Spinal Cord Hemisection Research Articles

C2 Spinal Cord Hemisection Reduces Mitochondrial Volume Density in Phrenic Motor Neurons

The diaphragm muscle (DIAm) is the major inspiratory pump for breathing. Neural control of the DIAm during breathing involves the recruitment of fatigue resistant motor units comprising smaller phrenic motor neurons (PhMNs), which innervate type I and IIa fibers. More fatigable DIAm motor units comprise larger PhMNs that innervate type IIx/IIb fibers, which generate greater forces but are more fatigable. The fatigable motor units are only infrequently recruited for expulsive behaviors of the DIAm. Thus, there are marked differences in the activation history of DIAm motor units. Consistent with their higher activity levels, previous studies from our lab have shown that smaller PhMNs and type I and IIa DIAm fibers have greater mitochondrial volume density (MVD). PhMNs located in the cervical spinal cord (C3-C5) receive direct excitatory input from medullary premotor neurons, which is primarily ipsilateral. Thus, C2 spinal cord hemisection (C2SH) disrupts the neural pathway essential for breathing and significantly reduces DIAm activity on the same side. Therefore, we hypothesized that C2SH significantly reduces MVD of smaller PhMNs. In Sprague Dawley rats, PhMNs on both sides were retrogradely labeled by intrapleural injection of cholera toxin B (CTB) 3 days prior to C2SH (right side). In addition, DIAm EMG activity was recorded on both sides by inserting bipolar electrodes. Mitochondria were labeled by intraspinal injection of MitoTracker Red and imaged in identified PhMNs using 3D confocal microscopy. A series of optical slices (0.5 mm) of PhMNs were deconvolved to improve contrast, then thresholded for MitoTracker labeling followed by 3D reconstruction, providing detailed images of mitochondrial morphometry and distribution within PhMNs. In sham rats, the MVD of smaller (lower somal surface area) PhMNs was higher compared to larger PhMNs. C2SH reduced DIAm EMG activity on the ipsilateral side across a 14-day period, while activity on the contralateral side increased. After 14 days, C2SH reduced MVD by ~25% in smaller PhMNs on the ipsilateral side. We conclude that the reduced activity of smaller PhMNs induced by C2SH induced mitochondrial morphological changes, most likely due to reduced mitochondrial biogenesis. These results indicate that reduced DIAm activity associated with mechanical ventilation may impose mitochondrial remodeling that reduces mitochondrial oxidative capacity in PhMNs. These adaptations may disrupt mitochondrial function and impair weaning from mechanical ventilation. Research support by NIH grants HL166204, HL146114 and AG44615. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Impact of Reduced Activity on Endurance of Rat Diaphragm Muscle

The diaphragm muscle (DIAm) as the major inspiratory pump is highly active. During breathing, fatigue resistant DIAm motor units are recruited that comprise type I and IIa fibers. Accordingly, these fibers have substantially higher mitochondrial volume density (MVD) and mitochondrial respiratory capacity (SDHmax) compared to more fatigable type IIx/IIb fibers. Cervical spinal cord injury disrupts descending inspiratory drive and markedly reduces eupneic activity of the DIAm. With C2 spinal cord hemisection (C2SH) DIAm activity on the ipsilateral side is reduced. Following a two-week period of C2SH, we found reduced MVD and SDHmax in type I and IIa DIAm fibers. As a result, we hypothesize that endurance of the DIAm is reduced. Female and male Sprague Dawley rats were used. EMG electrodes were placed in the left and right DIAm 3 days prior to C2SH surgery, and baseline recordings of eupneic DIAm activity were obtained in awake animals. The right C2 spinal cord was hemisected, and the extent of injury was confirmed by the absence of eupneic DIAm activity on the ipsilateral side while animals were anesthetized. Subsequently, DIAm EMG activity was recorded at 3, 7 and 14 days post C2SH. After two weeks, rats were anesthetized and the DIAm was excised. DIAm strips were cut and suspended in a tissue chamber containing Rees-Simpson solution maintained at 26oC, with thecostal margin clamped and the central tendon attached to a Cambridge Dual-Mode force/length transducer. DIAm strips were stimulated using platinum plate electrodes with supramaximal current (~250 mA) pulses (1 ms), and maximum specific force was determined at 75 Hz. The force-velocity relationship was then determined by systematically changing load, and maximum velocity (Vmax) was estimated based on curve fitting with the Hill-equation. Based on the force-velocity measurements peak power of the DIAm was calculated. Subsequently, the DIAm was repetitively stimulated and allowed to shorten at 30% maximum velocity (isovelocity approximating peak power output). Endurance of the DIAm was then determined as the duration that peak power output was sustained. Finally, the DIAm was rapidly frozen, and cross-sections were cut for fiber type and cross-sectional area (CSA) measurements. C2SH resulted in a marked decrease in eupneic DIAm EMG activity across the two-week period, which disproportionately affected type I and IIa DIAm fibers. However, there was no impact of C2SH on DIAm fiber type proportions or CSA. Following two-weeks C2SH, maximum specific force, Vmax, and peak power output of the DIAm were comparable between intact and injured sides. However, endurance of the DIAm was reduced on the injured side compared to the intact side. These results support our hypothesis that reduced activity of DIAm, reduces oxidative capacity of type I and IIa fibers and thereby reduces endurance. In clinical conditions such as mechanical ventilation DIAm activity is reduced and if maintained for longer periods of time this may affect DIAm endurance post weaning. Research supported by NIH grant: HL146114 (GCS). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Combined therapeutic acute intermittent hypercapnic hypoxia and exercise-based respiratory training improve breathing after chronic cervical spinal cord injury

Therapeutic acute intermittent hypoxia (tAIH) is simple, safe, and effective means of inducing respiratory motor plasticity and improving breathing in rodent models of acute cervical spinal cord injury (SCI). Unfortunately, tAIH effects on breathing function are less effective with chronic injury. On the other hand, tAIH restores non-respiratory (limb) motor function with chronic SCI, but only when combined with task specific training (TST). The effcacy of combined tAIH and respiratory TST on breathing ability in rodent models (or humans) with chronic SCI has not been investigated. Here, we explore the potential for tAIH and respiratory TST to increase breathing ability; we further differentiate automatic vs. volitional TST, since the ability to sustain independent breathing is dependent on automatic (and not volitional) breathing function, which relies on distinct neural pathways. Physical exercise increases breathing automatically in direct proportion to metabolic rate, regulating arterial blood gases by a mechanism independent of chemoreceptor feedback (i.e. “exercise hyperpnea”). Thus, in ongoing studies, we are exploring treadmill training as a form of task-specific respiratory training in rats with chronic cervical SCI. We focus on a refined tAIH protocol that combines hypoxia with hypercapnia during each episode (therapeutic acute intermittent hypercapnic hypoxia; or tAIHH), since preliminary data suggests that tAIHH is a more potent stimulus to respiratory motor plasticity vs. tAIH alone. Adult Sprague-Dawley rats with chronic C2 spinal cord hemisection were treated with either 4 weeks of either tAIHH (10, 5-min episodes of 10.5% O2, 4% inspired CO2) combined with treadmill exercise (30 min total run time, 30 cm/sec) or tAIHH alone. Breathing function was assessed in awake, freely moving rats using whole-body plethysmography. In preliminary analyses, breathing capacity (i.e., minute ventilation in response to maximal chemoreflex activation) was increased in chronically injured rats treated with daily tAIHH paired with treadmill training relative to rats treated with tAIHH alone (120.7mL/min/100g vs. 98.7mL/min/100g; p=0.0018). Similar improvements in breathing function were not observed with tAIHH alone. These are the first studies to investigate the therapeutic effcacy of combined tAIH and exercise-based respiratory training targeting automatic breathing. These initial results indicate that, similar to non-respiratory motor systems, pairing tAIHH with respiratory TST improves breathing function in rodents with chronic cervical SCI. Supported by: Craig H. Neilsen Foundation (SCIRP891379), and the McKnight Brain Institute. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

AAV6 mediated Gsx1 expression in neural stem progenitor cells promotes neurogenesis and restores locomotor function after contusion spinal cord injury

Genomic screened homeobox 1 (Gsx1 or Gsh1) is a neurogenic transcription factor required for the generation of excitatory and inhibitory interneurons during spinal cord development. In the adult, lentivirus (LV) mediated Gsx1 expression promotes neural regeneration and functional locomotor recovery in a mouse model of lateral hemisection spinal cord injury (SCI). The LV delivery method is clinically unsafe due to insertional mutations to the host DNA. In addition, the most common clinical case of SCI is contusion/compression. In this study, we identify that adeno-associated virus serotype 6 (AAV6) preferentially infects neural stem/progenitor cells (NSPCs) in the injured spinal cord. Using a rat model of contusion SCI, we demonstrate that AAV6 mediated Gsx1 expression promotes neurogenesis, increases the number of neuroblasts/immature neurons, restores excitatory/inhibitory neuron balance and serotonergic neuronal activity through the lesion core, and promotes locomotor functional recovery. Our findings support that AAV6 preferentially targets NSPCs for gene delivery and confirmed Gsx1 efficacy in clinically relevant rat model of contusion SCI.

Overview of Brown-Sequard Syndrome.

A neurologic syndrome called Brown-Séquard syndrome is brought on by spinal cord hemisection. On the side of the body, ipsilateral to the lesion, it presents as weakness or paralysis, proprioceptive abnormalities, and loss of pain and temperature perception. Brown-Séquard syndrome is an incomplete spinal cord syndrome with a range of severity in its clinical presentation.
 One of the most intricate and fascinating areas of the nervous system to study is the spinal cord. Its overwhelming clinical presentation, development problems, lesions, and intricate connections call for a deeper comprehension of its anatomical and physiological makeup.
 Like the brain, the extremely fragile and sensitive spinal cord is well-protected by the robust bony cage comprising the vertebral arch and vertebral bodies. Together, they create the spinal hollow, or long spinal column, which houses the spinal cord. Millions of neurons reside in the spinal cord, and their bundled fibers run as ascending or descending tracts. Thirty-one pairs of spinal nerves, primarily supplying the trunk and limbs, emerge from the spinal cord. These are mixed spinal nerves with a motor component that assists in controlling the trunk and voluntary muscles in all limbs. Additionally, they have a sensory component that aids in taking in the sensory data from these regions. Therefore, for a better understanding of the clinical presentation and its pathology in any spinal cord lesion, it is imperative to grasp the fundamentals of anatomy.
 In this short review, we have discussed the symptoms, management, and prevention of brown syndrome.

Injectable, Electroconductive, Free Radical Scavenging Silk Fibroin/Black Phosphorus/Glycyrrhizic Acid Nanocomposite Hydrogel for Enhancing Spinal Cord Repair.

Spinal cord injury (SCI) often leads to a severe permanent disability. A poor inflammatory microenvironment and nerve electric signal conduction block are the main reasons for difficulty in spinal cord nerve regeneration. In this study, black phosphorus (BP) and glycyrrhizic acid (GA) are integrated into methacrylate-modified silk fibroin (SF) to construct a bifunctional injectable hydrogel (SF/BP/GA) with appropriate conductivity and the ability to inhibit inflammation to promote neuronal regeneration after SCI. This work discovers that the SF/BP/GA hydrogel can reduce the oxidative damage mediated by oxygen free radicals, promote the polarization of macrophages toward the anti-inflammatory M2 phenotype, reduce the expression of inflammatory factors, and improve the inflammatory microenvironment. Moreover, it induces neural stem cell (NSC) differentiation and neurosphere formation, restores signal conduction at the SCI site in vivo, and ameliorates motor function in mice with spinal cord hemisection, revealing a significant neural repair effect. An injectable, electroconductive, free-radical-scavenging hydrogel is a promising therapeutic strategy for SCI repair.

Cervical spinal cord hemisection impacts sigh and the respiratory reset in male rats.

Cervical spinal cord injury impacts ventilatory and non-ventilatory functions of the diaphragm muscle (DIAm) and contributes to clinical morbidity and mortality in the afflicted population. Periodically, integrated brainstem neural circuit activity drives the DIAm to generate a markedly augmented effort or sigh-which plays an important role in preventing atelectasis and thus maintaining lung function. Across species, the general pattern of DIAm efforts during a normal sigh is variable in amplitude and the extent of post-sigh "apnea" (i.e., the post-sigh inter-breath interval). This post-sigh inter-breath interval acts as a respiratory reset, following the interruption of regular respiratory rhythm by sigh. We examined the impact of upper cervical (C2 ) spinal cord hemisection (C2 SH) on the transdiaphragmatic pressure (Pdi ) generated during sighs and the post-sigh respiratory reset in rats. Sighs were identified in Pdi traces by their characteristic biphasic pattern. We found that C2 SH results in a reduction of Pdi during both eupnea and sighs, and a decrease in the immediate post-sigh breath interval. These results are consistent with partial removal of descending excitatory synaptic inputs to phrenic motor neurons that results from C2 SH. Following cervical spinal cord injury, a reduction in the amplitude of Pdi during sighs may compromise the maintenance of normal lung function.

Osteopontin Promotes Angiogenesis in the Spinal Cord and Exerts a Protective Role Against Motor Function Impairment and Neuropathic Pain After Spinal Cord Injury.

Basic science study using a hemisection spinal cord injury (SCI) model. We sought to assess the effect of blocking osteopontin (OPN) upregulation on motor function recovery and pain behavior after SCI and to further investigate the possible downstream target of OPN in the injured spinal cord. OPN is a noncollagenous extracellular matrix protein widely expressed across different tissues. Its expression substantially increases following SCI. A previous study suggested that this protein might contribute to locomotor function recovery after SCI. However, its neuroprotective potential was not fully explored, nor were the underlying mechanisms. We constructed a SCI mouse model and analyzed the expression of OPN at different time points and the particular cell distribution in the injured spinal cord. Then, we blocked OPN upregulation with lentivirus-delivering siRNA targeting OPN specifically and examined its effect on motor function impairment and neuropathic pain after SCI. The underlying mechanisms were explored in the OPN-knockdown mice model and cultured vascular endothelial cells. The proteome study revealed that OPN was the most dramatically increased protein following SCI. OPN in the spinal cord was significantly increased three weeks after SCI. Suppressing OPN upregulation through siRNA exacerbated motor function impairment and neuropathic pain. In addition, SCI resulted in an increase in vascular endothelial growth factor (VEGF), AKT phosphorylation, and angiogenesis within the spinal cord, all of which were curbed by OPN reduction. Similarly, OPN knockdown suppressed VEGF expression, AKT phosphorylation, cell migration, invasion, and angiogenesis in cultured vascular endothelial cells. OPN demonstrates a protective influence against motor function impairment and neuropathic pain following SCI. This phenomenon may result from the proangiogenetic effect of OPN, possibly due to activation of the VEGF and/or AKT pathways.

[Translated article] Effects of duroplasty with bovine pericardium on fibrosis and extent of spinal cord injury: An experimental study in pigs

IntroductionTraumatic spinal cord injury (SCI) leads to increased intraspinal pressure that can be prevented by durotomy and duroplasty. The aim of the study was to evaluate fibrosis and neural damage in a porcine model of SCI after duroplasty and application of hyaluronic acid (HA) in the tissue cavity. Materials and methodsExperimental study. We created a porcine SCI model by durotomy and spinal cord hemisection of a cervical segment (1cm). Six pigs (Sus scrofa domestica) were used to evaluate three surgical scenarios: (1) control injury with dural reparative microsurgery, (2) duroplasty using bovine pericardium (BPD), and (3) previous method plus HA applied at the lesion. Animals were sacrificed one-month post-injury to assess fibrotic responses and neural tissue damage using conventional histological and immunohistochemical methods. ResultsIn the control case, dural suture prevented invasion of the lesion by extradural connective tissue, and the dura mater showed a 1-mm thickening in the perilesional area. The bovine pericardium patch blocked the entrance of extradural connective tissue, decreased dura-mater tension, and satisfactorily integrated within the receptor tissue. However, it also enhanced subdural and perilesional fibrosis, which was not inhibited by filling the lesion cavity with low- or high-molecular-weight HA. ConclusionsDuroplasty prevents collapse of the dura-mater over the spinal cord tissue, as well as invasion of the lesion by extramedullary fibrotic tissue, without creating additional neural damage. Nevertheless, it enhances the fibrotic response in the spinal cord lesion and the perilesional area. Additional antifibrotic strategies are needed to facilitate spinal cord repair.

Inhibition of aquaporin-4 and its subcellular localization attenuates below-level central neuropathic pain by regulating astrocyte activation in a rat spinal cord injury model

The mechanisms of central neuropathic pain (CNP) caused by spinal cord injury have not been sufficiently studied. We have found that the upregulation of astrocytic aquaporin-4 (AQP4) aggravated peripheral neuropathic pain after spinal nerve ligation in rats. Using a T13 spinal cord hemisection model, we showed that spinal AQP4 was markedly upregulated after SCI and mainly expressed in astrocytes in the spinal dorsal horn (SDH). Inhibition of AQP4 with TGN020 suppressed astrocyte activation, attenuated the development and maintenance of below-level CNP and promoted motor function recovery in vivo. In primary astrocyte cultures, TGN020 also changed cell morphology, diminished cell proliferation and suppressed astrocyte activation. Moreover, T13 spinal cord hemisection induced cell-surface abundance of the AQP4 channel and perivascular localization in the SDH. Targeted inhibition of AQP4 subcellular localization with trifluoperazine effectively diminished astrocyte activation in vitro and further ablated astrocyte activation, attenuated the development and maintenance of below-level CNP, and accelerated functional recovery in vivo. Together, these results provide mechanistic insights into the roles of AQP4 in the development and maintenance of below-level CNP. Intervening with AQP4, including targeting AQP4 subcellular localization, might emerge as a promising agent to prevent chronic CNP after SCI.

Acute inhibition of acid sensing ion channel 1a after spinal cord injury selectively affects excitatory synaptic transmission, but not intrinsic membrane properties, in deep dorsal horn interneurons.

Following a spinal cord injury (SCI), secondary damage mechanisms are triggered that cause inflammation and cell death. A key component of this secondary damage is a reduction in local blood flow that initiates a well-characterised ischemic cascade. Downstream hypoxia and acidosis activate acid sensing ion channel 1a (ASIC1a) to trigger cell death. We recently showed that administration of a potent venom-derived inhibitor of ASIC1a, Hi1a, leads to tissue sparing and improved functional recovery when delivered up to 8 h after ischemic stroke. Here, we use whole-cell patch-clamp electrophysiology in a spinal cord slice preparation to assess the effect of acute ASIC1a inhibition, via a single dose of Hi1a, on intrinsic membrane properties and excitatory synaptic transmission long-term after a spinal cord hemisection injury. We focus on a population of interneurons (INs) in the deep dorsal horn (DDH) that play a key role in relaying sensory information to downstream motoneurons. DDH INs in mice treated with Hi1a 1 h after a spinal cord hemisection showed no change in active or passive intrinsic membrane properties measured 4 weeks after SCI. DDH INs, however, exhibit significant changes in the kinetics of spontaneous excitatory postsynaptic currents after a single dose of Hi1a, when compared to naive animals (unlike SCI mice). Our data suggest that acute ASIC1a inhibition exerts selective effects on excitatory synaptic transmission in DDH INs after SCI via specific ligand-gated receptor channels, and has no effect on other voltage-activated channels long-term after SCI.

Lycium barbarum glycopeptide alleviates neuroinflammation in spinal cord injury via modulating docosahexaenoic acid to inhibiting MAPKs/NF-kB and pyroptosis pathways

BackgroundLycium barbarum polysaccharide (LBP) is an active ingredient extracted from Lycium barbarum that inhibits neuroinflammation, and Lycium barbarum glycopeptide (LbGp) is a glycoprotein with immunological activity that was purified and isolated from LBP. Previous studies have shown that LbGp can regulate the immune microenvironment, but its specific mechanism of action remains unclear.AimsIn this study, we aimed to explore the mechanism of action of LbGp in the treatment of spinal cord injury through metabolomics and molecular experiments.MethodsSD male rats were randomly assigned to three experimental groups, and after establishing the spinal cord hemisection model, LbGp was administered orally. Spinal cord tissue was sampled on the seventh day after surgery for molecular and metabolomic experiments. In vitro, LbGp was administered to mimic the inflammatory microenvironment by activating microglia, and its mechanism of action in suppressing neuroinflammation was further elaborated using metabolomics and molecular biology techniques such as western blotting and q-PCR.ResultsIn vivo and in vitro experiments found that LbGp can improve the inflammatory microenvironment by inhibiting the NF-kB and pyroptosis pathways. Furthermore, LbGp induced the secretion of docosahexaenoic acid (DHA) by microglia, and DHA inhibited neuroinflammation through the MAPK/NF-κB and pyroptosis pathways.ConclusionsIn summary, we hypothesize that LbGp improves the inflammatory microenvironment by regulating the secretion of DHA by microglia and thereby inhibiting the MAPK/NF-κB and pyroptosis pathways and promoting nerve repair and motor function recovery. This study provides a new direction for the treatment of spinal cord injury and elucidates the potential mechanism of action of LbGp.

Efecto de la reparación de la duramadre con pericardio bovino sobre la fibrosis y la extensión de la lesión medular: estudio experimental en cerdos

IntroductionTraumatic spinal cord injury (SCI) leads to increased intraspinal pressure that can be prevented by durotomy and duroplasty. The aim of the study was to evaluate fibrosis and neural damage in a porcine model of SCI after duroplasty and application of hyaluronic acid (HA) in the tissue cavity. Materials and methodsExperimental study. We created a porcine SCI model by durotomy and spinal cord hemisection of a cervical segment (1cm). Six pigs (Sus scrofa domestica) were used to evaluate three surgical scenarios: (1)control injury with dural reparative microsurgery, (2)duroplasty using bovine pericardium (BPD), and (3)previous method plus HA applied at the lesion. Animals were sacrificed one-month post-injury to assess fibrotic responses and neural tissue damage using conventional histological and immunohistochemical methods. ResultsIn the control case, dural suture prevented invasion of the lesion by extradural connective tissue, and the dura mater showed a 1-mm thickening in the perilesional area. The bovine pericardium patch blocked the entrance of extradural connective tissue, decreased dura-mater tension, and satisfactorily integrated within the receptor tissue. However, it also enhanced subdural and perilesional fibrosis, which was not inhibited by filling the lesion cavity with low- or high-molecular-weight HA. ConclusionsDuroplasty prevents collapse of the dura-mater over the spinal cord tissue, as well as invasion of the lesion by extramedullary fibrotic tissue, without creating additional neural damage. Nevertheless, it enhances the fibrotic response in the spinal cord lesion and the perilesional area. Additional antifibrotic strategies are needed to facilitate spinal cord repair.

Effects of Recombinant IL-13 Treatment on Gut Microbiota Composition and Functional Recovery after Hemisection Spinal Cord Injury in Mice.

In recent years, the gut-central nervous system axis has emerged as a key factor in the pathophysiology of spinal cord injury (SCI). Interleukin-13 (IL-13) has been shown to have anti-inflammatory and neuroprotective effects in SCI. The aim of this study was to investigate the changes in microbiota composition after hemisection injury and to determine whether systemic recombinant (r)IL-13 treatment could alter the gut microbiome, indirectly promoting functional recovery. The gut microbiota composition was determined by 16S rRNA gene sequencing, and correlations between gut microbiota alterations and functional recovery were assessed. Our results showed that there were no changes in alpha diversity between the groups before and after SCI, while PERMANOVA analysis for beta diversity showed significant differences in fecal microbial communities. Phylogenetic classification of bacterial families revealed a lower abundance of the Bacteroidales S24-7 group and a higher abundance of Lachnospiraceae and Lactobacillaceae in the post-SCI group. Systemic rIL-13 treatment improved functional recovery 28 days post-injury and microbiota analysis revealed increased relative abundance of Clostridiales vadin BB60 and Acetitomaculum and decreased Anaeroplasma, Ruminiclostridium_6, and Ruminococcus compared to controls. Functional assessment with PICRUSt showed that genes related to glyoxylate cycle and palmitoleate biosynthesis-I were the predominant signatures in the rIL-13-treated group, whereas sulfolactate degradation super pathway and formaldehyde assimilation-I were enriched in controls. In conclusion, our results indicate that rIL-13 treatment promotes changes in gut microbial communities and may thereby contribute indirectly to the improvement of functional recovery in mice, possibly having important implications for the development of novel treatment options for SCI.

Cerebrolysin recovers diaphragmatic function and reduces injury-associated astrogliosis following a cervical spinal cord hemi-section injury in rats

BackgroundSpinal cord injury (SCI) is widely considered the most disastrous medical condition. With no available treatment to date, SCI continues to cause disabilities to the patients and affect their own and their caregivers' quality of life. Cerebrolysin (CBL) is a neuropeptide preparation derived from purified brain proteins with suggested neuroprotective and neurotrophic properties. CBL showed earlier the ability to target multiple pathways that helped in the improvement of the recovery following different groups of neurological diseases and injuries, including ischemic stroke, traumatic brain injuries, and even neurodegenerative diseases. In the current study, the neuroprotective effect of CBL following a SCI is called into question using a rat model of spinal cord cervical hemi-section validated earlier by our lab and others. Using 32 rats divided into four groups randomly, CBL treatment was implemented for either 7 or 21 days duration, following the cervical spinal cord hemi-section.ResultsFollowing the CBL treatment, rats with cervical cord hemi-section showed functional improvement of diaphragmatic muscle as recorded by electromyography (EMG). In addition, the histopathological assessment of the spinal cord showed improved neuronal viability and reduced astrogliosis at the site of the injury compared to the non-treated group. 21-day treatment showed significant improvement when compared to the shorter 7-day regimen.ConclusionOur data suggest that CBL is capable of protecting and regenerating anterior horn motor neurons with functional recovery of diaphragmatic muscle functions in rats, suggesting the potential use of CBL for future regenerative and neuroprotective therapy following SCI.

CSF1R inhibition at chronic stage after spinal cord injury modulates microglia proliferation.

Traumatic spinal cord injury (SCI) induces irreversible autonomic and sensory-motor impairments. A large number of patients exhibit chronic SCI and no curative treatment is currently available. Microglia are predominant immune players after SCI, they undergo highly dynamic processes, including proliferation and morphological modification. In a translational aim, we investigated whether microglia proliferation persists at chronic stage after spinal cord hemisection and whether a brief pharmacological treatment could modulate microglial responses. We first carried out a time course analysis of SCI-induced microglia proliferation associated with morphological analysis up to 84 days post-injury (dpi). Second, we analyzed outcomes on microglia of an oral administration of GW2580, a colony stimulating factor-1 receptor tyrosine kinase inhibitor reducing selectively microglia proliferation. After SCI, microglia proliferation remains elevated at 84 dpi. The percentage of proliferative microglia relative to proliferative cells increases over time reaching almost 50% at 84 dpi. Morphological modifications of microglia processes are observed up to 84 dpi and microglia cell body area is transiently increased up to 42 dpi. A transient post-injury GW2580-delivery at two chronic stages after SCI (42 and 84 dpi) reduces microglia proliferation and modifies microglial morphology evoking an overall limitation of secondary inflammation. Finally, transient GW2580-delivery at chronic stage after SCI modulates myelination processes. Together our study shows that there is a persistent microglia proliferation induced by SCI and that a pharmacological treatment at chronic stage after SCI modulates microglial responses. Thus, a transient oral GW2580-delivery at chronic stage after injury may provide a promising therapeutic strategy for chronic SCI patients.

METTL3 Affects Spinal Cord Neuronal Apoptosis by Regulating Bcl-2 m6A Modifications After Spinal Cord Injury.

Spinal cord injury (SCI) is a severe type of neurological trauma. N6-methyladenosine (m6A) modification is one of the most common internal modifications of RNA. The role of METTL3, the predominant methylation enzyme of m6A modification, in SCI remains unclear. This study aimed to investigate the role of methyltransferase METTL3 in SCI. After establishing the oxygen-glucose deprivation (OGD) model of PC12 cells and rat spinal cord hemisection model, we found that the expression of METTL3 and the overall m6A modification level were significantly increased in neurons. The m6A modification was identified on B-cell lymphoma 2 (Bcl-2) messenger RNA (mRNA) by bioinformatics analysis, and m6A-RNA immunoprecipitation and RNA immunoprecipitation. In addition, METTL3 was blocked by the specific inhibitor STM2457 and gene knockdown, and then apoptosis levels were measured. In different models, we found that the expression of METTL3 and the overall m6A modification level were significantly increased in neurons. After inducing OGD, inhibition of METTL3 activity or expression increased the mRNA and protein levels of Bcl-2, inhibited neuronal apoptosis, and improved neuronal viability in the spinal cord. Inhibition of METTL3 activity or expression can inhibit the apoptosis of spinal cord neurons after SCI through the m6A/Bcl-2 signaling pathway.

Neuropathic pain following spinal cord hemisection induced by the reorganization in primary somatosensory cortex and regulated by neuronal activity of lateral parabrachial nucleus.

Neuropathic pain after spinal cord injury (SCI) remains a common and thorny problem, influencing the life quality severely. This study aimed to elucidate the reorganization of the primary sensory cortex (S1) and the regulatory mechanism of the lateral parabrachial nucleus (lPBN) in the presence of allodynia or hyperalgesia after left spinal cord hemisection injury (LHS). Through behavioral tests, we first identified mechanical allodynia and thermal hyperalgesia following LHS. We then applied two-photon microscopy to observe calcium activity in S1 during mechanical or thermal stimulation and long-term spontaneous calcium activity after LHS. By slice patch clamp recording, the electrophysiological characteristics of neurons in lPBN were explored. Finally, exploiting chemogenetic activation or inhibition of the neurons in lPBN, allodynia or hyperalgesia was regulated. The calcium activity in left S1 was increased during mechanical stimulation of right hind limb and thermal stimulation of tail, whereas in right S1 it was increased only with thermal stimulation of tail. The spontaneous calcium activity in right S1 changed more dramatically than that in left S1 after LHS. The lPBN was also activated after LHS, and exploiting chemogenetic activation or inhibition of the neurons in lPBN could induce or alleviate allodynia and hyperalgesia in central neuropathic pain. The neuronal activity changes in S1 are closely related to limb pain, which has accurate anatomical correspondence. After LHS, the spontaneously increased functional connectivity of calcium transient in left S1 is likely causing the mechanical allodynia in right hind limb and increased neuronal activity in bilateral S1 may induce thermal hyperalgesia in tail. This state of allodynia and hyperalgesia can be regulated by lPBN.

Therapeutic administration of mouse mast cell protease 6 improves functional recovery after traumatic spinal cord injury in mice by promoting remyelination and reducing glial scar formation.

Traumatic spinal cord injury (SCI) most often leads to permanent paralysis due to the inability of axons to regenerate in the adult mammalian central nervous system (CNS). In the past, we have shown that mast cells (MCs) improve the functional outcome after SCI by suppressing scar tissue formation at the lesion site via mouse mast cell protease 6 (mMCP6). In this study, we investigated whether recombinant mMCP6 can be used therapeutically to improve the functional outcome after SCI. Therefore, we applied mMCP6 locally via an intrathecal catheter in the subacute phase after a spinal cord hemisection injury in mice. Our findings showed that hind limb motor function was significantly improved in mice that received recombinant mMCP6 compared with the vehicle-treated group. In contrast to our previous findings in mMCP6 knockout mice, the lesion size and expression levels of the scar components fibronectin, laminin, and axon-growth-inhibitory chondroitin sulfate proteoglycans were not affected by the treatment with recombinant mMCP6. Surprisingly, no difference in infiltration of CD4+ T cells and reactivity of Iba-1+ microglia/macrophages at the lesion site was observed between the mMCP6-treated mice and control mice. Additionally, local protein levels of the pro- and anti-inflammatory mediators IL-1β, IL-2, IL-4, IL-6, IL-10, TNF-α, IFNγ, and MCP-1 were comparable between the two treatment groups, indicating that locally applied mMCP6 did not affect inflammatory processes after injury. However, the increase in locomotor performance in mMCP6-treated mice was accompanied by reduced demyelination and astrogliosis in the perilesional area after SCI. Consistently, we found that TNF-α/IL-1β-astrocyte activation was decreased and that oligodendrocyte precursor cell (OPC) differentiation was increased after recombinant mMCP6 treatment in vitro. Mechanistically, this suggests effects of mMCP6 on reducing astrogliosis and improving (re)myelination in the spinal cord after injury. In conclusion, these data show for the first time that recombinant mMCP6 is therapeutically active in enhancing recovery after SCI.

Novel regenerative drug, SPG302 promotes functional recovery of diaphragm muscle activity after cervical spinal cord injury.

Spinal cord hemisection at C2 (C2 SH), sparing the dorsal column is widely used to investigate the effects of reduced phrenic motor neuron (PhMN) activation on diaphragm muscle (DIAm) function, with reduced DIAm activity on the injured side during eupnoea. Following C2 SH, recovery of DIAm EMG activity may occur spontaneously over subsequent days/weeks. Various strategies have been effective at improving the incidence and magnitude of DIAm recovery during eupnoea, but little is known about the effects of C2 SH on transdiaphragmatic pressure (Pdi ) during other ventilatory and non-ventilatory behaviours. We employ SPG302, a novel type of pegylated benzothiazole derivative, to assess whether enhancing synaptogenesis (i.e., enhancing spared local connections) will improve the incidence and the magnitude of recovery of DIAm EMG activity and Pdi function 14 days post-C2 SH. In anaesthetised Sprague-Dawley rats, DIAm EMG and Pdi were assessed during eupnoea, hypoxia/hypercapnia and airway occlusion prior to surgery (C2 SH or sham), immediately post-surgery and at 14 days post-surgery. In C2 SH rats, 14 days of DMSO (vehicle) or SPG302 treatments (i.p. injection) occurred. At the terminal experiment, maximum Pdi was evoked by bilateral phrenic nerve stimulation. We show that significant EMG and Pdi deficits are apparent in C2 SH compared with sham rats immediately after surgery. In C2 SH rats treated with SPG302, recovery of eupneic, hypoxia/hypercapnia and occlusion DIAm EMG was enhanced compared with vehicle rats after 14 days. Treatment with SPG302 also ameliorated Pdi deficits following C2 SH. In summary, SPG302 is an exciting new therapy to explore for use in spinal cord injuries. KEY POINTS: Despite advances in our understanding of the effects of cervical hemisection (C2 SH) on diaphragm muscle (DIAm) EMG activity, very little is understood about the impact of C2 SH on the gamut of ventilatory and non-ventilatory transdiaphragmatic pressures (Pdi ). Recovery of DIAm activity following C2 SH is improved using a variety of approaches, but very few pharmaceuticals have been shown to be effective. One way of improving DIAm recovery is to enhance the amount of latent local spared connections onto phrenic motor neurons. A novel pegylated benzothiazole derivative enhances synaptogenesis in a variety of neurodegenerative conditions. Here, using a novel therapeutic SPG302, we show that 14 days of treatment with SPG302 ameliorated DIAm EMG and Pdi deficits compared with vehicle controls. Our results show that SPG302 is a compound with very promising potential for use in improving functional outcomes post-spinal cord injury.