been shown that CacyBP/SIP exhibits phosphatase activity towards ERK1/2 and p38 kinases. ObjectivesThe aim of our study was to analyze the localization and level of CacyBP/SIP and its substrates, phosphorylated ERK1/2 (p-ERK1/2) and phosphorylated p38 (p-p38) kinases, in an intact and transected rat spinal cord. MethodsTo achieve our goals we have performed Western blot/densitometric analysis and double immunofluorescence staining using rat spinal cord tissue, intact and after total transection at different time points. ResultsWe have observed a decrease in the level of CacyBP/SIP and an increase in the level of p-ERK1/2 and of p-p38 in fragments of the spinal cord excised 1 and 3 months after transection. Moreover, immunofluorescence staining has shown that CacyBP/SIP, p-ERK1/2 or p-p38 co-localized with a neuronal marker, NeuN, and with an oligodendrocyte marker, Olig2. ConclusionThe inverse correlation between CacyBP/SIP and p-ERK1/2 or p-p38 levels suggests that CacyBP/SIP may dephosphorylate p-ERK1/2 and p-p38 kinases and be involved in neural plasticity following spinal cord injury.