Blood-brain Barrier Tight Junction Research Articles

The Neuroprotection of 1,2,4-Triazole Derivative by Inhibiting Inflammation and Protecting BBB Integrity in Acute Ischemic Stroke.

The oxidative stress and neuroinflammation are important factors in acute ischemic stroke (AIS). Our former study showed the 1,2,4- triazole derivative (SYS18) had obviously neuroprotection by anti- oxidative stress on rat middle cerebral artery occlusion (MCAO) model. In this study, we continue to investigate its neuroprotection by anti-inflammatory effects and protecting BBB integrity in AIS. First, its effect on acute inflammation was evaluated by the mice model of increased peritoneal capillary permeability. Then, the MCAO cerebral edema models were built to evaluate its neuroprotection by reducing the neurological score, cerebral edema, improving the biochemical indicators, and pathological damage of brain tissue. At the same time, its protection on blood-brain barrier (BBB) integrity was proved by decreasing the BBB permeability and inhibiting glycocalyx degradation and regulating the BBB tight junction proteins expression of matrix metalloproteinase- 9 (MMP- 9) and claudin- 5 in brain tissue. Meanwhile, pharmacokinetic experiments showed that the compound had good BBB penetration. It has some advantages in the intensity of efficacy compared with the marketed drug edaravone. Based on these findings, SYS18 has a strong potential to become a neuroprotectant in the future.

Attentive graph neural network models for the prediction of blood brain barrier permeability.

The blood brain barrier's (BBB) unique endothelial cells and tight junctions selectively regulate passage of molecules to the central nervous system (CNS) to prevent pathogen entry and maintain neural homeostasis. Various neurological conditions and neurodegenerative diseases benefit from small molecules capable of BBB penetration (BBBP) to elicit a therapeutic effect. Predicting BBBP often involves in silico assessment of molecular properties such as lipophilicity (log P ) and polar surface area (PSA) using the CNS multiparameter optimization (MPO) method. This study curated an open-source dataset to benchmark rigorously machine learning (ML) and neural network (NN) models with each other and with MPO for predicting BBBP. Our analysis demonstrated that AI models, especially attentive NNs using stereochemical features, significantly outperform MPO in predicting BBBP. An attentive graph neural network (GNN), we refer to as CANDID-CNS™, achieved a 0.23-0.26 higher AUROC score than MPO on full test sets, and a 0.17-0.19 higher score on stereoisomers filtered subsets. Regarding stereoisomers that differ in BBBP, which MPO cannot distinguish, attentive GNNs correctly classify these with AUROC and MCC metrics comparable to or better than MPO's AUROC and MCC on less difficult test molecules. These findings suggest that integrating attentive GNN models into pharmaceutical drug discovery processes can substantially improve prediction rates, and thereby reduce the timeline, cost, and increase probability of success of designing brain penetrant therapeutics for the treatment of a wide variety of neurological and neurodegenerative diseases.

Liposomal Formulations of Anti-Alzheimer Drugs and siRNA for Nose-to-Brain Delivery: Design, Safety and Efficacy In Vitro

β-site amyloid precursor protein cleaving enzyme (BACE1) represents a key target for Alzheimer’s disease (AD) therapy because it is essential for producing the toxic amyloid β (Aβ) peptide that plays a crucial role in the disease’s development. BACE1 inhibitors are a promising approach to reducing Aβ levels in the brain and preventing AD progression. However, systemic delivery of such inhibitors to the brain demonstrates limited efficacy because of the presence of the blood-brain barrier (BBB). Nose-to-brain (NtB) delivery has the potential to overcome this obstacle. Liposomal drug delivery systems offer several advantages over traditional methods for delivering drugs and nucleic acids from the nose to the brain. The current study aims to prepare, characterize, and evaluate in vitro liposomal forms of donepezil, memantine, BACE-1 siRNA, and their combination for possible treatment of AD via NtB delivery. All the liposomal formulations were prepared using the rotary evaporation method. Their cellular internalization, cytotoxicity, and the suppression of beta-amyloid plaque and other pro-inflammatory cytokine expressions were studied. The Calu-3 Transwell model was used as an in vitro system for mimicking the anatomical and physiological conditions of the nasal epithelium and studying the suitability of the proposed formulations for possible NtB delivery. The investigation results show that liposomes provided the effective intracellular delivery of therapeutics, the potential to overcome tight junctions in BBB, reduced beta-amyloid plaque accumulation and pro-inflammatory cytokine expression, supporting the therapeutic potential of our approach.Graphical

The silent information regulator 1 agonist SRT1720 reduces experimental intracerebral hemorrhagic brain injury by regulating the blood-brain barrier integrity.

Intracerebral hemorrhage (ICH) is a significant public health matter that has no effective treatment. ICH-induced destruction of the blood-brain barrier (BBB) leads to neurological deterioration. Astrocytic sonic hedgehog (SHH) alleviates brain injury by maintaining the integrity of the BBB after ICH. Silent information regulator 1 (SIRT1) is neuroprotective in several central nervous system diseases via BBB regulation. It is also a possible influential factor of the SHH signaling pathway. Nevertheless, the role of SIRT1 on BBB and the underlying pathological process associated with the SHH signaling pathway after ICH remain unclear. We established an intracerebral hemorrhagic mouse model by collagenase injection. SRT1720 (a selective agonist of SIRT1) was used to evaluate the effect of SIRT1 on BBB integrity after ICH. SIRT1 expression was reduced in the mouse brain after ICH. SRT1720 attenuated neurobehavioral impairments and brain edema of ICH mouse. After ICH induction, SRT1720 improved BBB integrity and tight junction expressions in the mouse brain. The SHH signaling pathway-related factors smoothened and glioma-associated oncogene homolog-1 were increased with the intervention of SRT1720, while cyclopamine (a specific inhibitor of the SHH signaling pathway) reversed these effects. These findings suggest that SIRT1 protects from ICH by altering BBB permeability and tight junction expression levels. This process is associated with the SHH signaling pathway, suggesting that SIRT1 may be a potential therapeutic target for ICH.

Blood-Brain Barrier Permeability is Affected by Changes in Tight Junction Protein Expression at High-Altitude Hypoxic Conditions-this may have Implications for Brain Drug Transport.

Changes to blood-brain barrier structure and function may affect the delivery of drugs into the brain. It is worthwhile to exploring more study on how the blood-brain barrier changes in structure and function and how that affects drug transport in high-altitude hypoxic environment. The DIA high-throughput sequencing technique indicate that the rats blood-brain barrier has been identified to have 7252 proteins overall and 8 tight junction proteins, among which Claudin-7 was a plateau-specific tight junction protein under high-altitude hypoxia, and based on the interaction network study, 2421 proteins are found to interact with one another, with ZO-1 being the primary target. The results of the projected gene function analysis demonstrated that changes in tight junction proteins are related to the control of TRP channels by inflammatory mediators, the wnt signaling pathway, the ABC transporter system, and drug metabolism-CYP450 enzyme regulation. Additionally, the electron microscopy, the Evans blue combination with confocal laser scanning microscopy, and the Western Blot and RT-qPCR revealed that high-altitude hypoxic environment induces blood-brain barrier tight junctions to open, blood-brain barrier permeability increases, ZO-1, Occludin, Claudin-5 protein and mRNA expression decreased. Our research implies that structural and functional alterations in the blood-brain barrier induced by high altitude hypoxia may impact drug transport inside the central nervous system, and that drug transporters and drug-metabolizing enzymes may be key players in this process.

Dynamic changes in key factors of the blood-brain barrier in early diabetic mice.

Chronic hyperglycemia can result in damage to the hippocampus and dysfunction of the blood-brain barrier (BBB), potentially leading to neurological disorders. This study examined the histological structure of the hippocampus and the expression of critical genes associated with the BBB at 2 early stage time points in a streptozotocin-induced diabetes mellitus (DM) mouse model. Routine histology revealed vascular congestion and dilation of Virchow-Robin spaces in the hippocampal CA1 region of the DM group. Neuronal alterations included rounding and swelling and reduction in Nissl bodies and increased apoptosis. Compared to the control group, TJP1 mRNA expression in the DM group was significantly lower (P < .05 or P < .01), while mRNA levels of JAM3, TJP3, CLDN5, CLDN3, and OCLN initially increased and then decreased. At 7, 14, and 21 days, mRNA levels of the receptor for advanced glycation end products (AGER) were greater in the DM group than in the control group (P < .05 or P < .01). These findings indicate that early-stage diabetes may cause structural and functional impairments in hippocampal CA1 in mice. These abnormalities may parallel alterations in the expression of key BBB tight junction molecules and elevated AGER expression in early DM patients.

180 Dietary milk fat globule membrane alters intestinal microbiota of neonatal piglet following lipopolysaccharide challenge and marker of neuroinflammation

Abstract Early life stress exposure can increase morbidity and reduce animal wellbeing throughout life. Dysregulated gut function has been associated with neurological changes, and neurological changes affect gut homeostasis. Maternal milk is the gold-standard for yielding developmental benefits to offspring. Identification of milk components has led to an understanding of the bioactive nutrients important in neonatal development. Components of the milk fat globule membrane (MFGM) contribute immensely to neonatal gut health and neurodevelopment. However, mechanisms of actions are still being defined. The objective is to understand how dietary MFGM modulates the gut microbiota, blood-brain barrier tight junctions, and brain inflammatory markers following lipopolysaccharide (LPS) challenge. Piglets (n = 24, one-d-old) were assigned to soy (CON) or MFGM phospholipid supplemented diet (0.75% wt/wt) ± LPS (100 µg/kg body weight) in a 2 × 2 factorial design (n = 6 animals•diet-1•challenge-1.). Piglets were fed for 21 d and 6 animals/diet received saline or LPS injections 4 h before euthanasia. Colonic microbiota was characterized through 16s rRNA gene sequencing, while hypothalamus samples were collected to evaluate brain barrier function and inflammation-related genes using RT-qPCR. Microbiota data were processed using QIIME2 with DADA2 plugin and analyzed for alpha-diversity, beta-diversity, and differential abundance in R software. Gene transcription data were analyzed by a 2 × 2 experimental factorial design using the PROC MIXED procedure of SAS. Principal coordinates analysis indicated that MFGM-fed groups exhibited an observable separation from CON-fed groups in terms of microbial community structures (P &amp;lt; 0.05). Firmicutes_A, Bacteroidota, and Firmicutes_D were the predominant phyla across all treatments, with average proportions to be 52.21 %, 24.36 %, and 18.46 %, respectively. At genus level, MFGM + Saline group down-regulated Corynebacterium by 88.00 % (q &amp;lt; 0.05) and Unclassified Muribaculaceae by 73.33 % (0.050.05). However, there was an MFGM × LPS interaction (P &amp;lt; 0.05) observed for nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor alpha, in which MFGM attenuated the LPS-induced increase in mRNA relative abundance (3.62 and 2.39 ± 0.38). In conclusion, dietary MFGM altered neonatal piglet intestinal microbiota and an important signaling molecule of the neuroinflammatory response following LPS challenge.

Abstract WP324: MCA/FeCl 3 Thromboembolic Model, an Easily Reproducible Model for COVID-19-Induced Embolic Cerebrovascular Complications

COVID-19 neurological complications such as strokes and cognitive impairment have been reported. Yet the molecular mechanisms are under investigation. A mild and reproducible thromboembolic model will be a helpful tool for investigating COVID-thromboembolic complications. The aim of this study is to investigate the middle cerebral artery/ferric chloride (MCA/FeCl 3 ) thromboembolic model as an easily and reproducible model for COVID-19-induced cerebrovascular embolic complications. Methods: SARS-CoV-2 spike-protein of the alpha variant were injected intravenously (4 ug/animal) in K18 (transgenic knock-in humanized ACE2 in epithelial cells). Seven days later, MCA/FeCl 3 thromboembolic model was induced in K18 mice by exposing the MCA and placing filter paper wet with FeCl 3 . Laser speckle imaging was used for the assessment of blood flow and recanalization after 1,2,6, and 24 hrs. TTC was used to assess the infarct volume. Cognitive function was assessed using a novel object recognition test. Cerebral vascular density and blood-brain barrier (BBB) were assessed by immunohistochemistry staining using Iso-B4 lectin and Occludin-5 antibody. Results: SARS-CoV-2 spike-protein caused a significant increase in the time required for vascular recanalization as detected by laser speckle imaging compared to control K18 mice. (P&lt;0.05). In parallel, spike protein caused a significant increase in infarct size compared to control K18 mice (P&lt;0.05). Our immunohistochemistry showed that spike protein decreased vascular density and increased vascular rarefaction compared to control K18 mice (P&lt;0.05). A novel object recognition test showed that spike protein increased cognitive dysfunction compared to control (P&lt;0.05). Western blot analysis showed that spike protein increased inflammatory markers and decreased BBB tight junction protein compared to control K18 mice (P&lt;0.05). In conclusion , the MCA/FeCl 3 thromboembolic model is an easily reproducible mouse model to examine COVID-19-induced stroke and cerebrovascular complications.

Mitochondrial ferritin upregulation reduced oxidative stress and blood-brain-barrier disruption by maintaining cellular iron homeostasis in a neonatal rat model of germinal matrix hemorrhage

Germinal matrix hemorrhage (GMH) is a devasting neurological disease in premature newborns. After GMH, brain iron overload associated with hemoglobin degradation contributed to oxidative stress, causing disruption of the already vulnerable blood-brain barrier (BBB). Mitochondrial ferritin (FTMT), a novel mitochondrial outer membrane protein, is crucial in maintaining cellular iron homeostasis. We aimed to investigate the effect of FTMT upregulation on oxidative stress and BBB disruption associated with brain iron overload in rats. A total of 222 Sprague-Dawley neonatal rat pups (7 days old) were used to establish a collagenase-induced GMH model and an iron-overload model of intracerebral FeCl2 injection. Deferiprone was administered via gastric lavage 1 h after GMH and given daily until euthanasia. FTMT CRISPR Knockout and adenovirus (Ad)-FTMT were administered intracerebroventricularly 48 h before GMH and FeCl2 injection, respectively. Neurobehavioral tests, immunofluorescence, Western blot, Malondialdehyde measurement, and brain water content were performed to evaluate neurobehavior deficits, oxidative stress, and BBB disruption, respectively. The results demonstrated that brain expressions of iron exporter Ferroportin (FPN) and antioxidant glutathione peroxidase 4 (GPX4) as well as BBB tight junction proteins including Claudin-5 and Zona Occulta (ZO)-1 were found to be decreased at 72 h after GMH. FTMT agonist Deferiprone attenuated oxidative stress and preserved BBB tight junction proteins after GMH. These effects were partially reversed by FTMT CRISPR Knockout. Iron overload by FeCl2 injection resulted in oxidative stress and BBB disruption, which were improved by Ad-FTMT mediated FTMT overexpression. Collectively, FTMT upregulation is neuroprotective against brain injury associated with iron overload. Deferiprone reduced oxidative stress and BBB disruption by maintaining cellular iron homeostasis partially by the upregulating of FTMT after GMH. Deferiprone may be an effective treatment for patients with GMH.

Oxyberberine protects middle cerebral artery occlusion triggered cerebral injury through TLR4/NLRP3 pathway in rats

Cerebral ischemia is a life-threatening health concern that leads to severe neurological complications and fatalities worldwide. Although timely intervention with clot-removing agents curtails serious post-stroke neurological dysfunctions, no effective neuroprotective intervention is available for addressing post-recanalization neuroinflammation. Herein, for the first time we studied the effect of oxyberberine (OBB), a derivative of berberine, on transient middle cerebral artery occlusion (MCAO)-generated neurological consequences in Sprague-Dawley rats. The MCAO-operated rats exhibited significant somatosensory and sensorimotor dysfunctions in adhesive removal, foot fault, paw whisker, and rotarod assays at 1 and 3 days post-surgery. These MCAO-generated neurological deficits were prevented in OBB-treated (50 and 100 mg/kg) rats, and also coincided with a smaller infarct area (in 2,3,5-triphenyl tetrazolium chloride staining) and decreased neuronal death (in cresyl violet staining) in the ipsilateral hemisphere of these animals. The immunostaining of neuronal nuclear protein (NeuN) and glial-fibrillary acidic protein (GFAP) also echoes the neuroprotective nature of OBB. The increased expression of neuroinflammatory and blood-brain barrier tight junction proteins like toll-like receptor 4 (TLR4), TRAF-6, nuclear factor kappa B (NF-κB), pNF-κB, nNOS, ASC, and IKBα in the ipsilateral part of MCAO-operated rats were restored to normal following OBB treatment. We also observed the decline in plasma levels/mRNA transcription of TNF-α, IL-1β, NLRP3, IL-6, and matrix metalloproteinase-9 and increased expression of occludin and claudin in OBB-treated rats. These outcomes imply that OBB may prevent the MCAO-induced neurological consequences and neuroinflammation by interfering with TLR4 and NLRP3 signaling in rats.

Isomerization and phosphorylation significantly influence the effect of beta amyloid on blood‐brain barrier endothelial cells

AbstractBackgroundThe blood‐brain barrier (BBB) is typically compromised in Alzheimer’s disease (AD): its permeability and intracellular parameters of the endothelial cells are affected. AD‐related amyloid isoforms –Aβ42, isoD7‐Aβ42 and pS8‐Aβ42, inducing amyloidogenesis in a different mode – may have peripheral origin and affect the BBB cells. This study aimed to assess this effect.MethodMouse brain endothelial cells bEnd.3 underwent incubation with 10 µM of Aβ42, isoD7‐Aβ42 and pS8‐Aβ42 for 4, 24 and 48 hours to assess nitric oxide (NO), glutathione (GSH), reactive oxygen species (ROS), mitochondrial potential, Ca2+, cell viability by flow cytometry and mitochondrial functioning by Seahorse technology. To analyze the integrity of the cellular monolayer, the expression level of tight junction proteins and permeability of bEnd.3 monolayer to fluorescent tracers were assessed by Western Blotting and transwell‐modeling, correspondingly .ResultpS8‐Aβ42 and isoD7‐Aβ42 effects on the redox status of the cells and mitochondria functioning differ from the effects of Aβ42. Even at 4 hours, Aβ42 and isoD7‐Aβ42 induce immense changes in mitochondrial potential and NO level but pS8‐Aβ42 does not. Prolonged incubation with three isoforms leads to an increase in ROS, and greater increase is observed after incubation with isoD7‐Aβ42. pS8‐Aβ42 induces ROS increase later than other isoforms. Above all, incubation with pS8‐Aβ42 results in a less pronounced NO и GSH growth compared with Aβ42 и isoD7‐Aβ42 which induce activation of mitochondrial respiration and increase in mitochondrial potential, compared with pS8‐Aβ42. IsoD7‐Aβ42 is mostly toxic to bEnd.3 cells. Although Aβ42 and isoD7‐Aβ42 significantly affect redox parameters of bEnd.3 cells, their effects on BBB permeability and tight junction protein expression are less pronounced. Our data demonstrate that inhibition of NO synthases and NMDA receptors induces changes in BBB cells’ response to Aβ peptides.ConclusionAmyloid isoforms differently affect redox parameters of BBB cells significantly modulating mitochondria functioning. Iso‐Aβ42 induces higher cytotoxicity, thus, being a more pathogenic form. Post‐translational modifications of Aβ42 alter the amyloid effect on the BBB cells. It may result in different changes in BBB functioning and its permeability under amyloid isoforms.This research was funded by Russian Science Foundation (Grant No. #19‐74‐30007).

Fibrinogen in mice cerebral microvessels induces blood–brain barrier dysregulation with aging via a dynamin-related protein 1–dependent pathway

We previously reported evidence that oxidative stress during aging leads to adverse protein profile changes of brain cortical microvessels (MVs: end arterioles, capillaries, and venules) that affect mRNA/protein stability, basement membrane integrity, and ATP synthesis capacity in mice. As an extension of our previous study, we also found that proteins which comprise the blood–brain barrier (BBB) and regulate mitochondrial quality control were also significantly decreased in the mice’s cortical MVs with aging. Interestingly, the neuroinflammatory protein fibrinogen (Fgn) was increased in mice brain MVs, which corresponds with clinical reports indicating that the plasma Fgn concentration increased progressively with aging. In this study, protein–protein interaction network analysis indicated that high expression of Fgn is linked with downregulated expression of both BBB- and mitochondrial fission/fusion–related proteins in mice cortical MVs with aging. To investigate the mechanism of Fgn action, we observed that 2 mg/mL or higher concentration of human plasma Fgn changed cell morphology, induced cytotoxicity, and increased BBB permeability in primary human brain microvascular endothelial cells (HBMECs). The BBB tight junction proteins were significantly decreased with increasing concentration of human plasma Fgn in primary HBMECs. Similarly, the expression of phosphorylated dynamin-related protein 1 (pDRP1) and other mitochondrial fission/fusion–related proteins were also significantly reduced in Fgn-treated HBMECs. Interestingly, DRP1 knockdown by shRNA(h) resulted in the reduction of both BBB- and mitochondrial fission/fusion–related proteins in HBMECs. Our results suggest that elevated Fgn downregulates DRP1, leading to mitochondrial-dependent endothelial and BBB dysfunction in the brain microvasculature.

Estrogenic regulation of claudin 5 and tight junction protein 1 gene expression in zebrafish: A role on blood-brain barrier?

The blood-brain barrier (BBB) is a physical interface between the blood and the brain parenchyma, playing key roles in brain homeostasis. In mammals, the BBB is established thanks to tight junctions between cerebral endothelial cells, involving claudin, occludin, and zonula occludens proteins. Estrogens have been documented to modulate BBB permeability. Interestingly, in the brain of zebrafish, the estrogen-synthesizing activity is strong due to the high expression of Aromatase B protein, encoded by the cyp19a1b gene, in radial glial cells (neural stem cells). Given the roles of estrogens in BBB function, we investigated their impact on the expression of genes involved in BBB tight junctions. We treated zebrafish embryos and adult males with 17β-estradiol and observed an increased cerebral expression of tight junction and claudin 5 genes in adult males only. In females, treatment with the nuclear estrogen receptor antagonist (ICI182,780 ) had no impact. Interestingly, telencephalic injuries performed in males decreased tight junction gene expression that was partially reversed with 17β-estradiol. This was further confirmed by extravasation experiments of Evans blue showing that estrogenic treatment limits BBB leakage. We also highlighted the intimate links between endothelial cells and neural stem cells, suggesting that cholesterol and peripheral steroids could be taken up by endothelial cells and used as precursors for estrogen synthesis by neural stem cells. Together, our results show that zebrafish provides an alternative model to further investigate the role of steroids on the expression of genes involved in BBB integrity, both in constitutive and regenerative physiological conditions. The link we described between capillaries endothelial cells and steroidogenic neural cells encourages the use of this model in understanding the mechanisms by which peripheral steroids get into neural tissue and modulate neurogenic activity.

17-DMAG ameliorates neuroinflammation and BBB disruption via SOX5 mediated PI3K/Akt pathway after intracerebral hemorrhage in rats

Intracerebral hemorrhage (ICH) can result in secondary brain injury due to inflammation and breakdown of the blood–brain barrier (BBB), which are closely associated with patient prognosis. The potential of the heat shock protein 90 (Hsp90) inhibitor 17-DMAG in promoting neuroprotection has been observed in certain vascular diseases. However, the precise role of 17-DMAG treatment in ICH is not yet fully understood. In this study, we found that treatment with 17-DMAG (5 mg/kg) effectively reduced hematoma expansion and resulted in improved neurological outcomes. Meanwhile, the injection of 17-DMAG had a positive effect on reducing BBB disruption in rats with ICH. This effect was achieved by increasing the levels of BBB tight junction proteins (TJPs) such as zo-1, claudin-5, and occludin. As a result, the leakage of EB extravasation, brain edema and IgG in the peri-hematoma tissue were reduced. Furthermore, the injection of 17-DMAG decreased the infiltration of neutrophils into the brain tissues surrounding the hematoma in ICH rats and also reduced the production of proinflammatory cytokines IL-6 and TNF-α. Next, we used integrative mass spectrometry (MS) and molecular docking analysis to confirm that sex determining region Y-box protein 5 (SOX5) is a potential direct target of 17-DMAG in ICH. SOX5 encodes a positive regulator of the PI3K/Akt axis, and treatment with 17-DMAG resulted in a noticeable increase in SOX5 accumulation. To further investigate the role of SOX5, we employed virus-regulated SOX5 silencing and found that suppressing SOX5 blocked the ability of 17-DMAG to suppress neutrophil trafficking. Additionally, silencing SOX5 blocked the protective effects of 17-DMAG on the BBB by inhibiting PI3K, p-Akt, and BBB TJPs levels, which led to an increase in EB and IgG leakage in the peri-hematoma tissue after ICH. Similarly, when SOX5 was knocked down, the protective effects of 17-DMAG were lost. Overall, the results of our study indicate that the injection of 17-DMAG has the potential to mitigate neuroinflammation and prevent the disruption of the BBB caused by ICH, resulting in improved neurological outcomes in rats. These positive effects are attributed to the regulation of SOX5 and activation of the PI3K/Akt pathway. These findings highlight the possibility of targeting SOX5 and the PI3K/Akt pathway as a novel therapeutic approach for ICH.

Treatment with apoptosis inhibitor restores cognitive impairment in rats with myocardial infarction

We previously reported that apoptosis is responsible for cognitive impairment in rats with myocardial infarction (MI). Acute administration of an apoptosis inhibitor (Z-vad) effectively reduced brain inflammation in rats with cardiac ischemia/reperfusion injury. However, the beneficial effects of Z-vad on cognitive function, brain inflammation, mitochondrial function, cell death pathways, and neurogenesis in MI rats have not been investigated. Male rats were divided into sham or MI groups (left anterior descending coronary ligation). A successful MI was determined by a reduction of ejection fraction <50 %. Then, MI rats were allocated to receive vehicle, enalapril (10 mg/kg, a positive control), and Z-vad (1 mg/kg) for 4 weeks. Cardiac function, cognitive function, and molecular analysis were investigated. MI rats exhibited cardiac dysfunction, cognitive impairment, blood brain barrier (BBB) breakdown, dendritic spine loss, which were accompanied by an upregulation of oxidative stress, mitochondrial dysfunction, and apoptosis. Chronic treatment with Z-vad attenuated cardiac dysfunction following MI to the same extent as enalapril. Z-vad successfully improved cognitive function and restored dendritic spine density in MI rats through a reduction of systemic oxidative stress and brain mitochondrial dysfunction similar to enalapril. Moreover, Z-vad provided greater efficacy than enalapril in enhancing mitophagy, neurogenesis, synaptic proteins and reducing apoptosis in hippocampus of MI rats. Nevertheless, neither Z-vad nor enalapril increased BBB tight junction protein. In conclusion, treatment with an apoptosis inhibitor reduced cognitive impairment in MI rats via reducing oxidative stress, mitochondrial dysfunction, apoptosis, and restoring dendritic spine density, together with enhancing mitophagy and neurogenesis.

AMD3100-Mediated CXCR4 Inhibition Impairs Development of Primary Lymphoma of the Central Nervous System

A hallmark of primary lymphoma of the central nervous system (CNS; PCNSL) is the strong CXCR4 expression of the tumor cells, the function of which is still unknown. Invitro treatment of BAL17CNS lymphoma cells by AMD3100, which inhibits CXCR4-CXCL12 interactions, resulted in the significantly differential expression of 273 genes encoding proteins involved in cell motility, cell-cell signaling and interaction, hematological system development and function, and immunologic disease. Among the genes down-regulated was the one encoding CD200, a regulator of CNS immunologic activity. These data directly translated into the invivo situation; BAL17CNS CD200 expression was down-regulated by 89% (3% versus 28% CD200+ lymphoma cells) in AMD3100-treated versus untreated mice with BAL17CNS-induced PCNSL. Reduced lymphoma cell CD200 expression may contribute to the markedly increased microglial activation in AMD3100-treated mice. AMD3100 also maintained the structural integrity of blood-brain barrier tight junctions and the outer basal lamina of cerebral blood vessels. Subsequently, lymphoma cell invasion of the brain parenchyma was impaired, and maximal parenchymal tumor size was significantly reduced by 82% in the induction phase. Thus, AMD3100 qualified as a potentially attractive candidate to be included into the therapeutic concept of PCNSL. Beyond therapy, CXCR4-induced suppression of microglial activity is of general neuroimmunologic interest and identifies CD200 expressed by the lymphoma cells as a novel mechanism of immune escape in PCNSL.

Evidence that enolase-phosphatase 1 exacerbates early cerebral ischemia injury and blood–brain barrier breakdown by enhancing extracellular matrix destruction and inhibiting the interaction between ADI1 and MT1-MMP

Enolase-phosphatase 1 (ENOPH1) is a newly identified enzyme associated with stress responses and cell proliferation. Our previous study found that ENOPH1 mediates cerebral microvascular endothelial cell apoptosis under cerebral ischemia conditions. In this study, we systematically provide mechanistic insights into the regulation of ENOPH1 in blood–brain barrier (BBB) dysfuction induced by early ischemia. ENOPH1 knockout mice (ENOPH1 KO) and wild type (WT) mice were exposed to transient middle cerebral artery occlusion (tMCAO) for 90 min followed by 3 h of reperfusion in vivo, and brain microvascular endothelial cell lines (bEnd.3 cells) were exposed to oxygen-glucose deprivation (OGD) in vitro. BEnd.3 cells were transfected with ENOPH1 shRNA to knockdown ENOPH1 expression. Brain ischemic damage and nerve function was assessed with 2, 3, 5-triphenyltetrazolium chloride (TTC) staining and neurological scores. BBB permeability and tight junction (TJ) protein and adherens junction (AJ) proteins expression were analyzed by FITC-dextran staining, western blotting and coimmunofluorescence. The MMP-2/9 activity was analyzed by gelatin zymography. Differential protein expression was assessed by quantitative proteomics. The interaction between ADI1 and MT1-MMP was measured by coimmunoprecipitation assay and coimmunofluorescence. Knockout of ENOPH1 ameliorated cerebral ischemic injury, decreased BBB permeability, inhibited the activity of MMP-2/9, upregulated the expression of TJ/AJ proteins and reversed extracellular matrix destruction after ischemia in vivo. Mechanistic studies have shown that ENOPH1 silencing enhanced the interaction between ADI1 and MT1-MMP by promoting the nuclear translocation of ADI1 to inhibit MT1-MMP in bEnd.3 cells after OGD and decreasing the expression of Tnc and Fn1 to inhibit ECM degradation. Our results reveal that ENOPH1 increases the activity of MMP-2/9, then promotes TJ protein and extracellular matrix degradation, and eventually destroys the stability of the BBB. Therefore, ENOPH1 is a new therapeutic target for ischemic stroke.

Electroacupuncture Improves Blood-Brain Barrier and Hippocampal Neuroinflammation in SAMP8 Mice by Inhibiting HMGB1/TLR4 and RAGE/NADPH Signaling Pathways.

To investigate the molecular mechanisms underlying the beneficial effect of electroacupuncture (EA) in experimental models of Alzheimer's disease (AD) in vivo. Senescence-accelerated mouse prone 8 (SAMP8) mice were used as AD models and received EA at Yingxiang (LI 20, bilateral) and Yintang (GV 29) points for 20 days. For certain experiments, SAMP8 mice were injected intravenously with human fibrin (2 mg). The Morris water maze test was used to assess cognitive and memory abilities. The changes of tight junctions of blood-brain barrier (BBB) in mice were observed by transmission electron microscope. The expressions of fibrin, amyloid- β (Aβ), and ionized calcium-binding adapter molecule 1 (IBa-1) in mouse hippocampus (CA1/CA3) were detected by reverse transcription-quantitative polymerase chain reaction (qRT-PCR), Western blot or immunohistochemical staining. The expression of fibrin in mouse plasma was detected by enzyme-linked immunosorbent assay. The expressions of tight junction proteins zonula occludens-1 and claudin-5 in hippocampus were detected by qRT-PCR and immunofluorescence staining. Apoptosis of hippocampal neurons was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Fibrin was time-dependently deposited in the hippocampus of SAMP8 mice and this was inhibited by EA treatment (P<0.05 or P<0.01). Furthermore, EA treatment suppressed the accumulation of Aβ in the hippocampus of SAMP8 mice (P<0.01), which was reversed by fibrin injection (P<0.05 or P<0.01). EA improved SAMP8 mice cognitive impairment and BBB permeability (P<0.05 or P<0.01). Moreover, EA decreased reactive oxygen species levels and neuroinflammation in the hippocampus of SAMP8 mice, which was reversed by fibrin injection (P<0.05 or P<0.01). Mechanistically, EA inhibited the promoting effect of fibrin on the high mobility group box protein 1 (HMGB1)/toll-like receptor 4 (TLR4) and receptor for advanced glycation end products (RAGE)/nicotinamide adenine dinucleotide phosphate (NADPH) signaling pathways (P<0.01). EA may potentially improve cognitive impairment in AD via inhibition of fibrin/A β deposition and deactivation of the HMGB1/TLR4 and RAGE/NADPH signaling pathways.

The interplay between MMP-12 and t-PA in the brain after ischemic stroke

Tissue-type plasminogen activator (t-PA) expression is known to increase following transient focal cerebral ischemia and reperfusion. Previously, we reported downregulation of t-PA upon suppression of matrix metalloproteinase-12 (MMP-12), following transient focal cerebral ischemia and reperfusion. We now present data on the temporal expression of t-PA in the brain after transient ischemia, as well as the interaction between MMP-12 and t-PA, two proteases associated with the breakdown of the blood-brain barrier (BBB) and ischemic brain damage. We hypothesized that there might be reciprocal interactions between MMP-12 and t-PA in the brain after ischemic stroke. This hypothesis was tested using shRNA-mediated gene silencing and computational modeling. Suppression of t-PA following transient ischemia and reperfusion in rats attenuated MMP-12 expression in the brain. The overall effect of t-PA shRNA administration was to attenuate the degradation of BBB tight junction protein claudin-5, diminish BBB disruption, and reduce neuroinflammation by decreasing the expression of the microglia/macrophage pro-inflammatory M1 phenotype (CD68, iNOS, IL-1β, and TNFα). Reduced BBB disruption and subsequent lack of infiltration of macrophages (the main source of MMP-12 in the ischemic brain) could account for the decrease in MMP-12 expression after t-PA suppression. Computational modeling of in silico protein-protein interactions indicated that MMP-12 and t-PA may interact physically. Overall, our findings demonstrate that MMP-12 and t-PA interact directly or indirectly at multiple levels in the brain following an ischemic stroke. The present findings could be useful in the development of new pharmacotherapies for the treatment of stroke.

Spermidine/Spermine N1-Acetyltransferase 1 (SAT1)-A Potential Gene Target for Selective Sensitization of Glioblastoma Cells Using an Ionizable Lipid Nanoparticle to Deliver siRNA.

Spermidine/spermine N1-acetyltransferase 1 (SAT1) responsible for cell polyamine catabolism is overexpressed in glioblastoma multiforme (GB). Its role in tumor survival and promoting resistance towards radiation therapy has made it an interesting target for therapy. In this study, we prepared a lipid nanoparticle-based siRNA delivery system (LNP-siSAT1) to selectively knockdown (KD) SAT1 enzyme in a human glioblastoma cell line. The LNP-siSAT1 containing ionizable DODAP lipid was prepared following a microfluidics mixing method and the resulting nanoparticles had a hydrodynamic size of around 80 nm and a neutral surface charge. The LNP-siSAT1 effectively knocked down the SAT1 expression in U251, LN229, and 42MGBA GB cells, and other brain-relevant endothelial (hCMEC/D3), astrocyte (HA) and macrophage (ANA-1) cells at the mRNA and protein levels. SAT1 KD in U251 cells resulted in a 40% loss in cell viability. Furthermore, SAT1 KD in U251, LN229 and 42MGBA cells sensitized them towards radiation and chemotherapy treatments. In contrast, despite similar SAT1 KD in other brain-relevant cells no significant effect on cytotoxic response, either alone or in combination, was observed. A major roadblock for brain therapeutics is their ability to cross the highly restrictive blood-brain barrier (BBB) presented by the brain microcapillary endothelial cells. Here, we used the BBB circumventing approach to enhance the delivery of LNP-siSAT1 across a BBB cell culture model. A cadherin binding peptide (ADTC5) was used to transiently open the BBB tight junctions to promote paracellular diffusion of LNP-siSAT1. These results suggest LNP-siSAT1 may provide a safe and effective method for reducing SAT1 and sensitizing GB cells to radiation and chemotherapeutic agents.