Blood-spinal Cord Barrier Dysfunction Research Articles

An inhibitor of claudin-5 interactions, M01, alleviates neuroinflammation and vasogenic edema after blood-spinal cord barrier dysfunction.

Molecular docking modeling has confirmed that M01 (C30H28N4O5) acts as a potent inhibitor of claudin-5. Our prior data indicated that claudin-5 is essential to the structural integrity of the blood-spinal cord barrier (BSCB). The aim of this study was to investigate the effect of M01 on the integrity of the BSCB and its effect on neuroinflammation and vasogenic edema after blood-spinal cord barrier dysfunction in in-vitro and in-vivo models. Transwell chambers were used to construct an in-vitro model of the BSCB. Fluorescein isothiocyanate (FITC)-dextran permeability and leakage assays were performed to validate the reliability of the BSCB model. Semiquantitative analysis of inflammatory factor expression and nuclear factor-κB signaling pathway protein levels was performed using western blotting. The transendothelial electrical resistance of each group was measured, and the expression of a tight junction protein ZO-1 was determined via immunofluorescence confocal microscopy. Rat models of spinal cord injury were established by the modified Allen's weight-drop method. Histological analysis was carried out by hematoxylin and eosin staining. Locomotor activity was evaluated with Footprint analysis and the Basso-Beattie-Bresnahan scoring system. The M01 (10 μM) reduced the release of inflammatory factors and degradation of ZO-1 and improved the integrity of the BSCB by reversing vasogenic edema and leakage. M01 may represent a new strategy for the treatment of diseases related to BSCB destruction.

Blood-Spinal Cord Barrier: Its Role in Spinal Disorders and Emerging Therapeutic Strategies.

The blood-spinal cord barrier (BSCB) has been long thought of as a functional equivalent to the blood-brain barrier (BBB), restricting blood flow into the spinal cord. The spinal cord is supported by various disc tissues that provide agility and has different local immune responses compared to the brain. Though physiologically, structural components of the BSCB and BBB share many similarities, the clinical landscape significantly differs. Thus, it is crucial to understand the composition of BSCB and also to establish the cause-effect relationship with aberrations and spinal cord dysfunctions. Here, we provide a descriptive analysis of the anatomy, current techniques to assess the impairment of BSCB, associated risk factors and impact of spinal disorders such as spinal cord injury (SCI), amyotrophic lateral sclerosis (ALS), peripheral nerve injury (PNI), ischemia reperfusion injury (IRI), degenerative cervical myelopathy (DCM), multiple sclerosis (MS), spinal cavernous malformations (SCM) and cancer on BSCB dysfunction. Along with diagnostic and mechanistic analyses, we also provide an up-to-date account of available therapeutic options for BSCB repair. We emphasize the need to address BSCB as an individual entity and direct future research towards it.

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.

A pilot study of microRNA expression profiles of the spinal neuron in matrix metalloproteinase-9 knockout mice

Introduction: Matrix metalloproteinase-9 (MMP9) plays a key role in blood–spinal cord barrier dysfunction. MMP9 blockade leads to improved injured locomotor recovery. However, it is still unknown whether MMP9 deficiency affects gene expression or signal transduction pathways in the spinal neuron. Material and Methods: In this study, we first screen the MMP9 knockdown mice with high superoxide dismutase (SOD) expression and low MMP9 expression by polymerase chain reaction analysis. Then, the gene microarrays were used to screen differentially expressed genes in the spinal neuron from MMP-9 knockout and wild-type mice. There were six groups in this experiment, including three negative control groups: SOD_1, SOD_2, and SOD_3, and three experiment groups: SOD_ MMP9_1, SOD_ MMP9_2, and SOD_ MMP9_3. The gene ontology terms were used to predict the potential functions of these differentially expressed genes, and the Kyoto Encyclopedia of Genes and Genomes was used to analyze the potential functions of these target genes in the pathways. Results: We found that the gene expression in the spinal neuron from MMP9 knockout mice was significantly altered compared to wild-type mice. FoxO signaling, axon guidance, ubiquitin-mediated proteolysis, regulation of actin cytoskeleton, and proteoglycans were changed. Discussion and Conclusion: In summary, MMP9 plays a role in spinal neuron signaling and the underlying mechanism may through affecting several signaling pathways.

Mutant huntingtin protein expression and blood-spinal cord barrier dysfunction in huntington disease.

The aim of the study was to assess the distribution, frequency, and specific location of mutant huntingtin protein (mHTT) aggregates-the pathological hallmark of Huntington disease (HD)-within the various compartments of the spinal cord and their potential impact on the local vasculature and blood-spinal cord barrier (BSCB). We performed a series of postmortem immunohistochemical and immunofluorescent stainings, as well as Western blot analyses, on cervical and lumbar sections of the spinal cord in patients diagnosed with HD (n = 11 of all grades of disease severity) along with sex- and age-matched healthy controls (n = 9). We observed that mHTT was preferably expressed within the anterior horn of the gray matter, in both cervical and lumbar sections. At the cellular level, mHTT aggregates were more often encountered in the extracellular matrix but could also be observed within cell bodies and neurites as well as within the endothelium of blood vessels with an increase in the density of small blood vessels in cervical sections of HD cases. These vasculature changes were accompanied with features of BSCB leakage, as assessed by the presence of increased levels of fibrinogen in the surrounding parenchyma and enhanced leukocyte infiltration. This alteration in BSCB integrity may be explained, in part, by the dysregulation we found in some of the main proteins associated with it such as junctional adhesion molecule-1 and vascular endothelial cadherin. These observations have important implications for our understanding of HD pathology and may also have significant therapeutic implications. Ann Neurol 2017;82:981-994.

MiR-320a affects spinal cord edema through negatively regulating aquaporin-1 of blood-spinal cord barrier during bimodal stage after ischemia reperfusion injury in rats.

BackgroundSpinal cord edema is a serious complication and pathophysiological change after ischemia reperfusion (IR) injury. It has been demonstrated closely associated with bimodal disruption of blood–spinal cord barrier (BSCB) in our previous work. Aquaporin (AQP)1 plays important but contradictory roles in water homeostasis. Recently, microRNAs (miRs) effectively regulate numerous target mRNAs during ischemia. However, whether miRs are able to protect against dimodal disruption of BSCB by regulating perivascular AQP1 remains to be elucidated. ResultsSpinal water content and EB extravasation were suggested as a bimodal distribution in directly proportion to AQP1, since all maximal changes were detected at 12 and 48 h after reperfusion. Further TEM and double immunofluorescence showed that former disruption of BSCB at 12 h was attributed to cytotoxic edema by up-regulated AQP1 expressions in astrocytes, whereas the latter at 48 h was mixed with vasogenic edema with both endothelial cells and astrocytes involvement. Microarray analysis revealed that at 12 h post-injury, ten miRs were upregulated (>2.0 fold) and seven miRs were downregulated (<0.5 fold) and at 48 h, ten miRs were upregulated and eleven were downregulated compared to Sham-operated controls. Genomic screening and luciferase assays identified that miR-320a was a potential modulator of AQP1 in spinal cord after IR in vitro. In vivo, compared to rats in IR and negative control group, intrathecal infusion of miR-320a mimic attenuated IR-induced lower limb motor function deficits and BSCB dysfunction as decreased EB extravasation and spinal water content through down-regulating AQP1 expressions, whereas pretreated with miR-320a AMO reversed above effects. ConclusionThese findings indicate miR-320a directly and functionally affects spinal cord edema through negatively regulating AQP1 of BSCB after IR.

Presymptomatic activation of the PDGF-CC pathway accelerates onset of ALS neurodegeneration.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with unknown origins. Neurodegeneration in ALS mouse models occurs together with signs of disrupted blood-spinal cord barrier (BSCB) and regressed capillary network, but the molecular pathways contributing to these vascular pathologies remain unknown. We show that motor neurons of human sporadic ALS patients (n=12) have increased gene expression of PDGFC and its activator PLAT and presymptomatic activation of the PDGF-CC pathway in SOD1 (G93A) mice leads to BSCB dysfunction. Decrease of Pdgfc expression in SOD1 (G93A) mice restored vascular barrier properties, reduced motor neuron loss and delayed symptom onset by up to 3weeks. Similarly, lower expression levels of PDGFC or PLAT in motor neurons of sporadic ALS patients were correlated with older age at disease onset. PDGF-CC inhibition and restoration of BSCB integrity did not prevent capillary regression at disease end stage. Lower vessel density was found in spinal cords of sporadic ALS patients and the degree of regression in SOD1 (G93A) mice correlated with more aggressive progression after onset regardless of BSCB status. We conclude that PDGF-CC-induced BSCB dysfunction can contribute to timing of ALS onset, allow insight into disease origins and development of targeted novel therapies.

Role of the TLR4 pathway in blood-spinal cord barrier dysfunction during the bimodal stage after ischemia/reperfusion injury in rats

BackgroundSpinal cord ischemia-reperfusion (I/R) involves two-phase injury, including an initial acute ischemic insult and subsequent inflammatory reperfusion injury, resulting in blood-spinal cord barrier (BSCB) dysfunction involving the TLR4 pathway. However, the correlation between TLR4/MyD88-dependent and TLR4/TRIF-dependent pathways in BSCB dysfunction is not fully understood. The aim of this study is to characterize inflammatory responses in spinal cord I/R and the events that define its clinical progression with delayed neurological deficits, supporting a bimodal mechanism of injury.MethodsRats were intrathecally pretreated with TAK-242, MyD88 inhibitory peptide, or Resveratrol at a 12 h interval for 3 days before undergoing 14-minute occlusion of aortic arch. Evan’s Blue (EB) extravasation and water content were detected at 6, 12, 18, 24, 36, 48, and 72 h after reperfusion. EB extravasation, water content, and NF-κB activation were increased with time after reperfusion, suggesting a bimodal distribution, as maximal increasing were detected at both 12 and 48 h after reperfusion. The changes were directly proportional to TLR4 levels determined by Western blot. Double-labeled immunohistochemical analysis was also used to detect the relationship between different cell types of BSCB with TLR4. Furthermore, NF-κB and IL-1β were analyzed at 12 and 48 h to identify the correlation between MyD88-dependent and TRIF-dependent pathways.ResultsRats without functional TLR4 and MyD88 attenuated BSCB leakage and inflammatory responses at 12 h, suggesting the ischemic event was largely mediated by MyD88-dependent pathway. Similar protective effects observed in rats with depleted TLR4, MyD88, and TRIF receptor at 48 h infer that the ongoing inflammation which occurred in late phase was mainly initiated by TRIF-dependent pathway and such inflammatory response could be further amplified by MyD88-dependent pathway. Additionally, microglia appeared to play a major role in early phase of inflammation after I/R injury, while in late responding phase both microglia and astrocytes were necessary.ConclusionsThese findings indicate the relevance of TLR4/MyD88-dependent and TLR4/TRIF-dependent pathways in bimodal phases of inflammatory responses after I/R injury, corresponding with the clinical progression of injury and delayed onset of symptoms. The clinical usage of TLR4 signaling inhibitors at different phases may be a therapeutic option for the prevention of delayed injury.

The blood–spinal cord barrier: Morphology and Clinical Implications

The blood-spinal cord barrier (BSCB) is the functional equivalent of the blood-brain barrier (BBB) in the sense of providing a specialized microenvironment for the cellular constituents of the spinal cord. Even if intuitively the BSCB could be considered as the morphological extension of the BBB into the spinal cord, evidence suggests that this is not so. The BSCB shares the same principal building blocks with the BBB; nevertheless, it seems that morphological and functional differences may exist between them. Dysfunction of the BSCB plays a fundamental role in the etiology or progression of several pathological conditions of the spinal cord, such as spinal cord injury, amyotrophic lateral sclerosis, and radiation-induced myelopathy. This review summarizes current knowledge of the morphology of the BSCB, the methodology of studying the BSCB, and the potential role of BSCB dysfunction in selected disorders of the spinal cord, and finally summarizes therapeutic approaches to the BSCB.

Evaluating regional blood spinal cord barrier dysfunction following spinal cord injury using longitudinal dynamic contrast-enhanced MRI

BackgroundIn vivo preclinical imaging of spinal cord injury (SCI) in rodent models provides clinically relevant information in translational research. This paper uses multimodal magnetic resonance imaging (MRI) to investigate neurovascular pathology and changes in blood spinal cord barrier (BSCB) permeability following SCI in a mouse model of SCI.MethodsC57BL/6 female mice (n = 5) were subjected to contusive injury at the thoracic T11 level and scanned on post injury days 1 and 3 using anatomical, dynamic contrast-enhanced (DCE-MRI) and diffusion tensor imaging (DTI). The injured cords were evaluated postmortem with histopathological stains specific to neurovascular changes. A computational model was implemented to map local changes in barrier function from the contrast enhancement. The area and volume of spinal cord tissue with dysfunctional barrier were determined using semi-automatic segmentation.ResultsQuantitative maps derived from the acquired DCE-MRI data depicted the degree of BSCB permeability variations in injured spinal cords. At the injury sites, the damaged barriers occupied about 70% of the total cross section and 48% of the total volume on day 1, but the corresponding measurements were reduced to 55% and 25%, respectively on day 3. These changes implied spatio-temporal remodeling of microvasculature and its architecture in injured SC. Diffusion computations included longitudinal and transverse diffusivities and fractional anisotropy index. Comparison of permeability and diffusion measurements indicated regions of injured cords with dysfunctional barriers had structural changes in the form of greater axonal loss and demyelination, as supported by histopathologic assessments.ConclusionThe results from this study collectively demonstrated the feasibility of quantitatively mapping regional BSCB dysfunction in injured cord in mouse and obtaining complementary information about its structural integrity using in vivo DCE-MRI and DTI protocols. This capability is expected to play an important role in characterizing the neurovascular changes and reorganization following SCI in longitudinal preclinical experiments, but with potential clinical implications.

Pathophysiology of Blood-Spinal Cord Barrier in Traumatic Injury and Repair

Blood-spinal cord barrier (BSCB) plays an important role in the regulation of the fluid microenvironment of the spinal cord. Trauma to the spinal cord impairs the BSCB permeability to proteins leading to vasogenic edema formation. Several endogenous neurochemical mediators and growth factors contribute to trauma induced BSCB disruption. Studies carried out in our laboratory suggest that those drugs and neurotrophic factors capable to attenuate the BSCB dysfunction following trauma are neuroprotective in nature. Whereas, agents that do not exert any influence on the BSCB disruption failed to reduce cell injury. These observations are in line with the idea that BSCB disruption plays an important role in the pathophysiology of spinal cord injuries. The probable mechanism(s) of trauma induced BSCB dysfunction and its contribution to cell injuries are discussed.

Matrix Metalloproteinases Limit Functional Recovery after Spinal Cord Injury by Modulation of Early Vascular Events

Inflammation in general and proteinases generated as a result are likely mediators of early secondary pathogenesis after spinal cord injury. We report that matrix metalloproteinase-9 (MMP-9) plays an important role in blood-spinal cord barrier dysfunction, inflammation, and locomotor recovery. MMP-9 was present in the meninges and neurons of the uninjured cord. MMP-9 increased rapidly after a moderate contusion spinal cord injury, reaching a maximum at 24 hr, becoming markedly reduced by 72 hr, and not detectable at 7 d after injury. It was seen in glia, macrophages, neutrophils, and vascular elements in the injured spinal cord at 24 hr after injury. The natural tissue inhibitors of MMPs were unchanged over this time course. MMP-9-null mice exhibited significantly less disruption of the blood-spinal cord barrier, attenuation of neutrophil infiltration, and significant locomotor recovery compared with wild-type mice. Similar findings were observed in mice treated with a hydroxamic acid MMP inhibitor from 3 hr to 3 d after injury, compared with the vehicle controls. Moreover, the area of residual white matter at the lesion epicenter was significantly greater in the inhibitor-treated group. This study provides evidence that MMP-9 plays a key role in abnormal vascular permeability and inflammation within the first 3 d after spinal cord injury, and that blockade of MMPs during this critical period attenuates these vascular events and leads to improved locomotor recovery. Our findings suggest that early inhibition of MMPs may be an efficacious strategy for the spinal cord-injured patient.

Upregulation of vascular endothelial growth factor is associated with radiation-induced blood-spinal cord barrier breakdown.

The pathogenesis of radiation-induced injury to the central nervous system (CNS) remains unclear. Dysfunction of the blood-brain barrier (BBB) is associated with radiation-induced white matter lesions. The aim of this study was to determine if vascular endothelial growth factor (VEGF) is implicated in radiation-induced BBB disruption. Adult rats were irradiated with a single dose of 8 or 22 Gy to the spinal cord from C2 to T2. At various times up to 20 weeks following irradiation, blood-spinal cord barrier (BSCB) permeability was assessed using immunohistochemistry with anti-albumin antibody. Cell proliferation was assessed using bromodeoxyuridine (BrdU), and endothelial cell identity was assessed morphologically and using immunostaining for factor VIII-related antigen. Expression of VEGF protein and message was assessed using immunohistochemistry and in situ hybridization respectively. In the unirradiated rat spinal cord, there was no evidence of albumin immunoreactivity and little evidence of VEGF expression. After a dose of 22 Gy, focal albumin staining in white matter was observed at 16 weeks. Diffuse staining was seen at 20 weeks and was associated with necrosis and demyelination in white matter. This was associated with a significant increase in white matter glial cells that showed immunoreactivity and in situ hybridization signal for VEGE VEGF expressing cells showed dual immunoreactivity for glial fibrillary acidic protein. No increase in VEGF positive cells was observed in gray matter after 22 Gy. After a dose of 8 Gy, there was no increase in VEGF expression or albumin immunostaining in either white or gray matter. Microvessel endothelial cell density showed a trend towards a decrease with time after 22 Gy as compared with 8 Gy or unirradiated controls. BrdU immunostaining provided no evidence for endothelial cell proliferation in control or in the irradiated spinal cord. It is concluded that radiation-induced BSCB dysfunction is associated with upregulation of VEGF in astrocytes without associated endothelial proliferation. The temporal and spatial association of VEGF upregulation with the white matter lesions suggests a role of VEGF in radiation-induced late CNS injury.