Blood-spinal Cord Barrier Integrity Research Articles

Exosomes derived from bone marrow mesenchymal stem cells protect the injured spinal cord by inhibiting pericyte pyroptosis.

Mesenchymal stem cell (MSC) transplantation is a promising treatment strategy for spinal cord injury, but immunological rejection and possible tumor formation limit its application. The therapeutic effects of MSCs mainly depend on their release of soluble paracrine factors. Exosomes are essential for the secretion of these paracrine effectors. Bone marrow mesenchymal stem cell-derived exosomes (BMSC-EXOs) can be substituted for BMSCs in cell transplantation. However, the underlying mechanisms remain unclear. In this study, a rat model of T10 spinal cord injury was established using the impact method. Then, 30 minutes and 1 day after spinal cord injury, the rats were administered 200 μL exosomes via the tail vein (200 μg/mL; approximately 1 × 106 BMSCs). Treatment with BMSC-EXOs greatly reduced neuronal cell death, improved myelin arrangement and reduced myelin loss, increased pericyte/endothelial cell coverage on the vascular wall, decreased blood-spinal cord barrier leakage, reduced caspase 1 expression, inhibited interleukin-1β release, and accelerated locomotor functional recovery in rats with spinal cord injury. In the cell culture experiment, pericytes were treated with interferon-γ and tumor necrosis factor-α. Then, Lipofectamine 3000 was used to deliver lipopolysaccharide into the cells, and the cells were co-incubated with adenosine triphosphate to simulate injury in vitro. Pre-treatment with BMSC-EXOs for 8 hours greatly reduced pericyte pyroptosis and increased pericyte survival rate. These findings suggest that BMSC-EXOs may protect pericytes by inhibiting pyroptosis and by improving blood-spinal cord barrier integrity, thereby promoting the survival of neurons and the extension of nerve fibers, and ultimately improving motor function in rats with spinal cord injury. All protocols were conducted with the approval of the Animal Ethics Committee of Zhengzhou University on March 16, 2019.

ALS-causing SOD1 mutants regulate occludin phosphorylation/ubiquitination and endocytic trafficking via the ITCH/Eps15/Rab5 axis

It is increasingly recognized that blood-spinal cord barrier (BSCB) breakdown is a hallmark of amyotrophic lateral sclerosis (ALS). BSCB integrity is disrupted prior to disease onset. Occludin, as the functional component of the endothelial barrier, is downregulated in mouse models expressing ALS-linked superoxide dismutase-1 (SOD1) mutants. However, the molecular mechanisms underlying the regulation of occludin expression remain elusive. Here, using SOD1G93A transgenic mice and endothelial cells expressing SOD1 mutants of different biochemical characteristics, we found that the SOD1 mutation disrupted endothelial barrier integrity and that the occludin expression level was downregulated with disease progression. Our mechanistic studies revealed that abnormal reactive oxygen species (ROS) in mutant SOD1-expressing cells induced occludin phosphorylation, which facilitated the subsequent occludin ubiquitination mediated by the E3 ligase ITCH. Moreover, ubiquitinated occludin interacted with Eps15 to initiate its internalization, then trafficked to Rab5-positive vesicles and be degraded by proteasomes, resulting in a reduction in cell surface localization and total abundance. Notably, either ITCH or Eps15 knockdown was sufficient to rescue occludin degradation and ameliorate endothelial barrier disruption. In conclusion, our study reveals a novel mechanism of occludin degradation mediated by ALS-causing SOD1 mutants and demonstrates a role for occludin in regulating BSCB integrity.

Nuclear heme oxygenase-1 improved the hypoxia-mediated dysfunction of blood-spinal cord barrier via the miR-181c-5p/SOX5 signaling pathway.

Our previous study demonstrated that adenovirus-delivered GFP nuclear heme oxygenase-1 (nuclear HO-1, NHO-1) fragments lacking 23 amino acids at the C-terminus (Ad-GFP-HO-1C[INCREMENT]23) showed the potential therapeutic effects mediated by its improvement of the blood-spinal cord barrier (BSCB) integrity. However, the NHO-1-mediated molecular mechanism in regulating the BSCB function remains unclear. The BSCB model in vitro was established via a coculture of primary rat brain microvascular endothelial cells (RBMECs) and spinal cord astrocytes on transwell system. NHO-1 markedly reduced the disruption of the BSCB integrity induced by hypoxia. And NHO-1 significantly attenuated the expression of miR-181c-5p, but increased the expression level of SOX5 protein. miR-181c-5p was shown as an essential miRNA for increasing the BSCB permeability under hypoxia condition. Furthermore, we identified that miR-181c-5p could regulate the expression of SOX5 through binding to the 3'-UTR of its mRNA. And the decreased BSCB permeability and upregulation of tight junction (TJ) protein expression induced by NHO-1 could be partly reversed by the inhibition of SOX5 or miR-181c-5p (+). The present study results provide a better understanding of the molecular mechanisms induced by NHO-1 in improving the BSCB integrity, which is associated with the regulation of miR-181c-5p/SOX5/TJ signaling pathway.

Delayed administration of high dose human immunoglobulin G enhances recovery after traumatic cervical spinal cord injury by modulation of neuroinflammation and protection of the blood spinal cord barrier

Background/introductionThe neuroinflammatory response plays a major role in the secondary injury cascade after traumatic spinal cord injury (SCI). To date, systemic anti-inflammatory medications such as methylprednisolone sodium succinate (MPSS) have shown promise in SCI. However, systemic immunosuppression can have detrimental side effects. Therefore, immunomodulatory approaches including the use of human immunoglobulin G (hIgG) could represent an attractive alternative. While emerging preclinical data suggests that hIgG is neuroprotective after SCI, the optimal time window of administration and the mechanism of action remain incompletely understood. These knowledge gaps were the focus of this research study. MethodsFemale adult Wistar rats received a clip compression-contusion SCI at the C7/T1 level of the spinal cord. Injured rats were randomized, in a blinded manner, to receive a single intravenous bolus of hIgG (2 g/kg) or control buffer at 15 minutes (min), 1 hour (h) or 4 h post-SCI. At 24 h and 8 weeks post-SCI, molecular, histological and neurobehavioral analyses were undertaken. ResultsAt all 3 administration time points, hIgG (2 g/kg) resulted in significantly better short-term and long-term outcomes as compared to control buffer. No significant differences were observed when comparing outcomes between the different time points of administration. At 24 h post-injury, hIgG (2 g/kg) administration enhanced the integrity of the blood spinal cord barrier (BSCB) by increasing expression of tight junction proteins and reducing inflammatory enzyme expression. Improvements in BSCB integrity were associated with reduced immune cell infiltration, lower amounts of albumin and Evans Blue in the injured spinal cord and greater expression of anti-inflammatory cytokines. Furthermore, hIgG (2 g/kg) increased expression of neutrophil chemoattractants in the spleen and sera. After hIgG (2 g/kg) treatment, there were more neutrophils in the spleen and fewer neutrophils in the blood. hIgG also co-localized with endothelial cell ligands that mediate neutrophil extravasation into the injured spinal cord. Importantly, short-term effects of delayed hIgG (2 g/kg) administration were associated with enhanced tissue and neuron preservation, as well as neurobehavioral and sensory recovery at 8 weeks post-SCI. Discussion and conclusionhIgG (2 g/kg) shows promise as a therapeutic approach for SCI. The anti-inflammatory effects mediated by hIgG (2 g/kg) in the injured spinal cord might be explained in twofold. First, hIgG might antagonize neutrophil infiltration into the spinal cord by co-localizing with endothelial cell ligands that mediate various steps in neutrophil extravasation. Second, hIgG could traffic neutrophils towards the spleen by increasing expression of neutrophil chemoattractants in the spleen and sera. Overall, we demonstrate that delayed administration of hIgG (2 g/kg) at 1 and 4-h post-injury enhances short-term and long-term benefits after SCI by modulating local and systemic neuroinflammatory cascades.

Retraction Note to: Retinoic Acid Prevents Disruption of Blood-Spinal Cord Barrier by Inducing Autophagic Flux After Spinal Cord Injury.

This article has been retracted. Please see the Retraction Notice for more detail: https://doi.org/10.1007/s11064-020-03149-1 .

Spinal cannabinoid receptor 2 activation reduces hypersensitivity associated with bone cancer pain and improves the integrity of the blood–spinal cord barrier

BackgroundDisruption of the blood–spinal cord barrier (BSCB) can facilitate inflammation that results in pain hypersensitivity. Proinflammatory cytokines produced by activated microglia and astrocytes damage the BSCB. This study aims to...

Multiplexed chemogenetics in astrocytes and motoneurons restore blood-spinal cord barrier in ALS.

Blood-spinal cord barrier (BSCB) disruption is thought to contribute to motoneuron (MN) loss in amyotrophic lateral sclerosis (ALS). It is currently unclear whether impairment of the BSCB is the cause or consequence of MN dysfunction and whether its restoration may be directly beneficial. We revealed that SOD1 G93A , FUS ΔNLS , TDP43 G298S , and Tbk1 +/- ALS mouse models commonly shared alterations in the BSCB, unrelated to motoneuron loss. We exploit PSAM/PSEM chemogenetics in SOD1 G93A mice to demonstrate that the BSCB is rescued by increased MN firing, whereas inactivation worsens it. Moreover, we use DREADD chemogenetics, alone or in multiplexed form, to show that activation of Gi signaling in astrocytes restores BSCB integrity, independently of MN firing, with no effect on MN disease markers and dissociating them from BSCB disruption. We show that astrocytic levels of the BSCB stabilizers Wnt7a and Wnt5a are decreased in SOD1 G93A mice and strongly enhanced by Gi signaling, although further decreased by MN inactivation. Thus, we demonstrate that BSCB impairment follows MN dysfunction in ALS pathogenesis but can be reversed by Gi-induced expression of astrocytic Wnt5a/7a.

Gallic acid attenuates blood-spinal cord barrier disruption by inhibiting Jmjd3 expression and activation after spinal cord injury

After spinal cord injury (SCI), blood-spinal cord barrier (BSCB) disruption results in secondary injury including apoptotic cell death of neurons and oligodendrocytes, thereby leads to permanent neurological deficits. Recently, we reported that the histone H3K27me3 demethylase Jmjd3 plays a role in regulating BSCB integrity after SCI. Here, we investigated whether gallic acid (GA), a natural phenolic compound that is known to be anti-inflammatory, regulates Jmjd3 expression and activation, thereby attenuates BSCB disruption following the inflammatory response and improves functional recovery after SCI. Rats were contused at T9 and treated with GA (50 mg/kg) via intraperitoneal injection immediately, 6 h and 12 h after SCI, and further treated for 7 d with the same dose once a day. To elucidate the underlying mechanism, we evaluated Jmjd3 activity and expression, and assessed BSCB permeability by Evans blue assay after SCI. GA significantly inhibited Jmjd3 expression and activation after injury both in vitro and in vivo. GA also attenuated the expression and activation of matrix metalloprotease-9, which is well known to disrupt the BSCB after SCI. Consistent with these findings, GA attenuated BSCB disruption and reduced the infiltration of neutrophils and macrophages compared with the vehicle control. Finally, GA significantly alleviated apoptotic cell death of neurons and oligodendrocytes and improved behavior functions. Based on these data, we propose that GA can exert a neuroprotective effect by inhibiting Jmjd3 activity and expression followed the downregulation of matrix metalloprotease-9, eventually attenuating BSCB disruption after SCI.

A validated quantitative method for the assessment of neuroprotective barrier impairment in neurodegenerative disease models.

The blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB) are highly specialized structures that limit molecule entry from the blood and maintain homeostasis within the central nervous system (CNS). BBB and BSCB breakdown are associated with multiple neurodegenerative diseases. Given the key role of neuroprotective barrier impairment in neurodegeneration, it is important to identify an effective quantitative method to assess barrier integrity in animal models. In this study, we developed and validated a quantitative method for assessing BBB and BSCB integrity using sodium fluorescein, a compound that outperformed other fluorescent dyes. We demonstrated using this method that multiple CNS regions progressively increase in permeability in models of Huntington's disease and amyotrophic lateral sclerosis, whereas biphasic disruption occurred in a mouse model of Alzheimer's disease with disease progression. Collectively, we report a quantitative fluorometric marker with validated reproducible experimental methods that allows the effective assessment of BBB and BSCB integrity in animal models. This method could be useful to further the understanding of the contribution of these neuroprotective barriers to neurodegeneration processes.

CO-Releasing Molecule (CORM)-3 Ameliorates Spinal Cord-Blood Barrier Disruption Following Injury to the Spinal Cord.

Spinal cord injury (SCI) is a clinical tough neurological problem without efficient cure currently. Blood-spinal cord barrier (BSCB) interruption is not only a crucial pathological feature for SCI process but is a possible target for future SCI treatments; however, few treatments have been developed to intervene BSCB. In the present study, we intravenously injected CO-releasing molecule3 (CORM-3), a classical exogenous CO donor, to the rats experiencing SCI and assessed its protection on BSCB integrity in rats. Our results demonstrated that the exogenous increasing of CO by CORM-3 blocked the tight junction (TJ) protein degeneration and neutrophils infiltration, subsequently suppressed the BSCB damage and improved the motor recovery after SCI. And we certified that the CO-induced down-regulation of MMP-9 expression and activity in neutrophil might be associated with the NF-κB signaling. Taken together, our study indicates that CO-releasing molecule (CORM)-3 ameliorates BSCB after spinal cord injury.

Tetramethylpyrazine Protects Blood-Spinal Cord Barrier Integrity by Modulating Microglia Polarization Through Activation of STAT3/SOCS3 and Inhibition of NF-кB Signaling Pathways in Experimental Autoimmune Encephalomyelitis Mice.

We previously reported that tetramethylpyrazine (TMP) alleviates experimental autoimmune encephalomyelitis (EAE) by decreasing glia activation. Activated microglia has been shown to mediate blood-spinal cord barrier (BSCB) disruption, which is a primary and continuous pathological characteristic of multiple sclerosis (MS). Therefore, in this study, we further investigated whether TMP protects the BSCB integrity by inhibition of glia activation to alleviate EAE. Extravasation of evans blue was used to detect the BSCB disruption. Tumor necrosis factor-α (TNF-α)/interlukine-1β (IL-1β) and interlukine-4 (IL-4)/interlukine-10 (IL-10) were determined by enzyme-linked immunosorbent assay. BV2 glial cells stimulated by interferon-γ (IFN-γ) were co-cultured with human brain microvascular endothelial cells to investigate the effect of TMP on the BSCB disruption. Flow cytometry was used to analyze the microglia phenotype. Western blot was performed to reveal the signaling pathways involved in the microglia activation. In this study, most importantly, we found that TMP protects the BSCB integrity by modulating microglia polarization from M1 phenotype to M2 phenotype through activation of STAT3/SOCS3 and inhibition of NF-кB signaling pathways. Moreover, TMP significantly preserves the tight junction proteins, reduces the secretion of pro-inflammatory cytokines (TNF-α, IL-1β) and increases the secretion of anti-inflammatory cytokines (IL-4, IL-10) from IFN-γ-stimulated BV2 microglia cells. Consequently, protection of the BSCB integrity leads to alleviation of clinical symptoms and demyelination in EAE mice. Therefore, TMP might be an effective therapeutic agent for cerebral disorders with BBB or BSCB disruption, such as ischemic stroke, MS, and traumatic brain injury.

CORM-2-Solid Lipid Nanoparticles Maintain Integrity of Blood-Spinal Cord Barrier After Spinal Cord Injury in Rats.

Spinal cord injury (SCI) is a devastating condition of the central nervous system that can lead to permanent motor and sensory deficits. Carbon monoxide-releasing molecule-2 (CORM-2) has been shown to have anti-inflammatory, anti-apoptotic, and angiogenic properties that may be useful for the treatment of SCI. However, it has a short carbon monoxide (CO) release half-life (approximately 1min). To address this challenge, we developed a CORM-2-incorporated solid lipid nanoparticle (CORM-2-SLN) and evaluated its ameliorating effects for preventing blood-spinal cord barrier (BSCB) disruption and endothelial cell death following SCI. After a moderate compression injury of the spinal cord (compression with a 35-g impounder for 5min), groups of rats were treated with a CORM-2-solution and CORM-2-SLNs at an equal dose of 10mg/kg each via an intraperitoneal injection for 8 consecutive days. Behavior analysis was performed and animals were later sacrificed at different time points and evaluated for whether the CORM-2-SLNs prevented BSCB disruption and rescued endothelial cell damage following SCI. The CORM-2-SLN-treated group showed significantly diminished extravasation of Evans Blue dye with enhanced expression of tight junction proteins following SCI. Likewise, significantly diminished endothelial cell markers after SCI were optimally stabilized at 21days. Additionally, lipopolysaccharide (LPS)-induced loss of tight junction integrity was significantly preserved after CORM-2-SLN treatment in human cerebral microvascular endothelial cell line (hCMEC/D3). Clinically, CORM-2-SLNs were associated with a significantly improved functional recovery, as compared with the CORM-2-solution. CORM-2-SLNs may help potentially to maintain BSCB integrity following SCI.

Exogenous activation of cannabinoid-2 receptor modulates TLR4/MMP9 expression in a spinal cord ischemia reperfusion rat model

BackgroundCannabinoid-2 receptor (CB2R) plays an important role in the cascading inflammation following ischemic injury. The toll-like receptors 4 (TLR4)/matrix metalloproteinase 9 (MMP9) signal pathway is involved in blood-brain barrier dysfunction induced by ischemia stroke. The aim of this study is to investigate the roles of exogenous activation of CB2R on attenuating neurological deficit and blood-spinal cord barrier (BSCB) disruption during rat spinal cord ischemia reperfusion (I/R) injury, through modulation of the TLR4/MMP9 axis.MethodsAnimals were intraperitoneally pretreated with TLR4 inhibitor TAK-242, CB2R agonist JWH-133 with or without CB2R antagonist AM630, or equivalent volume of vehicle 1 h before undergoing 14-min occlusion of descending aorta or sham operation. One, two, three, and 7 days after reperfusion, hindlimb locomotor function was evaluated with Basso, Beattie, and Bresnahan (BBB) Locomotor Scale, BSCB integrity was detected by measurement of Evans blue (EB) extravasation and spinal cord edema. The protein expression levels of CB2R, tight junction protein Zonula occluden-1 (ZO-1), TLR4, MMP9, MyD88, NF-κB p65, and NF-κB p-p65 were determined by western blot. The MMP9 activity was analyzed by gelatin zymography. Double immunofluorescence staining was used to identify the perivascular localization of CB2R, TLR4, MMP9, and reactive astrocytes, as well as the colocalization of CB2R, TLR4, and MMP9 with reactive astrocytes.ResultsJWH-133 pretreatment attenuated hindlimb motor functional deficit and BSCB leakage, along with preventing downregulation of ZO-1 and upregulation of TLR4/MMP9, similar to the effects of TAK-242 preconditioning. JWH-133 or TAK-242 pretreatment reduced the perivascular expression of TLR4/MMP9 and reactive astrocytes following injury. JWH-133 pretreatment also downregulated MyD88/NF-κB level, MMP9 activity, and the astrocytic TLR4/MMP9 after I/R injury.ConclusionsExogenous activation of CB2R by JWH-133 attenuated neurological deficit and BSCB disruption after spinal cord I/R injury via inhibition of TLR4/MMP9 expression.

C-X-C Motif Chemokine 10 Contributes to the Development of Neuropathic Pain by Increasing the Permeability of the Blood-Spinal Cord Barrier.

Neuropathic pain is among the most debilitating forms of chronic pain. Studies have suggested that chronic pain pathogenesis involves neuroimmune interactions and blood–spinal cord barrier (BSCB) disruption. However, the underlying mechanisms are poorly understood. We modeled neuropathic pain in rats by inducing chronic constriction injury (CCI) of the sciatic nerve and analyzed the effects on C-X-C motif chemokine 10 (CXCL10)/CXCR3 activation, BSCB permeability, and immune cell migration from the circulation into the spinal cord. We detected CXCR3 expression in spinal neurons and observed that CCI induced CXCL10/CXCR3 activation, BSCB disruption, and mechanical hyperalgesia. CCI-induced BSCB disruption enabled circulating T cells to migrate into the spinal parenchyma. Intrathecal administration of an anti-CXCL10 antibody not only attenuated CCI-induced hyperalgesia, but also reduced BSCB permeability, suggesting that CXCL10 acts as a key regulator of BSCB integrity. Moreover, T cell migration may play a critical role in the neuroimmune interactions involved in the pathogenesis of CCI-induced neuropathic pain. Our results highlight CXCL10 as a new potential drug target for the treatment of nerve injury–induced neuropathic pain.

Elevated TRPV4 Levels Contribute to Endothelial Damage and Scarring in Experimental Spinal Cord Injury.

Currently, the role of transient receptor potential vanilloid type 4 (TRPV4), a nonselective cation channel in the pathology of spinal cord injury (SCI), is not recognized. Herein, we report the expression and contribution of TRPV4 in the pathology of scarring and endothelial and secondary damage after SCI. TRPV4 expression increased during the inflammatory phase in female rats after SCI and was expressed primarily by cells at endothelial-microglial junctions. Two-photon microscopy of intracellular-free Ca2+ levels revealed a biphasic increase at similar time points after SCI. Expression of TRPV4 at the injury epicenter, but not intracellular-free Ca2+, progressively increases with the severity of the injury. Activation of TRPV4 with specific agonist altered the organization of endothelial cells, affected tight junctions in the hCMEC/D3 BBB cell line in vitro, and increases the scarring in rat spinal cord as well as induced endothelial damage. By contrast, suppression of TRPV4 with a specific antagonist or in female Trpv4 KO mouse attenuated inflammatory cytokines and chemokines, prevented the degradation of tight junction proteins, and preserve blood-spinal cord barrier integrity, thereby attenuate the scarring after SCI. Likewise, secondary damage was reduced, and behavioral outcomes were improved in Trpv4 KO mice after SCI. These results suggest that increased TRPV4 expression disrupts endothelial cell organization during the early inflammatory phase of SCI, resulting in tissue damage, vascular destabilization, blood-spinal cord barrier breakdown, and scarring. Thus, TRPV4 inhibition/knockdown represents a promising therapeutic strategy to stabilize/protect endothelial cells, attenuate nociception and secondary damage, and reduce scarring after SCI.SIGNIFICANCE STATEMENT TRPV4, a calcium-permeable nonselective cation channel, is widely expressed in both excitable and nonexcitable cells. Spinal cord injury (SCI) majorly caused by trauma/accidents is associated with changes in osmolarity, mechanical injury, and shear stress. After SCI, TRPV4 was increased and were found to be linked with the severity of injury at the epicenter at the time points that were reported to be critical for repair/treatment. Activation of TRPV4 was damaging to endothelial cells that form the blood-spinal cord barrier and thus contributes to scarring (glial and fibrotic). Importantly, inhibition/knockdown of TRPV4 prevented these effects. Thus, the manipulation of TRPV4 signaling might lead to new therapeutic strategies or combinatorial therapies to protect endothelial cells and enhance repair after SCI.

Vascularization of self-assembled peptide scaffolds for spinal cord injury repair

The disruption of the blood-spinal cord barrier (BSCB) following spinal cord injury contributes to inflammation and glial scarring that inhibits axon growth and diminishes the effectiveness of conduits transplanted to the injury site to promote this growth. The purpose of this study is to evaluate whether scaffolds containing microvessels that exhibit BSCB integrity reduce inflammation and scar formation at the injury site and lead to increased axon growth. For these studies, a self-assembling peptide scaffold, RADA-16I, is used due to its established permissiveness to axon growth and ability to support vascularization. Immunocytochemistry and permeability transport assays verify the formation of tight-junction containing microvessels within the scaffold. Peptide scaffolds seeded with different concentrations of microvascular cells are then injected into a spinal contusion injury in rats to evaluate how microvessels affect axon growth and neurovascular interaction. The effect of the vascularized scaffold on inflammation and scar formation is evaluated by quantifying histological sections stained with ED-1 and GFAP, respectively. Our results indicate that the peptide scaffolds containing microvessels reduce inflammation and glial scar formation and increase the density of axons growing into the injury/transplant site. These results demonstrate the potential benefit of scaffold vascularization to treat spinal cord injury. Statement of SignificanceThis study evaluates the benefit of transplanting microvascular cells within a self-assembling peptide scaffold, RADA-16I, that has shown promise for facilitating regeneration in the central nervous system in previous studies. Our results indicate that vasculature featuring tight junctions that give rise to the blood-spinal cord barrier can be formed within the peptide scaffold both in vitro and in a rat model of a subacute contusion spinal cord injury. Histological analysis indicates that the presence of the microvessels encourages axon infiltration into the site of injury and reduces the area of astrocyte activation and inflammation. Overall, these results demonstrate the potential of vascularizing scaffolds for the repair of spinal cord injury.

5-hydroxytryptamine 1F Receptor Agonist Induces Mitochondrial Biogenesis and Promotes Recovery from Spinal Cord Injury.

Spinal cord injury (SCI) is characterized by vascular disruption leading to ischemia, decreased oxygen delivery, and loss of mitochondrial homeostasis. This mitochondrial dysfunction results in loss of cellular functions, calcium overload, and oxidative stress. Pharmacological induction of mitochondrial biogenesis (MB) may be an effective approach to treat SCI. LY344864, a 5-hydroxytryptamine 1F (5-HT1F) receptor agonist, is a potent inducer of MB in multiple organ systems. To assess the efficacy of LY344864-induced MB on recovery post-SCI, female mice were subjected to moderate force-controlled impactor-induced contusion SCI followed by daily LY344864 administration for 21 days. Decreased mitochondrial DNA and protein content was present in the injury site 3 days post-SCI. LY344864 treatment beginning 1 h after injury attenuated these decreases, indicating MB. Additionally, injured mice treated with LY344864 displayed decreased Evan's Blue dye accumulation in the spinal cord compared with vehicle-treated mice 7 days after injury, suggesting restoration of vascular integrity. LY344864 also increased locomotor capability, with treated mice reaching a Basso-Mouse Scale score of 3.4 by 21 days, whereas vehicle-treated mice exhibited a score of 1.9. Importantly, knockout of the 5-HT1F receptor blocked LY344864-induced recovery. Remarkably, a similar degree of locomotor restoration was observed when treatment initiation was delayed until 8 h after injury. Furthermore, cross-sectional analysis of the spinal cord 21 days after injury revealed decreased lesion volume with delayed LY344864 treatment initiation, emphasizing the potential clinical applicability of this therapeutic approach. These data provide evidence that induction of MB via 5-HT1F receptor agonism may be a promising strategy for the treatment of SCI. SIGNIFICANCE STATEMENT: Treatment with LY344864 induces mitochondrial biogenesis in both the naive and injured mouse spinal cord. In addition, treatment with LY344864 beginning after impactor-induced contusion spinal cord injury improves mitochondrial homeostasis, blood-spinal cord barrier integrity, and locomotor function within 7 days. Importantly, similar locomotor results are observed whether treatment is initiated at 1 h after injury or 8 h after injury. These data indicate the potential for pharmacological induction of mitochondrial biogenesis through a 5-hydroxytryptamine 1F agonist as a novel therapeutic approach for spinal cord injury.

Brilliant Blue G Inhibits Inflammasome Activation and Reduces Disruption of Blood-Spinal Cord Barrier Induced by Spinal Cord Injury in Rats.

BackgroundBrilliant blue G (BBG) is a P2X7 receptor inhibitor that has been reported to improve spinal cord injury (SCI) in previous studies, but the specific mechanism has been unclear. In this study, we investigated the effects of BBG on inflammasomes and blood–spinal cord barrier (BSCB) permeability after SCI.Material/MethodsThe experimental rats were randomly divided into 3 groups: sham, SCI, and SCI+BBG. The expression of P2X7 and inflammasome-related proteins was measured by Western blot and immunohistochemistry, while IL-1β and IL-18 levels were measured by using an enzyme-linked immunosorbent assay (ELISA) kit. The permeability of the BSCB was evaluated by Evans Blue (EB) exosmosis, and histological alterations were observed by hematoxylin-eosin staining. Motor function recovery was assessed by the Basso, Beattie, Bresnahan (BBB) scale after SCI.ResultsThe expression levels of P2X7, NLRP3, ASC, cleaved XIAP, caspase-1, caspase-11, IL-1β, and IL-18 were increased significantly after SCI, and BBG administration inhibited this increase at 72 h after SCI. BBG administration significantly reduced EB leakage at 24 h after SCI. Furthermore, treatment with BBG significantly attenuated histological alterations and improved motor function recovery after SCI.ConclusionsBBG administration promoted motor function recovery and alleviated tissue injury, and these effects might be related to the suppression of inflammasomes and the maintenance of BSCB integrity.

Inhibiting aberrant p53-PUMA feedback loop activation attenuates ischaemia reperfusion-induced neuroapoptosis and neuroinflammation in rats by downregulating caspase 3 and the NF-\u03baB cytokine pathway

BackgroundIschaemia reperfusion (IR) induces multiple pathophysiological changes. In addition to its classical role in regulating tumourigenesis, the feedback loop formed by p53 and its driven target p53-upregulated modulator of apoptosis (PUMA) was recently demonstrated to be the common node tightly controlling various cellular responses during myocardial IR. However, the roles of the p53-PUMA feedback loop in the spinal cord remain unclear. This study aimed to elucidate the roles of p53-PUMA feedback interactions in the spinal cord after IR, specifically investigating their regulation of caspase 3-mediated apoptosis and nuclear factor (NF)-κB-mediated cytokine release.MethodsSD rats subjected to 12 min of aortic arch occlusion served as IR models. Neurological assessment as well as p53 and PUMA mRNA and protein expression analyses were performed at 12-h intervals during a 48-h reperfusion period. The cellular distributions of p53 and PUMA were determined via double immunofluorescence staining. The effects of the p53-PUMA feedback loop on modulating hind-limb function; the number of TUNEL-positive cells; and protein levels of caspase 3, NF-κB and cytokines interleukin (IL)-1β and tumour necrosis factor (TNF)-α, were evaluated by intrathecal treatment with PUMA-specific or scramble siRNA and pifithrin (PFT)-α. Blood-spinal cord barrier (BSCB) breakdown was examined by Evans blue (EB) extravasation and water content analyses.ResultsIR induced significant behavioural deficits as demonstrated by deceased Tarlov scores, which displayed trends opposite those of PUMA and p53 protein and mRNA expression. Upregulated PUMA and p53 fluorescent labels were widely distributed in neurons, astrocytes and microglia. Injecting si-PUMA and PFT-α exerted significant anti-apoptosis effects as shown by the reduced number of TUNEL-positive cells, nuclear abnormalities and cleaved caspase 3 levels at 48 h post-IR. Additionally, p53 colocalized with NF-κB within the cell. Similarly, injecting si-PUMA and PFT-α exerted anti-inflammatory effects as shown by the decreased NF-κB translocation and release of IL-1β and TNF-α. Additionally, injecting si-PUMA and PFT-α preserved the BSCB integrity as determined by decreased EB extravasation and spinal water content. However, injecting si-Con did not induce any of the abovementioned effects.ConclusionsInhibition of aberrant p53-PUMA feedback loop activation by intrathecal treatment with si-PUMA and PFT-α prevented IR-induced neuroapoptosis, inflammatory responses and BSCB breakdown by inactivating caspase 3-mediated apoptosis and NF-κB-mediated cytokine release.

Hydrogen Sulfide Ameliorates Blood-Spinal Cord Barrier Disruption and Improves Functional Recovery by Inhibiting Endoplasmic Reticulum Stress-Dependent Autophagy.

Spinal cord injury (SCI) induces the disruption of blood-spinal cord barrier (BSCB), which elicits neurological deficits by triggering secondary injuries. Hydrogen sulfide (H2S) is a gaseous mediator that has been reported to have neuroprotective effect in the central nervous system. However, the relationship between H2S and BSCB disruption during SCI remains unknown. Therefore, it is interesting to evaluate whether the administration of NaHS, a H2S donor, can protect BSCB integrity against SCI and investigate the potential mechanisms underlying it. In present study, we found that SCI markedly activated endoplasmic reticulum (ER) stress and autophagy in a rat model of complete crushing injury to the spinal cord at T9 level. NaHS treatment prevented the loss of tight junction (TJ) and adherens junction (AJ) proteins both in vivo and in vitro. However, the protective effect of NaHS on BSCB restoration was significantly reduced by an ER stress activator (tunicamycin, TM) and an autophagy activator (rapamycin, Rapa). Moreover, SCI-induced autophagy was remarkably blocked by the ER stress inhibitor (4-phenylbutyric acid, 4-PBA). But the autophagy inhibitor (3-Methyladenine, 3-MA) only inhibited autophagy without obvious effects on ER stress. Finally, we had revealed that NaHS significantly alleviated BSCB permeability and improved functional recovery after SCI, and these effects were markedly reversed by TM and Rapa. In conclusion, our present study has demonstrated that NaHS treatment is beneficial for SCI recovery, indicating that H2S treatment is a potential therapeutic strategy for promoting SCI recovery.