Rodent Spinal Cord Research Articles

Astrocytic Ryk signaling coordinates scarring and wound healing after spinal cord injury.

Wound healing after spinal cord injury involves highly coordinated interactions among multiple cell types, which is poorly understood. Astrocytes play a central role in creating a border against the non-neural lesion core. To do so, astrocytes undergo dramatic morphological changes by first thickening the processes and then elongating and overlap them. We show here show that the expression of a cell-surface receptor, Ryk, is induced in astrocytes after injury in both rodent and human spinal cord. Astrocyte-specific knockout of Ryk dramatically elongated the reactive astrocytes and accelerated the formation of the border and reduced the size of the scar. Astrocyte-specific knockout of Ryk also accelerated the injury responses of multiple cell types, including the resolution of neuroinflammation. Single cell transcriptomics analyses revealed a broad range of changes cell signaling among astrocytes, microglia, fibroblasts, endothelial cell, etc, after astrocyte-specific Ryk knockout, suggesting that Ryk not only regulates the injury response of astrocytes but may also regulate signals which coordinate the responses of multiple cell types. The elongation is mediated by NrCAM, a cell adhesion molecule induced by astrocyte-specific conditional knockout of Ryk after spinal cord injury. Our findings suggest a promising therapeutic target to accelerate wound healing and promote neuronal survival and enhance functional recovery.

Implantation of Optoelectronic Devices in the Rodent Spinal Cord.

Neuromodulation can provide diagnostic, modulatory, and therapeutic applications. While extensive work has been conducted in the brain, modulation of the spinal cord remains relatively unexplored. The inherently delicate and mobile spinal cord tissue imposes constraints that make the precise implantation of neural probes challenging. Despite recent advances in neuromodulation devices, particularly flexible bioelectronics, opportunities to expand their use in the spinal cord have been limited by the surgical complexities of device implantation. Here, we provide a series of surgical protocols tailored specifically for the implantation of a custom-made optoelectronic device that interfaces with the spinal cord in rodents. The steps to place and anchor an optical shank on a specific segment of the spinal cord via two different surgical implantation methods are detailed here. These methods are optimized for a diverse range of devices and applications, which may or may not require direct contact with the spinal cord for optical stimulation. To elucidate the methodology, the vertebral anatomy is referenced first to identify prominent landmarks before making a skin incision. The surgical steps to secure an optical shank over the cervical spine in rodents are demonstrated. Procedures are then outlined for securing the optoelectronic device connected to the optical shank in a subcutaneous space away from the spinal cord, minimizing unnecessary direct contact. Behavioral studies comparing animals receiving the implants to those undergoing sham surgeries indicate that the optical shanks did not adversely affect hindlimb or forelimb function seven days post-implantation. The present work broadens the neuromodulation toolkit for use in future studies aimed at investigating various spinal cord interventions.

Immature functional development of lumbar locomotor networks in adult Irf8-/- mice.

To date, research on the role of the brainstem and spinal cord in motor behavior has relied on in vitro preparations of the neonatal rodent spinal cord, with or without the brainstem; their spatial and temporal scope are subject to technical limitations imposed by low oxygen tension in deep tissues. Therefore, we created an arterially perfused in situ preparation that allowed us to investigate functional interactions in the CNS from the neonatal to adult period. Decerebrated rodents were kept alive via total artificial cardiopulmonary bypass for extracorporeal circulation; the plasma oxygen and ion components needed for survival were supplied through the blood vessels. Interferon regulatory factor 8 (IRF8) is a transcription factor that promotes myeloid cell development and stimulates innate immune responses. In the brain, IRF8 is expressed only in microglia and directs the expression of many genes that serve microglial functions. Recent evidence indicates that IRF8 affects behavior and modulates Alzheimer's disease progression in a mouse model. However, whether this immune deficiency arising from the absence of IRF8 influences the development of the neuronal network in the spinal cord is unknown. We applied the above methodology to mice of all ages and electrophysiologically explored whether the absence of IRF8 influences the development of lumbar central pattern generator (CPG) networks. In mice of all ages, bilateral neuronal discharges by the normal CPG networks activated by the modulated sympathetic tone via descending pathways at high flow rates became organized into discharge episodes punctuated by periods of quiescence. Similar discharge episodes were generated by the adult CPG networks (≥P14 days) activated by drug application. However, discharge episodes elicited by activating the neonatal-juvenile CPG networks (<P14 days) occurred alternately on the left and right sides. Interestingly, discharge episodes elicited by the CPG networks in adult IRF8 knockout mice (P11-12 weeks) consisted of those elicited by the CPG networks of both periods. Thus, it was suggested that growing up with immunodeficiency due to loss of IRF8 might interfere with the normal development of functions exerted by the lumbar CPG network because IRF8 plays a role in the normal development of the lumbar CPG network.

Astrocyte PERK and IRE1 Signaling Contributes to Morphine Tolerance and Hyperalgesia through Upregulation of Lipocalin-2 and NLRP3 Inflammasome in the Rodent Spinal Cord.

Endoplasmic reticulum stress plays a crucial role in the pathogenesis of neuroinflammation and chronic pain. This study hypothesized that PRKR-like endoplasmic reticulum kinase (PERK) and inositol-requiring enzyme type 1 (IRE1) regulate lipocalin-2 (LCN2) and Nod-like receptor family pyrin domain containing 3 (NLRP3) expression in astrocytes, thereby contributing to morphine tolerance and hyperalgesia. The study was performed in Sprague-Dawley rats and C57/Bl6 mice of both sexes. The expression of LCN2 and NLRP3 was assessed by Western blotting. The tail-flick, von Frey, and Hargreaves tests were used to evaluate nociceptive behaviors. Chromatin immunoprecipitation was conducted to analyze the binding of activating transcription factor 4 (ATF4) to the promoters of LCN2 and TXNIP. Whole-cell patch-clamp recordings were used to evaluate neuronal excitability. Pharmacologic inhibition of PERK and IRE1 attenuated the development of morphine tolerance and hyperalgesia in male (tail latency on day 7, 8.0 ± 1.13 s in the morphine + GSK2656157 [10 μg] group vs. 5.8 ± 0.65 s in the morphine group; P = 0.04; n = 6 rats/group) and female (tail latency on day 7, 6.0 ± 0.84 s in the morphine + GSK2656157 [10 μg] group vs. 3.1 ± 1.09 s in the morphine group; P = 0.0005; n = 6 rats/group) rats. Activation of PERK and IRE1 upregulated expression of LCN2 and NLRP3 in vivo and in vitro. Chromatin immunoprecipitation analysis showed that ATF4 directly bound to the promoters of the LCN2 and TXNIP. Lipocalin-2 induced neuronal hyperexcitability in the spinal cord and dorsal root ganglia via melanocortin-4 receptor. Astrocyte endoplasmic reticulum stress sensors PERK and IRE1 facilitated morphine tolerance and hyperalgesia through upregulation of LCN2 and NLRP3 in the spinal cord.

Low-Intensity Pulsed Ultrasound Neuromodulation of a Rodent's Spinal Cord Suppresses Motor Evoked Potentials.

Here we investigate the ability of low-intensity ultrasound (LIUS) applied to the spinal cord to modulate the transmission of motor signals. Male adult Sprague-Dawley rats (n = 10, 250-300 g, 15 weeks old) were used in this study. Anesthesia was initially induced with 2% isoflurane carried by oxygen at 4 L/min via a nose cone. Cranial, upper extremity, and lower extremity electrodes were placed. A thoracic laminectomy was performed to expose the spinal cord at the T11 and T12 vertebral levels. A LIUS transducer was coupled to the exposed spinal cord, and motor evoked potentials (MEPs) were acquired each minute for either 5- or 10-minutes of sonication. Following the sonication period, the ultrasound was turned off and post-sonication MEPs were acquired for an additional 5 minutes. Hindlimb MEP amplitude significantly decreased during sonication in both the 5- (p < 0.001) and 10-min (p = 0.004) cohorts with a corresponding gradual recovery to baseline. Forelimb MEP amplitude did not demonstrate any statistically significant changes during sonication in either the 5- (p = 0.46) or 10-min (p = 0.80) trials. LIUS applied to the spinal cord suppresses MEP signals caudal to the site of sonication, with recovery of MEPs to baseline after sonication. LIUS can suppress motor signals in the spinal cord and may be useful in treating movement disorders driven by excessive excitation of spinal neurons.

P.145 Uncovering differences in oligodendrogenesis between human and rodent spinal cord stem cells

Background: Spinal cord regeneration in pre-clinical rodent studies is feasible by promoting oligodendrocyte regrowth, which is necessary for axon myelination. It is uncertain whether human neural stem/progenitor cells (NSPCs) are capable of differentiation into oligodendrocytes, similar to rat. In this study, we compare the functional and transcriptional features of primary spinal cord NSPCs from adult humans and rats. Methods: Oligodendrogenesis between human &amp; rat NSPCs from adult donors and rats was cultured using the Neurosphere assay. NSPCs were exposed to 1% FBS to trigger differentiation &amp; PDGF-AA to promote oligodendrocyte formation. Immunocytochemistry &amp; RNA sequencing compared transcriptomes and analyzing differentially expressed genes. Results: Human NSPCs showed a reduced potential for oligodendrocyte generation compared to rat NSPCs (0.013±0.01% and 0.029±0.01% O4+ after one and two weeks in humans; 4.9±0.4% and 6.3±0.6% O4+ after one and two weeks in rats). PDGF-AA treatment at 40 ng/µL for one week was able to effectively promote oligodendrocyte differentiation in rat NSPCs, but had a minimal effect on human NSPCs (8.5±1.4 fold increase in O4+). OLIG1/2, SOX10, and CNP were enriched in rat NSPCs. Conclusions: We compared oligodendrogenesis potential between human and rat NSPCs and found significantly lower capacity in human NSPCs, possibly hindering successful myelinating techniques.

Differential effects of NOX2 and NOX4 inhibition after rodent spinal cord injury.

Reactive oxygen species (ROS) are a contributing factor to impaired function and pathology after spinal cord injury (SCI). The NADPH oxidase (NOX) enzyme is a key source of ROS; there are several NOX family members, including NOX2 and NOX4, that may play a role in ROS production after SCI. Previously, we showed that a temporary inhibition of NOX2 by intrathecal administration of gp91ds-tat immediately after injury improved recovery in a mouse SCI model. However, chronic inflammation was not affected by this single acute treatment, and other NOX family members were not assessed. Therefore, we aimed to explore the effect of genetic knockout (KO) of NOX2 or acute inhibition of NOX4 with GKT137831. A moderate SCI contusion injury was performed in 3 month old NOX2 KO and wild-type (WT) mice, who received no treatment or GKT137831/vehicle 30 minutes post-injury. Motor function was assessed using the Basso Mouse Scale (BMS), followed by evaluation of inflammation and oxidative stress markers. NOX2 KO mice, but not GKT137831 treated mice, demonstrated significantly improved BMS scores at 7, 14, and 28 days post injury (DPI) in comparison to WT mice. However, both NOX2 KO and GKT137831 significantly reduced ROS production and oxidative stress markers. Furthermore, a shift in microglial activation toward a more neuroprotective, anti-inflammatory state was observed in KO mice at 7 DPI and a reduction of microglial markers at 28 days. While acute alterations in inflammation were noted with GKT137831 administration, this was not sustained through 28 days. In vitro analysis also showed that while GKT137831 reduced ROS production by microglia, it did not translate to changes in pro-inflammatory marker expression within these cells. These data demonstrate that NOX2 and NOX4 play a role in post-injury ROS, but a single dose of NOX4 inhibitor fails to enhance long-term recovery.

Validation of Spinal Cord Injury Sensory Deficit Model with Head-Fixed Rat

Background/Objective: Numerous studies have reported restoring motor function through spinal cord stimulation in a rodent spinal cord injury model; however, replicating sensory information through spinal cord stimulation has not been thoroughly tested.We have previously trained rats to detect artificial sensations which lead to the question if this can be generated in a spinal cord injured rat. Our study begins to address this question by first validating a spinal cord injury model to test. Our hypothesis is if a decrease in the percent correct response compared to pre-procedure is observed, then this will verify sensory loss in this dorsal column lesion model of spinal cord injury.&#x0D; Methods: We use sensory detection in a head-fixed rat to verify a spinal cord injury sensory deficit model. We begin by placing a water-deprived rat into a custom-made head-fix set up where two water spouts are placed in front of the rat. Vibration stimuli is applied to the hind paw via a vibration motor set at 200 hz ± 50 hz. The rat is trained to associate stimulus with left spout and no stimulus with right spout. Correct percent response is recorded and once rat demonstrates detection, the rat undergoes a dorsal column hemi-section procedure. Post procedure, the rat will be placed back in the head-fix set up to repeat the behavioral experiment.&#x0D; Results: Thus far, only habituation training has been completed with the rat. The following steps include detection training, dorsal column hemi section, and re doing the detection training to measure a difference in correct response rate.&#x0D; Conclusion and Potential Impact:This is a crucial step for spinal cord injury research as this confirms sensory loss and will allow further testing to restore sensation. After validation, we can begin testing to restore sensation using stimulation of the spinal cord.

Single channel properties of pannexin-1 and connexin-43 hemichannels and P2X7 receptors in astrocytes cultured from rodent spinal cords.

Astrocytes express surface channels involved in purinergic signaling. Among these channels, pannexin-1 (Px1) and connexin-43 (Cx43) hemichannels (HCs) release ATP that acts directly, or through its derivatives, on neurons and glia via purinergic receptors. Although HCs are functional, that is, open and close under physiological and pathological conditions, single channel properties of Px1 HCs in astrocytes have not been defined. Here, we developed a dual voltage clamp technique in HeLa cells expressing human Px1-YFP, and then applied this system to rodent spinal astrocytes to compare their single channel properties with other surface channels, that is, Cx43 HCs and P2X7 receptors (P2X7Rs). Channels were recorded in cell attached patches and evoked with ramp cycles applied through another pipette in whole cell voltage clamp. The mean unitary conductances of Px1 HCs were comparable in HeLa Px1-YFP cells and spinal astrocytes, ~42 and ~48 pS, respectively. Based on their unitary conductance, voltage-dependence, and unitary activity after pharmacological and gene silencing, Px1 HCs in astrocytes could be distinguished from Cx43 HCs and P2X7Rs. Channel activity of Px1 HCs and P2X7Rs was greater than that of Cx43 HCs in control astrocytes during ramps. Unitary activity of Px1 HCs was decreased and that of Cx43 HCs and P2X7Rs increased in astrocytes treated with fibroblast growth factor 1 (FGF-1). In summary, we resolved single channel properties of three different surface channels involved in purinergic signaling in spinal astrocytes, which were differentially modulated by FGF-1, a growth factor involved in neurodevelopment, inflammation and repair.

Blood Flow Changes Associated with Spinal Cord Injury Assessed by Non-linear Doppler Contrast-Enhanced Ultrasound.

Contrast-enhanced ultrasound (CEUS) is clinically used to image the microcirculation at lower imaging frequencies (<2 MHz). Recently, plane-wave acquisitions and Doppler processing have revealed improved microbubble sensitivity, enabling CEUS use at higher frequencies (15 MHz) and the ability to image simultaneously blood flow in the micro- and macrocirculations. We used this approach to assess acute and chronic blood flow changes within contused spinal cord in a rodent spinal cord injury model. Immediately after spinal cord injury, we found significant differences in perfusion deficit between moderate and severe injuries (1.73 ± 0.1 mm2 vs. 3.2 ± 0.3 mm2, respectively), as well as a delay in microbubble arrival time in tissue adjacent to the injury site (0.97 ± 0.1 s vs. 1.54 ± 0.1 s, respectively). Acutely, morphological changes to central sulcal arteries were observed where vessels rostral to the contusion were displaced 4.8 ± 2.2° and 8.2 ± 3.1° anteriorly, and vessels caudal to the contusion 17.8 ± 3.9° and 24.2 ± 4.1° posteriorly, respectively, for moderate and severe injuries. Significant correlation of the acute perfusion deficit and arrival time were found with the chronic assessment of locomotive function and histological estimate of spared spinal cord tissue.

Delayed administration of elezanumab, a human anti-RGMa neutralizing monoclonal antibody, promotes recovery following cervical spinal cord injury

Spinal cord injury (SCI) elicits a cascade of degenerative events including cell death, axonal degeneration, and the upregulation of inhibitory molecules which limit repair. Repulsive guidance molecule A (RGMa) is an axon growth inhibitor which is also involved in neuronal cell death and differentiation. SCI causes upregulation of RGMa in the injured rodent, non-human primate, and human spinal cord. Recently, we showed that delayed administration of elezanumab, a high affinity human RGMa-specific monoclonal antibody, promoted neuroprotective and regenerative effects following thoracic SCI. Since most human traumatic SCI is at the cervical level, and level-dependent anatomical and molecular differences may influence pathophysiological responses to injury and treatment, we examined the efficacy of elezanumab and its therapeutic time window of administration in a clinically relevant rat model of cervical impact-compression SCI. Pharmacokinetic analysis of plasma and spinal cord tissue lysate showed comparable levels of RGMa antibodies with delayed administration following cervical SCI. At 12w after SCI, elezanumab promoted long term benefits including perilesional sparing of motoneurons and increased neuroplasticity of key descending pathways involved in locomotion and fine motor function. Elezanumab also promoted growth of corticospinal axons into spinal cord gray matter and enhanced serotonergic innervation of the ventral horn to form synaptic connections caudal to the cervical lesion. Significant recovery in grip and trunk/core strength, locomotion and gait, and spontaneous voiding ability was found in rats treated with elezanumab either immediately post-injury or at 3 h post-SCI, and improvements in specific gait parameters were found when elezanumab was delayed to 24 h post-injury. We also developed a new locomotor score, the Cervical Locomotor Score, a simple and sensitive measure of trunk/core and limb strength and stability during dynamic locomotion.

Experimental Protocols and Analytical Procedures for Studying Synaptic Transmission in Rodent Spinal Cord Dorsal Horn.

Synaptic modulation and plasticity are key mechanisms underlying pain transmission in the spinal cord and supra-spinal centers. The study and understanding of these phenomena are fundamental to investigating both acute nociception and maladaptive changes occurring in chronic pain. This article describes experimental protocols and analytical methods utilized in electrophysiological studies to investigate synaptic modulation and plasticity at the first station of somatosensory processing, the spinal cord dorsal horn. Protocols useful for characterizing the nature of synaptic inputs, the site of modulation (pre- versus postsynaptic), and the presence of short-term synaptic plasticity are presented. These methods can be employed to study the physiology of acute nociception, the pathological mechanisms of persistent inflammatory and neuropathic pain, and the pharmacology of receptors and channels involved in pain transmission. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Spinal cord dissection and acute slice preparation Basic Protocol 2: Stimulation of the dorsal root and extracellular recording (compound action potentials and field potentials) Basic Protocol 3: Patch-clamp recording from dorsal horn neurons: action potential firing patterns and evoked synaptic inputs Basic Protocol 4: Analysis of parameters responsible for changes in synaptic efficacy Basic Protocol 5: Recording and analysis of currents mediated by astrocytic glutamate.

BET protein inhibition promotes non-myeloid cell mediated neuroprotection after rodent spinal cord contusion

Spinal cord injuries (SCI) often lead to multiple neurological deficits as a result from the initial trauma and also the secondary damage that follows. Despite abundant preclinical data proposing anti-inflammatory therapies to minimize secondary injury and improve functional recovery, the field still lacks an effective neuroprotective treatment. Epigenetic proteins, such as bromodomain and extraterminal domain (BET) proteins, are emerging as new targets to regulate inflammation. More importantly, pharmacological inhibition of BET proteins suppresses pro-inflammatory gene transcription after SCI. In this study, we tested the therapeutic potential of inhibiting BET proteins after SCI with clinically relevant compounds, and investigated the role of the BET protein BRD4 in macrophages during progression of SCI pathology. Systemic inhibition of BET proteins with I-BET762 significantly reduced lesion size 8 weeks after a contusion injury in rats. However, we observed no histological or locomotor improvements after SCI when we deleted Brd4 in macrophages through the use of myeloid-specific Brd4 knockout mice or after macrophage-targeted pharmacological BET inhibition. Taken together, our data indicate that systemic I-BET762 treatment is neuroprotective, and the histopathological improvement observed is likely to be a result of effects on non-macrophage targets. Expanding our understanding on the role of BET proteins after SCI is necessary to identify novel therapeutic targets that can effectively promote repair after SCI.

197 Gold Nanoparticle Enhanced X-ray Nanotomography Reveals Region-Specific CSF Pathways Through the Brain and Spinal Cord that are Altered in Post-hemorrhagic Hydrocephalus

INTRODUCTION: Cerebrospinal fluid (CSF) is essential for the healthy development and function of the brain, but understanding the mechanisms of its distribution in and around the central nervous system (CNS) remains a major challenge. To address this need, we developed a new technique, gold nanoparticle-enhanced x-ray nanotomography (AuNp-XRM), to achieve micrometer-scale resolution visualization of CSF pathways across the entire brain and spinal cord of rodents with further applications to a model of interventricular hemorrhage/post-hemorrhagic hydrocephalus (IVH/PHH). METHODS: P4 rodents were injected with aCSF or hemoglobin to create the control and IVH conditions. 72 hours later, animals underwent MRIs to determine onset of PHH. Animals were then injected with 1.9 nm and 15 nm gold nanoparticles and imaged using XRM. Following XRM, the brains, spines and meninges were harvested for histology. RESULTS: Using AuNp-XRM, we demonstrate visualization of previously characterized glymphatic, cranial nerve, and choroid plexus-mediated CSF circulation patterns, and present evidence to further identify CSF interactions with the leptomeninges, choroid plexus, and parenchyma of the brain and spinal cord. We also identified an intraparenchymal CSF circulation that targets stem-cell rich and cholinergic neuronal populations. CSF distribution to these areas was mediated by flow of CSF through the paravascular spaces of perforators originating from the large blood vessels of the basal cisterns and was significantly decreased in an animal model of PHH. CONCLUSION: Our findings using AuNp-XRM show that cholinergic cell populations in the brain and spinal cord differentially handle CSF, and that PHH alters CSF distribution to these areas of the brain. These region-specific patterns of CSF distribution may have critical relevance to normal brain development, brain development following PHH, and other neuropathological conditions.

Translational PET Imaging of Spinal Cord Injury with the Serotonin Transporter Tracer [11C]AFM.

The descending raphespinal serotonin (5-HT) system contributes to neural activities required for locomotion. The presynaptic serotonin transporter (SERT) is a marker of 5-HT innervation. In this study, we explored the use of PET imaging with the SERT radioligand [11C]AFM as a biomarker of 5-HT axon damage after spinal cord injury (SCI) in a rodent model and its translation to imaging SCI in humans. PET imaging with [11C]AFM was performed in healthy rats under baseline and citalopram blocking conditions and a mid-thoracic transection rat model of SCI. The lumbar-to-cervical activity (L/C) ratio was calculated for the healthy and SCI animals to assess SERT binding decrease after SCI. Finally, translation of [11C]AFM PET was attempted to explore its potential to image SCI in humans. Intense uptake in the brain and intact spinal cord was observed at 30-60min post-injection of [11C]AFM in healthy rats. About 65% of [11C]AFM uptake in the spinal cord was blocked by citalopram. In the SCI rat model, the cervical uptake of [11C]AFM was similar to that in healthy rats, but the lumbar uptake was dramatically reduced, resulting in about half the L/C ratio in SCI rats compared to healthy rats. In contrast, [11C]AFM uptake in the human spinal cord showed no obvious decrease after treatment with citalopram. In the human subjects with SCI, decreases in [11C]AFM uptake were also not obvious in the section of spinal cord caudal to the injury point. [11C]AFM PET imaging of SERT provides a useful preclinical method to non-invasively visualize the rodent spinal cord and detect SERT changes in SCI rodent models. However, there appears to be little detectable specific binding signal for [11C]AFM in the human spinal cord. An SERT tracer with higher affinity and lower non-specific binding signal is needed to image the spinal cord in humans and to assess the axonal status in SCI patients.

The role of aquaporin 4 (AQP4) in spinal cord injury.

Aquaporin-4 (AQP-4) is an aquaporin composed of six helical transmembrane domains and two highly conserved ASN-pro-ALA (NPA) motifs. It is strongly expressed in rodent and human spinal cord tissues and plays a key role in the pathological process after SCI. After SCI, edema, glial scarring, and inflammation can accelerate the progression of injury and lead to deterioration of function. Many studies have reported that AQP-4 plays an important role in SCI. In particular, it plays an important role in secondary pathological processes (spinal cord edema, glial scar formation, and inflammatory response) after SCI. Loss of AQP-4 has been associated with reduced spinal edema and improved prognosis after SCI in mice. In addition, downregulation of AQP-4 reduces glial scar formation and the inflammatory response after SCI. There is a consensus from numerous studies that AQP-4 may be a potential target for SCI therapy, which guides the ongoing investigation for molecular therapy of SCI. Here, we review the structure of AQP-4, its expression in normal and damaged spinal cord, and its role in SCI, as well as discuss the theoretical basis for the treatment of SCI.

Investigating the Feasibility of In Vivo Perfusion Imaging Methods for Spinal Cord Using Hyperpolarized [13C]t-Butanol and [13C,15N2]Urea

This study examined the feasibility of using two novel agents, hyperpolarized [13C]t-butanol and [13C,15N2]urea, for assessing in vivo perfusion of the intact spinal cord in rodents. Due to their distinct permeabilities to blood brain barrier (BBB), we hypothesized that [13C]t-butanol and [13C,15N2]urea exhibit unique 13C signal characteristics in the spinal cord. Dynamic 13C t-butanol MRI data were acquired from healthy Long-Evans rats using a symmetric, ramp-sampled, partial-Fourier 13C echo-planar imaging sequence after the injection of hyperpolarized [13C]t-butanol solution. In subsequent scans, dynamic 13C urea MRI data were acquired after the injection of hyperpolarized [13C,15N2]urea. The SNRs of t-butanol and urea were calculated for regions corresponding to spine, supratentorial brain, and blood vessels and plotted over time. Mean peak SNR and AUC were calculated from the dynamic plots for each region and compared between t-butanol and urea. In spine and supratentorial brain, the mean peak SNR and AUC of t-butanol were significantly higher than those of urea (p < 0.05). In contrast, urea was predominantly contained within vasculature and exhibited significantly higher levels of mean peak SNR and AUC compared to t-butanol in blood vessels (p < 0.05). This study has demonstrated the feasibility of using hyperpolarized [13C]t-butanol and [13C,15N2]urea for assessing in vivo perfusion in cervical spinal cord. Due to differences in blood-brain barrier permeability, t-butanol rapidly crossed the blood-brain barrier and diffused into spine and brain tissue, while urea predominantly remained in vasculature. The results from this study suggest that this technique may provide unique non-invasive imaging tracers that are able to directly monitor hemodynamic processes in the normal and injured spinal cord.

The presynaptic glycine transporter GlyT2 is regulated by the Hedgehog pathway in vitro and in vivo

The identity of a glycinergic synapse is maintained presynaptically by the activity of a surface glycine transporter, GlyT2, which recaptures glycine back to presynaptic terminals to preserve vesicular glycine content. GlyT2 loss-of-function mutations cause Hyperekplexia, a rare neurological disease in which loss of glycinergic neurotransmission causes generalized stiffness and strong motor alterations. However, the molecular underpinnings controlling GlyT2 activity remain poorly understood. In this work, we identify the Hedgehog pathway as a robust controller of GlyT2 expression and transport activity. Modulating the activation state of the Hedgehog pathway in vitro in rodent primary spinal cord neurons or in vivo in zebrafish embryos induced a selective control in GlyT2 expression, regulating GlyT2 transport activity. Our results indicate that activation of Hedgehog reduces GlyT2 expression by increasing its ubiquitination and degradation. This work describes a new molecular link between the Hedgehog signaling pathway and presynaptic glycine availability.

Design and construction of an interchangeable RF coil system for rodent spinal cord MR imaging at 9.4 T

Rodent models of spinal cord injury (SCI) have been widely used in pre-clinical studies. Injuries may occur at different levels of the lumbar and thoracic cord, and the number of segments injured and their depths may vary along the spine. It is thereby challenging to build one universal RF coil that exhibits optimal performance for all spinal cord imaging applications, especially in an animal scanner with small in-bore space and limited hardware configurations. We developed an interchangeable RF coil system for a 9.4 T small animal MRI scanner, in which the users can select an optimal coil specialized for imaging specific parts of a rat spine. We also developed the associated animal management device for immobilization and positioning. The whole system allows ease of RF coil exchange, animal fixation, and positioning, and thus reduces the animal preparation time before the MRI scan significantly. Compared to a commercial general-purpose 2-cm-diameter coil that was used in our previous studies, the specialized coil optimized for Sprague-Dawley rat lumbar spinal cord imaging exhibits up to 2.4 times SNR improvement.

Nicotine Neurotoxicity Involves Low Wnt1 Signaling in Spinal Locomotor Networks of the Postnatal Rodent Spinal Cord.

The postnatal rodent spinal cord in-vitro is a useful model to investigate early pathophysiological changes after injury. While low dose nicotine (1 µM) induces neuroprotection, how higher doses affect spinal networks is unknown. Using spinal preparations of postnatal wild-type Wistar rat and Wnt1Cre2:Rosa26Tom double-transgenic mouse, we studied the effect of nicotine (0.5–10 µM) on locomotor networks in-vitro. Nicotine 10 µM induced motoneuron depolarization, suppressed monosynaptic reflexes, and decreased fictive locomotion in rat spinal cord. Delayed fall in neuronal numbers (including motoneurons) of central and ventral regions emerged without loss of dorsal neurons. Conversely, nicotine (0.5–1 µM) preserved neurons throughout the spinal cord and strongly activated the Wnt1 signaling pathway. High-dose nicotine enhanced expression of S100 and GFAP in astrocytes indicating a stress response. Excitotoxicity induced by kainate was contrasted by nicotine (10 µM) in the dorsal area and persisted in central and ventral regions with no change in basal Wnt signaling. When combining nicotine with kainate, the activation of Wnt1 was reduced compared to kainate/sham. The present results suggest that high dose nicotine was neurotoxic to central and ventral spinal neurons as the neuroprotective role of Wnt signaling became attenuated. This also corroborates the risk of cigarette smoking for the foetus/newborn since tobacco contains nicotine.