Blood-spinal Cord Barrier Permeability Research Articles

Mertk Reduces Blood-Spinal Cord Barrier Permeability Through the Rhoa/Rock1/P-MLC Pathway After Spinal Cord Injury.

Disruption of the blood-spinal cord barrier (BSCB) is a critical event in the secondary injury following spinal cord injury (SCI). Mertk has been reported to play an important role in regulating inflammation and cytoskeletal dynamics. However, the specific involvement of Mertk in BSCB remains elusive. Here, we demonstrated a distinct role of Mertk in the repair of BSCB. Mertk expression is decreased in endothelial cells following SCI. Overexpression of Mertk upregulated tight junction proteins (TJs), reducing BSCB permeability and subsequently inhibiting inflammation and apoptosis. Ultimately, this led to enhanced neural regeneration and functional recovery. Further experiments revealed that the RhoA/Rock1/P-MLC pathway plays a key role in the effects of Mertk. These findings highlight the role of Mertk in promoting SCI recovery through its ability to mitigate BSCB permeability and may provide potential targets for SCI repair.

465 Focused Ultrasound-Mediated Blood-Spinal Cord Barrier Permeabilization: A Promising Targeted Approach for Treating Chronic Spinal Cord Injury in Rat Models

INTRODUCTION: Focused ultrasound (FUS) with microbubble contrast agents can temporarily disrupt the blood-brain barrier (BBB), enabling drug delivery. FUS may also offer a minimally invasive method for delivering therapeutic agents to the affected area in chronic spinal cord injury (SCI) by transiently opening the blood-spinal cord barrier (BSCB). METHODS: Nine female Sprague-Dawley rats underwent a T9-T11 laminectomy with contusion SCI and were randomly allocated into FUS or control group. Three weeks later, FUS was administered intravenously with microbubbles. A pressure sweep was performed until ultra-harmonics were captured using a wideband hydrophone. We administered 2% Evans blue (EB) immediately after FUS confirmed the BSCB permeability. Transcardial perfusion was performed 2 hours later, and spinal cords were collected. Spectrophotometry was used to determine the EB concentration. Statistical analysis was performed using a student t-test. RESULTS: Five rats were treated with FUS while four were used as controls. Successful feedback control was achieved with FUS, and ultra-harmonics were observed in the FUS group at a mean peak pressure of 0.5 MPa. Macroscopic identification of EB in the spinal cord sections of animals exposed to FUS confirmed the localized opening of the BSCB. Spectrophotometry revealed an increase in EB concentration in the FUS group compared to controls: Mean EB concentration was 0.03 μg in the FUS group versus 0.016 μg in controls at 1-cm above the injury (p < 0.001); 0.27 μg versus 0.093 μg at the injury site (p < 0.001); and 0.25 μg versus 0.18 μg at 1-cm below the injury(p < 0.001). CONCLUSIONS: FUS increases the permeability of the BSCB in a chronic contusion SCI rat model. This suggests that FUS-mediated BSCB permeabilization may hold promise as a targeted approach for improving therapeutic outcomes in the treatment of chronic SCI.

Inhibition of UTX/KDM6A improves recovery of spinal cord injury by attenuating BSCB permeability and macrophage infiltration through the MLCK/p-MLC pathway

Spinal cord injury (SCI) can prompt an immediate disruption to the blood–spinal cord barrier (BSCB). Restoring the integrity of this barrier is vital for the recovery of neurological function post-SCI. The UTX protein, a histone demethylase, has been shown in previous research to promote vascular regeneration and neurological recovery in mice with SCI. However, it is unclear whether UTX knockout could facilitate the recovery of the BSCB by reducing its permeability. In this study, we systematically studied BSCB disruption and permeability at different time points after SCI and found that conditional UTX deletion in endothelial cells (ECs) can reduce BSCB permeability, decrease inflammatory cell infiltration and ROS production, and improve neurological function recovery after SCI. Subsequently, we used RNA sequencing and ChIP-qPCR to confirm that conditional UTX knockout in ECs can down-regulate expression of myosin light chain kinase (MLCK), which specifically mediates myosin light chain (MLC) phosphorylation and is involved in actin contraction, cell retraction, and tight junctions (TJs) protein integrity. Moreover, we found that MLCK overexpression can increase the ratio of p-MLC/MLC, further break TJs, and exacerbate BSCB deterioration. Overall, our findings indicate that UTX knockout could inhibit the MLCK/p-MLC pathway, resulting in decreased BSCB permeability, and ultimately promoting neurological recovery in mice. These results suggest that UTX is a promising new target for treating SCI.Graphical

Small extracellular vesicles derived from umbilical cord mesenchymal stem cells repair blood-spinal cord barrier disruption after spinal cord injury through down-regulation of Endothelin-1 in rats

Spinal cord injury could cause irreversible neurological dysfunction by destroying the blood-spinal cord barrier (BSCB) and allowing blood cells like neutrophils and macrophages to infiltrate the spinal cord. Small extracellular vesicles (sEVs) derived from mesenchymal stem cells (MSCs) found in the human umbilical cord have emerged as a potential therapeutic alternative to cell-based treatments. This study aimed to investigate the mechanism underlying the alterations in the BSCB permeability by human umbilical cord MSC-derived sEVs (hUC-MSCs-sEVs) after SCI. First, we used hUC-MSCs-sEVs to treat SCI rat models, demonstrating their ability to inhibit BSCB permeability damage, improve neurological repair, and reduce SCI-induced upregulation of prepro-endothelin-1 (prepro-ET-1) mRNA and endothelin-1 (ET-1) peptide expression. Subsequently, we confirmed that hUC-MSCs-sEVs could alleviate cell junction destruction and downregulate MMP-2 and MMP-9 expression after SCI, contributing to BSCB repair through ET-1 inhibition. Finally, we established an in vitro model of BSCB using human brain microvascular endothelial cells and verified that hUC-MSCs-sEVs could increase the expression of junction proteins in endothelial cells after oxygen-glucose deprivation by ET-1 downregulation. This study indicates that hUC-MSCs-sEVs could help maintain BSCB’s structural integrity and promote functional recovery by suppressing ET-1 expression.

Role of the blood-spinal cord barrier: An adheren junction regulation mechanism that promotes chronic postsurgical pain

Chronic postsurgical pain (CPSP) is a serious postoperative complication with high incidence, and its pathogenesis involves neuroimmune interactions and the breakdown of the blood-spinal cord barrier (BSCB), the decreased level of adheren junction (AJ)-related proteins is an important cause of BSCB injury. Vascular endothelial-cadherin (VE-cadherin) and p120 catenin (p120) constitute the endothelial barrier adheren junction. The Src/p120/VE-cadherin pathway is involved in the regulation of the endothelial barrier function. However, the role of the BSCB-AJ regulatory mechanism in CPSP has not been reported. In this study, we established a skin/muscle incision and retraction (SMIR) model and evaluated the paw withdrawal threshold (PWT), the effects of an Src inhibitor and p120 knockdown on p-Src, p120 and VE-cadherin expression, as well as BSCB-AJ function in rat spinal cord were observed to explore the regulation of BSCB-AJ function by the p-Src/p120/VE-cadherin pathway in promoting SMIR-induced CPSP. The levels of p-Src, p120 and VE-cadherin in the spinal cord were detected by Western blot. Meanwhile, BSCB permeability test was used to detect the changes in BCSB function. Finally, the spatial and temporal localization of p120 in spinal cord was detected by immunofluorescence. Our findings indicated that p-Src/p120/VE-cadherin could induce BSCB-AJ dysfunction and promote the development of CPSP.

Neurovascular Unit Compensation from Adjacent Level May Contribute to Spontaneous Functional Recovery in Experimental Cervical Spondylotic Myelopathy.

The progression and remission of cervical spondylotic myelopathy (CSM) are quite unpredictable due to the ambiguous pathomechanisms. Spontaneous functional recovery (SFR) has been commonly implicated in the natural course of incomplete acute spinal cord injury (SCI), while the evidence and underlying pathomechanisms of neurovascular unit (NVU) compensation involved in SFR remains poorly understood in CSM. In this study, we investigate whether compensatory change of NVU, in particular in the adjacent level of the compressive epicenter, is involved in the natural course of SFR, using an established experimental CSM model. Chronic compression was created by an expandable water-absorbing polyurethane polymer at C5 level. Neurological function was dynamically assessed by BBB scoring and somatosensory evoked potential (SEP) up to 2 months. (Ultra)pathological features of NVUs were presented by histopathological and TEM examination. Quantitative analysis of regional vascular profile area/number (RVPA/RVPN) and neuroglial cells numbers were based on the specific EBA immunoreactivity and neuroglial biomarkers, respectively. Functional integrity of blood spinal cord barrier (BSCB) was detected by Evan blue extravasation test. Although destruction of the NVU, including disruption of the BSCB, neuronal degeneration and axon demyelination, as well as dramatic neuroglia reaction, were found in the compressive epicenter and spontaneous locomotor and sensory function recovery were verified in the modeling rats. In particular, restoration of BSCB permeability and an evident increase in RVPA with wrapping proliferated astrocytic endfeet in gray matter and neuron survival and synaptic plasticity were confirmed in the adjacent level. TEM findings also proved ultrastructural restoration of the NVU. Thus, NVU compensation changes in the adjacent level may be one of the essential pathomechanisms of SFR in CSM, which could be a promising endogenous target for neurorestoration.

Perlecan Improves Blood Spinal Cord Barrier Repair Through the Integrin β1/ROCK/MLC Pathway After Spinal Cord Injury.

Spinal cord injury (SCI) can lead to the destruction of the blood-spinal cord barrier (BSCB), causing various inflammatory cytokines, neutrophils, and macrophages to infiltrate the lesion area, resulting in secondary injury. Basement membranes (BMs) are maintained by all types of cells in the BSCB and contribute to BSCB maintenance. Perlecan is an important constituent of vascular BMs, maintaining vascular integrity and neuroprotection. However, it is not clear whether Perlecan is involved in BSCB repair after SCI. In this study, we found that Perlecan was specifically expressed in the BMs in the spinal cord and underwent degradation/remodeling after SCI. Subsequently, a CRISPR/Cas9-based SAM system was used to overexpress Perlecan in the injured spinal cord, resulting in significantly enhanced locomotor recovery and neural regeneration. Overexpression of Perlecan reduced BSCB permeability along with the neuroinflammatory response. Interestingly, Perlecan inhibited stress fiber formation by interacting with integrin β1 and inhibiting downstream ROCK/MLC signaling, resulting in reduced tight junctions (TJs) disassembly and improved BSCB integrity. Furthermore, the integrin receptor antagonist GRGDSP abolished the effects of Perlecan overexpression on BSCB permeability and TJs integrity. Overall, our findings suggest that Perlecan reduces BSCB permeability and the neuroinflammatory response by interacting with integrin β1 and inhibiting the downstream ROCK/MLC pathway to promote neurological recovery after SCI.

Elevated intraspinal pressure drives edema progression after acute compression spinal cord injury in rabbits

Elevated intraspinal pressure (ISP) following traumatic spinal cord injury (tSCI) can be an important factor for secondary SCI that may result in greater tissue damage and functional deficits. Our present study aimed to investigate the dynamic changes in ISP after different degrees of acute compression SCI in rabbits with closed canals and explore its influence on spinal cord pathophysiology. Closed balloon compression injuries were induced with different inflated volumes (40 μl, 50 μl or no inflation) at the T7/8 level in rabbits. ISP was monitored by a SOPHYSA probe at the epicenter within 7 days post-SCI. Edema progression, spinal cord perfusion and damage severity were evaluated by serial multisequence MRI scans, somatosensory evoked potentials (SEPs) and behavioral scores. Histological and blood spinal cord barrier (BSCB) permeability results were subsequently analyzed. The results showed that the ISP waveforms comprised three peaks, significantly increased after tSCI, peaked at 72 h (21.86 ± 3.13 mmHg) in the moderate group or 48 h (31.71 ± 6.02 mmHg) in the severe group and exhibited “slow elevated and fast decreased” or “fast elevated and slow decreased” dynamic changes in both injured groups. Elevated ISP after injury was correlated with spinal cord perfusion and edema progression, leading to secondary lesion enlargement. The secondary damage aggravation can be visualized by diffusion tensor tractography (DTT). Moreover, the BSCB permeability was significantly increased at the epicenter and rostrocaudal segments at 72 h after SCI; by 14 days, notable permeability was still observed at the caudal segment in the severely injured rabbits. Our results suggest that the ISP of rabbits with closed canals increased after acute compression SCI and exhibited different dynamic change patterns in moderately and severely injured rabbits. Elevated ISP exacerbated spinal cord perfusion, drove edema progression and led to secondary lesion enlargement that was strongly associated with BSCB disruption. For severe tSCI, early intervention targeting elevated ISP may be an indispensable choice to rescue spinal cord function.

Levetiracetam Attenuates the Spinal Cord Injury Induced by Acute Trauma via Suppressing the Expression of Perforin.

The spinal cord injury (SCI) is one of the major reasons causing the motor dysfunctions of the patients. At present, few therapeutic strategies can effectively improve the symptom of SCI. Levetiracetam has been confirmed to alleviate the injury of nerve cells, while its functions in SCI remains unclear. In this study, C57BL/6J mice were used to establish SCI models to observe the effect of levetiracetam on SCI. The mice were fed with 180 mg/kg levetiracetam when suffering from SCI, and Basso mouse score (BMS) and CatWalk-assisted gait analysis were used to observe the motor functions of the mice. Nissl staining and TUNEL staining were used to observe the injury of nerve cells. The abundance of inflammatory factors was measured by ELISA. The permeability of blood-spinal cord barrier (BSCB) in mice was detected with macrophage infiltration analysis. Moreover, the abundance of perforin in the tissues was detected by western blot. The results showed that the SCI mice treated with levetiracetam exhibited lighter motor dysfunction compared with the mice treated with saline. Levetiracetam can effectively reduce the inflammatory reactions and alleviate apoptosis of the nerve cells. Moreover, levetiracetam remarkably decreased the BSCB permeability of SCI mice. Besides, it was also found that levetiracetam can significantly inhibit the expression of perforin. In conclusion, this study suggests that levetiracetam can attenuate the injury of BSCB to block the progression of SCI via suppressing the expression of perforin.

Fecal Microbiota Transplantation Exerts Neuroprotective Effects in a Mouse Spinal Cord Injury Model by Modulating the Microenvironment at the Lesion Site.

ABSTRACTThe primary traumatic event that causes spinal cord injury (SCI) is followed by a progressive secondary injury featured by vascular disruption and ischemia, inflammatory responses and the release of cytotoxic debris, which collectively add to the hostile microenvironment of the lesioned cord and inhibit tissue regeneration and functional recovery. In a previous study, we reported that fecal microbiota transplantation (FMT) promotes functional recovery in a contusion SCI mouse model; yet whether and how FMT treatment may impact the microenvironment at the injury site are not well known. In the current study, we examined individual niche components and investigated the effects of FMT on microcirculation, inflammation and trophic factor secretion in the spinal cord of SCI mice. FMT treatment significantly improved spinal cord tissue sparing, vascular perfusion and pericyte coverage and blood-spinal cord-barrier (BSCB) integrity, suppressed the activation of microglia and astrocytes, and enhanced the secretion of neurotrophic factors. Suppression of inflammation and upregulation of trophic factors, jointly, may rebalance the niche homeostasis at the injury site and render it favorable for reparative and regenerative processes, eventually leading to functional recovery. Furthermore, microbiota metabolic profiling revealed that amino acids including β-alanine constituted a major part of the differentially detected metabolites between the groups. Supplementation of β-alanine in SCI mice reduced BSCB permeability and increased the number of surviving neurons, suggesting that β-alanine may be one of the mediators of FMT that participates in the modulation and rebalancing of the microenvironment at the injured spinal cord.IMPORTANCE FMT treatment shows a profound impact on the microenvironment that involves microcirculation, blood-spinal cord-barrier, activation of immune cells, and secretion of neurotrophic factors. Analysis of metabolic profiles reveals around 22 differentially detected metabolites between the groups, and β-alanine was further chosen for functional validation experiments. Supplementation of SCI mice with β-alanine significantly improves neuronal survival, and the integrity of blood-spinal cord-barrier at the lesion site, suggesting that β-alanine might be one of the mediators following FMT that has contributed to the recovery.

Pharmacologic treatment with OKN-007 reduces alpha-motor neuron loss in spinal cord of aging mice.

Aging is associated with molecular and functional declines in multiple physiologic systems. We have previously reported age-related changes in spinal cord that included a decline in α-motor neuron numbers, axonal loss, and demyelination associated with increased inflammation and blood-spinal cord barrier (BSCB) permeability. These changes may influence other pathologies associated with aging, in particular loss of muscle mass and function (sarcopenia), which we and others have shown is accompanied by neuromuscular junction disruption and loss of innervation. Interventions to protect and maintain motor neuron viability and function in aging are currently lacking and could have a significant impact on improving healthspan. Here we tested a promising compound, OKN-007, that has known antioxidant, anti-inflammatory and neuroprotective properties, as a potential intervention in age-related changes in the spinal cord. OKN-007 is a low molecular weight disulfonyl derivative of (N-tert Butyl-α-phenylnitrone) (PBN) that can easily cross the blood-brain barrier. We treated middle age (16 month) wild-type male mice with OKN-007 in drinking water at a dose of 150 mg/kg/day until 25 months of age. OKN-007 treatment exerted a number of beneficial effects in the aging spinal cord, including a 35% increase in the number of lumbar α-motor neurons in OKN-treated old mice compared to age-matched controls. Brain spinal cord barrier permeability, which is increased in aging spinal cord, was also blunted by OKN-007 treatment. Age-related changes in microglia proliferation and activation are blunted by OKN-007, while we found no effect on astrocyte proliferation. Transcriptome analysis identified expression changes in a number of genes that are involved in neuronal structure and function and revealed a subset of genes whose changes in response to aging are reversed by OKN-007 treatment. Overall, our findings suggest that OKN-007 exerts neuroprotective and anti-inflammatory effects on the aging spinal cord and support OKN-007 as a potential therapeutic to improve α-motor neuron health.

Sodium tanshinone IIA sulfonate promotes spinal cord injury repair by inhibiting blood spinal cord barrier disruption in vitro and in vivo.

Spinal cord injury (SCI) leads to microvascular damage and the destruction of the blood spinal cord barrier (BSCB), which can progress into secondary injuries, such as apoptosis and necrosis of neurons and glia, culminating in permanent neurological deficits. BSCB restoration is the primary goal of SCI therapy, although very few drugs can repair damaged barrier structure and permeability. Sodium tanshinone IIA sulfonate (STS) is commonly used to treat cardiovascular disease. However, the therapeutic effects of STS on damaged BSCB during the early stage of SCI remain uncertain. Therefore, we exposed spinal cord microvascular endothelial cells to H2 O2 and treated them with different doses of STS. In addition to protecting the cells from H2 O2 -induced apoptosis, STS also reduced cellular permeability. In the in vivo model of SCI, STS reduced BSCB permeability, relieved tissue edema and hemorrhage, suppressed MMP activation and prevented the loss of tight junction and adherens junction proteins. Our findings indicate that STS treatment promotes SCI recovery, and should be investigated further as a drug candidate against traumatic SCI.

Bazedoxifene, a Selective Estrogen Receptor Modulator, Promotes Functional Recovery in a Spinal Cord Injury Rat Model.

In research on various central nervous system injuries, bazedoxifene acetate (BZA) has shown two main effects: neuroprotection by suppressing the inflammatory response and remyelination by enhancing oligodendrocyte precursor cell differentiation and oligodendrocyte proliferation. We examined the effects of BZA in a rat spinal cord injury (SCI) model. Anti-inflammatory and anti-apoptotic effects were investigated in RAW 264.7 cells, and blood-spinal cord barrier (BSCB) permeability and angiogenesis were evaluated in a human brain endothelial cell line (hCMEC/D3). In vivo experiments were carried out on female Sprague Dawley rats subjected to moderate static compression SCI. The rats were intraperitoneally injected with either vehicle or BZA (1mg/kg pre-SCI and 3 mg/kg for 7 days post-SCI) daily. BZA decreased the lipopolysaccharide-induced production of proinflammatory cytokines and nitric oxide in RAW 264.7 cells and preserved BSCB disruption in hCMEC/D3 cells. In the rats, BZA reduced caspase-3 activity at 1 day post-injury (dpi) and suppressed phosphorylation of MAPK (p38 and ERK) at dpi 2, hence reducing the expression of IL-6, a proinflammatory cytokine. BZA also led to remyelination at dpi 20. BZA contributed to improvements in locomotor recovery after compressive SCI. This evidence suggests that BZA may have therapeutic potential to promote neuroprotection, remyelination, and functional outcomes following SCI.

Human Bone Marrow Mesenchymal Stem Cell-Derived Exosomes Attenuate Blood-Spinal Cord Barrier Disruption via the TIMP2/MMP Pathway After Acute Spinal Cord Injury.

After spinal cord injury (SCI), destruction of the blood-spinal cord barrier (BSCB) results in infiltration of blood cells, such as neutrophils and macrophages, leading to permanent neurological dysfunction. Previous studies have shown that human bone marrow mesenchymal stem cell (BMSC)-derived exosomes have a beneficial neuroprotective effect in SCI models. However, whether BMSC-Exos contribute to the integrity of the BSCB has not been clarified. The purpose of this study was to investigate the mechanism of BMSC-Exo-induced changes in the permeability of the BSCB after SCI. Here, we first used BMSC-Exos to treat an SCI rat model, showing that BMSC-Exos can inhibit BSCB permeability damage and improve spontaneous repair. Next, we found that tissue inhibitors of matrix metalloproteinase 2 (TIMP2) have been shown to play an important role in the function of BMSC-Exos by inhibiting the matrix metalloproteinase (MMP) pathway, thereby reducing the reduction of cell junction proteins. Therefore, we constructed siTIMP2 to knock out TIMP2 in BMSC-Exos, which caused the activity of BMSC-Exos to be significantly weakened. Finally, we constructed an in vitro model of BSCB with HBMECs and verified that TIMP2 in BMSC-Exos in vitro can also alleviate BSCB damage. This proof-of-principle study demonstrates that BMSC-Exos can preserve the integrity of the BSCB and improve functional recovery after SCI through the TIMP2/MMP signaling pathway.

Blocking the EGFR/p38/NF-κB signaling pathway alleviates disruption of BSCB and subsequent inflammation after spinal cord injury

Epidermal growth factor receptor (EGFR) activation is involved in blood spinal cord barrier (BSCB) disruption and secondary injury after spinal cord injury (SCI). However, the underlying mechanisms of EGFR activation mediating BSCB disruption and secondary injury after SCI remain unclear. An in vitro model of oxygen and glucose deprivation/reoxygenation (OGD/R) induced BSCB damage and in vivo rat SCI model were employed to define the role of EGFR/p38/NF-κB signal pathway activation and its induced inflammatory injury in main cellular components of BSCB. Genetic regulation (lentivirus delivered shRNA and overexpression system) or chemical intervention (agonist or inhibitor) were applied to activate or inactivate EGFR and p38 in astrocytes and microvascular endothelial cells (MEC) under which conditions, the expression of pro-inflammatory factors (TNF-α, iNOS, COX-2, and IL-1β), tight junction (TJ) protein (ZO-1 and occludin), nuclear translocation of NF-κB and permeability of BSCB were analyzed. The pEGFR was increased in astrocytes and MEC which induced the activation of EGFR and p38 and NF-κB nuclear translocation. The activation of EGFR and p38 increased the TNF-α, iNOS, COX-2, and IL-1β responsible for the inflammatory injury and reduced the ZO-1 and occludin which caused BSCB disruption. While EGFR or p38 inactivation inhibited NF-κB nuclear translocation, and markedly attenuated the production of pro-inflammatory factors and the loss of TJ protein. This study suggests that the EGFR activation in main cellular components of BSCB after SCI mediates BSCB disruption and secondary inflammatory injury via the EGFR/p38/NF-κB pathway.

Baicalin attenuates blood-spinal cord barrier disruption and apoptosis through PI3K/Akt signaling pathway after spinal cord injury.

Baicalin is a natural active ingredient isolated from Scutellariae Radix that can cross the blood-brain barrier and exhibits neuroprotective effects on multiple central nervous system diseases. However, the mechanism behind the neuroprotective effects remains unclear. In this study, rat models of spinal cord injury were established using a modified Allen's impact method and then treated with intraperitoneal injection of Baicalin. The results revealed that Baicalin greatly increased the Basso, Beattie, Bresnahan Locomotor Rating Scale score, reduced blood-spinal cord barrier permeability, decreased the expression of Bax, Caspase-3, and nuclear factor κB, increased the expression of Bcl-2, and reduced neuronal apoptosis and pathological spinal cord injury. SH-SY5Y cell models of excitotoxicity were established by application of 10 mM glutamate for 12 hours and then treated with 40 µM Baicalin for 48 hours to investigate the mechanism of action of Baicalin. The results showed that Baicalin reversed tight junction protein expression tendencies (occludin and ZO-1) and apoptosis-related protein expression (Bax, Bcl-2, Caspase-3, and nuclear factor-κB), and also led to up-regulation of PI3K and Akt phosphorylation. These effects on Bax, Bcl-2, and Caspase-3 were blocked by pretreatment with the PI3K inhibitor LY294002. These findings suggest that Baicalin can inhibit blood-spinal cord barrier permeability after spinal cord injury and reduce neuronal apoptosis, possibly by activating the PI3K/Akt signaling pathway. This study was approved by Animal Ethics Committee of Xi'an Jiaotong University on March 6, 2014.

Small extracellular vesicles released by infused mesenchymal stromal cells target M2 macrophages and promote TGF-β upregulation, microvascular stabilization and functional recovery in a rodent model of severe spinal cord injury.

Intravenous (IV) infusion of bone marrow‐derived mesenchymal stem/stromal cells (MSCs) stabilizes the blood‐spinal cord barrier (BSCB) and improves functional recovery in experimental models of spinal cord injury (SCI). Although IV delivered MSCs do not traffic to the injury site, IV delivered small extracellular vesicles (sEVs) derived from MSCs (MSC‐sEVs) do and are taken up by a subset of M2 macrophages. To test whether sEVs released by MSCs are responsible for the therapeutic effects of MSCs, we tracked sEVs produced by IV delivered DiR‐labelled MSCs (DiR‐MSCs) after transplantation into SCI rats. We found that sEVs were released by MSCs in vivo, trafficked to the injury site, associated specifically with M2 macrophages and co‐localized with exosome markers. Furthermore, while a single MSC injection was sufficient to improve locomotor recovery, fractionated dosing of MSC‐sEVs over 3 days (F‐sEVs) was required to achieve similar therapeutic effects. Infusion of F‐sEVs mimicked the effects of single dose MSC infusion on multiple parameters including: increased expression of M2 macrophage markers, upregulation of transforming growth factor‐beta (TGF‐β), TGF‐β receptors and tight junction proteins, and reduction in BSCB permeability. These data suggest that release of sEVs by MSCs over time induces a cascade of cellular responses leading to improved functional recovery.

Technical Note: Quantification of blood-spinal cord barrier permeability after application of magnetic resonance-guided focused ultrasound in spinal cord injury.

To demonstrate that magnetic resonance-guided focused ultrasound (MRgFUS) facilitates blood-spinal cord barrier (BSCB) permeability and develop observer-independent MRI quantification of BSCB permeability after MRgFUS for spinal cord injury (SCI). Noninjured Sprague-Dawley rats (n = 3) underwent MRgFUS and were administered Evans blue post-MRgFUS to confirm BSCB opening. Absorbance was measured by spectrophotometry and correlated with its corresponding image intensity. Rats (n = 21) underwent T8-T10 laminectomy and extradural compression of the spinal cord (23g weighted aneurysm-type clip, 1 min). The intervention group (n = 11) was placed on a preclinical MRgFUS system, administered microbubbles (Optison, 0.2 mL/kg), and received 3 MRgFUS sonications (25 ms bursts, 1 Hz pulses for 3 min, 3 acoustic W, approximately 1.0-2.1 MPa peak pressure as measured via hydrophone). The sham group (n = 10) received equivalent procedures with no sonications. T1w MRI was obtained both pre- and post-MRgFUS BSCB opening. Spinal cords were segmented manually or semiautomatically and a Pearson correlation with P ≤ 0.001 was used to correlate the two segmentation methods. MRgFUS sonication and control regions intensity values were evaluated with a paired t-test with a P ≤ 0.01. Semiautomatic segmentation reduced computational time by 95% and was correlated with manual segmentation (Pearson = 0.92, P < 0.001, n = 71 regions). In the noninjured rat group, Evans blue absorbance correlated with image intensity in the MRgFUS and control regions (Pearson = 0.82, P = 0.02, n = 6). In rats that underwent the SCI procedure, an increase in signal intensity in the MRgFUS targeted region relative to control was seen in all SCI rats (10.65 ± 12.4%, range: 0.96-43.9%, n = 11, P = 0.002). SCI sham MRgFUS revealed no change (0.63 ± 0.52%, 95% CI 0.320.95, n = 10). This result was significant between both groups (P = 0.003). The implemented semiautomatic segmentation procedure improved data analysis efficiency. Quantitative methods using contrast-enhanced MRI with histological validation are sensitive for detection of blood-spinal cord barrier opening induced by magnetic resonance-guided focused ultrasound.

Dl-3-n-butylphthalide Attenuates Spinal Cord Injury via Regulation of MMPs and Junction Proteins in Mice.

As a serious trauma of the neurological system, spinal cord injury (SCI) results in permanent disability, gives rise to immediate vascular damage and a wide range of matters that induce the breakage of blood spinal cord barrier (BSCB). SCI activates the expression of MMP-2/9, which are considered to accelerate the disruption of BSCB. Recent research shows that Dl-3-n-butylphthalide (NBP) exerted protective effects on blood spinal cord barrier in animals after SCI, but the underlying molecular mechanism of NBP on the BSCB undergoing SCI is unknown. Here, our research show that NBP inhibited the expression of MMP-2/9, then improved the permeability of BSCB following SCI. After the T9 level of spinal cord performed with a moderate injury, NBP was managed by intragastric administration and further performed once a day. NBP remarkably improved the permeability of BSCB and junction proteins degration, then promoted locomotion recovery. The protective effect of NBP on BSCB destruction is related to the regulation of MMP-2/9 induced by SCI. Moreover, NBP obviously inhibited the MMP-2/9 expression and junction proteins degradation in microvascular endothelial cells. In conclusion, our results indicate that MMP-2/9 are relevant to the breakdown of BSCB, NBP impairs BSCB destruction through inhibiting MMP-2/9 and promotes functional recovery subjected to SCI. NBP is likely to become a new nominee as a therapeutic to treat SCI via a transigent BSCB.

Investigating the blood-spinal cord barrier in preclinical models: a systematic review of in vivo imaging techniques.

This study is a systematic review. To evaluate current in vivo techniques used in the investigation of the blood-spinal cord barrier (BSCB). Search of English language literature for animal studies that investigated the BSCB in vivo. Data extraction included animal model/type, protocol for BSCB evaluation, and study outcomes. Descriptive syntheses are provided. A total of 40 studies were included, which mainly investigated rodent models of experimental autoimmune encephalomyelitis (EAE) or spinal cord injury (SCI). The main techniques used were magnetic resonance imaging (MRI) and intravital microscopy (IVM). MRI served as a reliable tool to longitudinally track BSCB permeability changes with dynamic contrast enhancement (DCE) using gadolinium, or assess inflammatory infiltrations with targeted alternative contrast agents. IVM provided high-resolution visualization of cellular and molecular interactions across the microvasculature, commonly with either epi-fluorescence or two-photon microscopy. MRI and IVM techniques enabled the evaluation of therapeutic interventions and mechanisms that drive spinal cord dysfunction in EAE and SCI. A small number of studies demonstrated the feasibility of DCE-computed tomography, ultrasound, bioluminescent, and fluorescent optical imaging methods to evaluate the BSCB. Technique-specific limitations and multiple protocols for image acquisition and data analyses are described for all techniques. There are few in vivo investigations of the BSCB. Additional studies are needed in less commonly studied spinal cord disorders, and to establish standardized protocols for data acquisition and analysis. Further development of techniques and multimodal approaches could overcome current imaging limitations to the spinal cord. These advancements might promote wider adoption of techniques, and can provide greater potential for clinical translation.