Intrauterine inflammation and postnatal intravenous dopamine alter the neurovascular unit in preterm newborn lambs.

BACKGROUND: Intrauterine inflammation is considered a major cause of brain injury in preterm infants, leading to long-term neurodevelopmental deficits. A potential contributor to this brain injury is dysregulation of neurovascular coupling. We have shown that intrauterine inflammation induced by intra-amniotic lipopolysaccharide (LPS) in preterm lambs, and postnatal dopamine administration, disrupts neurovascular coupling and the functional cerebral haemodynamic responses, potentially leading to impaired brain development. In this study, we aimed to characterise the structural changes of the neurovascular unit following intrauterine LPS exposure and postnatal dopamine administration in the brain of preterm lambs using cellular and molecular analyses. METHODS: At 119-120 days of gestation (term = 147 days), LPS was administered into the amniotic sac in pregnant ewes. At 126-7 days of gestation, the LPS-exposed lambs were delivered, ventilated and given either a continuous intravenous infusion of dopamine at 10 µg/kg/min or isovolumetric vehicle solution for 90 min (LPS, n = 6; LPSDA, n = 6). Control preterm lambs not exposed to LPS were also administered vehicle or dopamine (CTL, n = 9; CTLDA, n = 7). Post-mortem brain tissue was collected 3-4 h after birth for immunohistochemistry and RT-qPCR analysis of components of the neurovascular unit. RESULTS: LPS exposure increased vascular leakage in the presence of increased vascular density and remodelling with increased astrocyte "end feet" vessel coverage, together with downregulated mRNA levels of the tight junction proteins Claudin-1 and Occludin. Dopamine administration decreased vessel density and size, decreased endothelial glucose transporter, reduced neuronal dendritic coverage, increased cell proliferation within vessel walls, and increased pericyte vascular coverage particularly within the cortical and deep grey matter. Dopamine also downregulated VEGFA and Occludin tight junction mRNA, and upregulated dopamine receptor DRD1 and oxidative protein (NOX1, SOD3) mRNA levels. Dopamine administration following LPS exposure did not exacerbate any effects induced by LPS. CONCLUSION: LPS exposure and dopamine administration independently alters the neurovascular unit in the preterm brain. Alterations to the neurovascular unit may predispose the developing brain to further injury.

SLC22A17 as a Cell Death-Linked Regulator of Tight Junctions in Cerebral Ischemia.

BACKGROUND: Beyond neuronal injury, cell death pathways may also contribute to vascular injury after stroke. We examined protein networks linked to major cell death pathways and identified SLC22A17 (solute carrier family 22 member 17) as a novel mediator that regulates endothelial tight junctions after ischemia and inflammatory stress. METHODS: Protein-protein interactions and brain enrichment analyses were performed using STRING, Cytoscape, and a human tissue-specific expression RNA-seq database. In vivo experiments were performed using mouse models of transient focal cerebral ischemia. Human stroke brain tissues were used to detect SLC22A17 by immunostaining. In vitro experiments were performed using human brain endothelial cultures subjected to inflammatory stress. Immunostaining and Western blot were used to assess responses in SLC22A17 and endothelial tight junctional proteins. Water content, dextran permeability, and electrical resistance assays were used to assess edema and blood-brain barrier (BBB) integrity. Gain and loss-of-function studies were performed using lentiviral overexpression of SLC22A17 or short interfering RNA against SLC22A17, respectively. RESULTS: Protein-protein interaction analysis showed that core proteins from apoptosis, necroptosis, ferroptosis, and autophagy cell death pathways were closely linked. Among the 20 proteins identified in the network, the iron-handling solute carrier SLC22A17 emerged as the mediator enriched in the brain. After cerebral ischemia in vivo, endothelial expression of SLC22A17 increases in both human and mouse brains along with BBB leakage. In human brain endothelial cultures, short interfering RNA against SLC22A17 prevents TNF-α (tumor necrosis factor alpha)-induced ferroptosis and downregulation in tight junction proteins and disruption in transcellular permeability. Notably, SLC22A17 could repress the transcription of tight junctional genes. Finally, short interfering RNA against SLC22A17 ameliorates BBB leakage in a mouse model of focal cerebral ischemia. CONCLUSIONS: Using a combination of cell culture, human stroke samples, and mouse models, our data suggest that SLC22A17 may play a role in the control of BBB function after cerebral ischemia. These findings may offer a novel mechanism and target for ameliorating BBB injury and edema after stroke.

Increased Expression of VCAM1 on Brain Endothelial Cells Drives Blood-Brain Barrier Impairment Following Chronic Cerebral Hypoperfusion.

Chronic cerebral hypoperfusion (CCH)-triggered blood-brain barrier (BBB) dysfunction is a core pathological change occurring in vascular dementia (VD). Despite the recent advances in the exploration of the structural basis of BBB impairment and the routes of entry of harmful compounds after a BBB leakage, the molecular mechanisms inducing BBB impairment remain largely unknown in terms of VD. Here, we employed a CCH-induced VD model and discovered increased vascular cell adhesion molecule 1 (VCAM1) expression on the brain endothelial cells (ECs). The expression of VCAM1 was directly correlated with the severity of BBB impairment. Moreover, the VCAM1 expression was associated with different regional white matter lesions. Furthermore, a compound that could block VCAM1 activation, K-7174, was also found to alleviate BBB leakage and protect the white matter integrity, whereas pharmacological manipulation of the BBB leakage did not affect the VCAM1 expression. Thus, our results demonstrated that VCAM1 is an important regulator that leads to BBB dysfunction following CCH. Blocking VCAM1-mediated BBB impairment may thus offer a new strategy to treat CCH-related neurodegenerative diseases.

Chronic Traumatic Encephalopathy as the Course of Alzheimer's Disease.

This editorial investigates chronic traumatic encephalopathy (CTE) as a course of Alzheimer's disease (AD). CTE is a debilitating neurodegenerative disease that is the result of repeated mild traumatic brain injury (TBI). Many epidemiological studies show that experiencing a TBI in early or middle life is associated with an increased risk of dementia later in life. Chronic traumatic encephalopathy (CTE) and Alzheimer's disease (AD) present a series of similar neuropathological features that were investigated in this work like recombinant tau into filaments or the accumulation and aggregation of Aß protein. However, these two conditions differ from each other in brain-blood barrier damage. The purpose of this review was to evaluate information about CTE and AD from various articles, focusing especially on new therapeutic possibilities for the improvement in cognitive skills.

HIV-Associated Neurocognitive Disorder: A Look into Cellular and Molecular Pathology.

Despite combined antiretroviral therapy (cART) limiting HIV replication to undetectable levels in the blood, people living with HIV continue to experience HIV-associated neurocognitive disorder (HAND). HAND is associated with neurocognitive impairment, including motor impairment, and memory loss. HIV has been detected in the brain within 8 days of estimated exposure and the mechanisms for this early entry are being actively studied. Once having entered into the central nervous system (CNS), HIV degrades the blood-brain barrier through the production of its gp120 and Tat proteins. These proteins are directly toxic to endothelial cells and neurons, and propagate inflammatory cytokines by the activation of immune cells and dysregulation of tight junction proteins. The BBB breakdown is associated with the progression of neurocognitive disease. One of the main hurdles for treatment for HAND is the latent pool of cells, which are insensitive to cART and prolong inflammation by harboring the provirus in long-lived cells that can reactivate, causing damage. Multiple strategies are being studied to combat the latent pool and HAND; however, clinically, these approaches have been insufficient and require further revisions. The goal of this paper is to aggregate the known mechanisms and challenges associated with HAND.

Early Blood-Brain Barrier Impairment as a Pathological Hallmark in a Novel Model of Closed-Head Concussive Brain Injury (CBI) in Mice.

Concussion, caused by a rotational acceleration/deceleration injury mild enough to avoid structural brain damage, is insufficiently captured in recent preclinical models, hampering the relation of pathophysiological findings on the cellular level to functional and behavioral deficits. We here describe a novel model of unrestrained, single vs. repetitive concussive brain injury (CBI) in male C56Bl/6j mice. Longitudinal behavioral assessments were conducted for up to seven days afterward, alongside the evaluation of structural cerebral integrity by in vivo magnetic resonance imaging (MRI, 9.4 T), and validated ex vivo by histology. Blood-brain barrier (BBB) integrity was analyzed by means of fluorescent dextran- as well as immunoglobulin G (IgG) extravasation, and neuroinflammatory processes were characterized both in vivo by positron emission tomography (PET) using [18F]DPA-714 and ex vivo using immunohistochemistry. While a single CBI resulted in a defined, subacute neuropsychiatric phenotype, longitudinal cognitive testing revealed a marked decrease in spatial cognition, most pronounced in mice subjected to CBI at high frequency (every 48 h). Functional deficits were correlated to a parallel disruption of the BBB, (R2 = 0.29, p < 0.01), even detectable by a significant increase in hippocampal uptake of [18F]DPA-714, which was not due to activation of microglia, as confirmed immunohistochemically. Featuring a mild but widespread disruption of the BBB without evidence of macroscopic damage, this model induces a characteristic neuro-psychiatric phenotype that correlates to the degree of BBB disruption. Based on these findings, the BBB may function as both a biomarker of CBI severity and as a potential treatment target to improve recovery from concussion.

Effects of Sodium Nitroprusside on Lipopolysaccharide-Induced Inflammation and Disruption of Blood-Brain Barrier.

In various neurodegenerative conditions, inflammation plays a significant role in disrupting the blood-brain barrier (BBB), contributing to disease progression. Nitric oxide (NO) emerges as a central regulator of vascular function, with a dual role in inflammation, acting as both a pro- and anti-inflammatory molecule. This study investigates the effects of the NO donor sodium nitroprusside (SNP) in protecting the BBB from lipopolysaccharide (LPS)-induced inflammation, using bEnd.3 endothelial cells as a model system. Additionally, Raw 264.7 macrophages were employed to assess the effects of LPS and SNP on their adhesion to a bEnd.3 cell monolayer. Our results show that LPS treatment induces oxidative stress, activates the JAK2/STAT3 pathway, and increases pro-inflammatory markers. SNP administration effectively mitigates ROS production and IL-6 expression, suggesting a potential anti-inflammatory role. However, SNP did not significantly alter the adhesion of Raw 264.7 cells to bEnd.3 cells induced by LPS, probably because it did not have any effect on ICAM-1 expression, although it reduced VCAM expression. Moreover, SNP did not prevent BBB disruption. This research provides new insights into the role of NO in BBB disruption induced by inflammation.

Gain-of-function mutations of TRPV4 acting in endothelial cells drive blood-CNS barrier breakdown and motor neuron degeneration in mice.

Blood-CNS barrier disruption is a hallmark of numerous neurological disorders, yet whether barrier breakdown is sufficient to trigger neurodegenerative disease remains unresolved. Therapeutic strategies to mitigate barrier hyperpermeability are also limited. Dominant missense mutations of the cation channel transient receptor potential vanilloid 4 (TRPV4) cause forms of hereditary motor neuron disease. To gain insights into the cellular basis of these disorders, we generated knock-in mouse models of TRPV4 channelopathy by introducing two disease-causing mutations (R269C and R232C) into the endogenous mouse Trpv4 gene. TRPV4 mutant mice exhibited weakness, early lethality, and regional motor neuron loss. Genetic deletion of the mutant Trpv4 allele from endothelial cells (but not neurons, glia, or muscle) rescued these phenotypes. Symptomatic mutant mice exhibited focal disruptions of blood-spinal cord barrier (BSCB) integrity, associated with a gain of function of mutant TRPV4 channel activity in neural vascular endothelial cells (NVECs) and alterations of NVEC tight junction structure. Systemic administration of a TRPV4-specific antagonist abrogated channel-mediated BSCB impairments and provided a marked phenotypic rescue of symptomatic mutant mice. Together, our findings show that mutant TRPV4 channels can drive motor neuron degeneration in a non-cell autonomous manner by precipitating focal breakdown of the BSCB. Further, these data highlight the reversibility of TRPV4-mediated BSCB impairments and identify a potential therapeutic strategy for patients with TRPV4 mutations.

Therapeutic effects of orexin-A in sepsis-associated encephalopathy in mice.

BACKGROUND: Sepsis-associated encephalopathy (SAE) causes acute and long-term cognitive deficits. However, information on the prevention and treatment of cognitive dysfunction after sepsis is limited. The neuropeptide orexin-A (OXA) has been shown to play a protective role against neurological diseases by modulating the inflammatory response through the activation of OXR1 and OXR2 receptors. However, the role of OXA in mediating the neuroprotective effects of SAE has not yet been reported. METHODS: A mouse model of SAE was induced using cecal ligation perforation (CLP) and treated via intranasal administration of exogenous OXA after surgery. Mouse survival, in addition to cognitive and anxiety behaviors, were assessed. Changes in neurons, cerebral edema, blood-brain barrier (BBB) permeability, and brain ultrastructure were monitored. Levels of pro-inflammatory factors (IL-1ß, TNF-α) and microglial activation were also measured. The underlying molecular mechanisms were investigated by proteomics analysis and western blotting. RESULTS: Intranasal OXA treatment reduced mortality, ameliorated cognitive and emotional deficits, and attenuated cerebral edema, BBB disruption, and ultrastructural brain damage in mice. In addition, OXA significantly reduced the expression of the pro-inflammatory factors IL-1ß and TNF-α, and inhibited microglial activation. In addition, OXA downregulated the expression of the Rras and RAS proteins, and reduced the phosphorylation of P-38 and JNK, thus inhibiting activation of the MAPK pathway. JNJ-10,397,049 (an OXR2 blocker) reversed the effect of OXA, whereas SB-334,867 (an OXR1 blocker) did not. CONCLUSION: This study demonstrated that the intranasal administration of moderate amounts of OXA protects the BBB and inhibits the activation of the OXR2/RAS/MAPK pathway to attenuate the outcome of SAE, suggesting that OXA may be a promising therapeutic approach for the management of SAE.

Variants in the MS4A cluster interact with soluble TREM2 expression on biomarkers of neuropathology.

Recent evidence suggests that Alzheimer's disease (AD) genetic risk variants (rs1582763 and rs6591561) of the MS4A locus are genome-wide significant regulators of soluble TREM2 levels such that the minor allele of the protective variant (rs1582763) is associated with higher sTREM2 and lower AD risk while the minor allele of (rs6591561) relates to lower sTREM2 and higher AD risk. Our group previously found that higher sTREM2 relates to higher Aß40, worse blood-brain barrier (BBB) integrity (measured with the CSF/plasma albumin ratio), and higher CSF tau, suggesting strong associations with amyloid abundance and both BBB and neurodegeneration complicate interpretation. We expand on this work by leveraging these common variants as genetic tools to tune the interpretation of high CSF sTREM2, and by exploring the potential modifying role of these variants on the well-established associations between CSF sTREM2 as well as TREM2 transcript levels in the brain with AD neuropathology. Biomarker analyses leveraged data from the Vanderbilt Memory & Aging Project (n = 127, age = 72 ± 6.43) and were replicated in the Alzheimer's Disease Neuroimaging Initiative (n = 399, age = 73 ± 7.39). Autopsy analyses were performed leveraging data from the Religious Orders Study and Rush Memory and Aging Project (n = 577, age = 89 ± 6.46). We found that the protective variant rs1582763 attenuated the association between CSF sTREM2 and Aß40 (ß = -0.44, p-value = 0.017) and replicated this interaction in ADNI (ß = -0.27, p = 0.017). We did not observe this same interaction effect between TREM2 mRNA levels and Aß peptides in brain (Aß total ß = -0.14, p = 0.629; Aß1-38, ß = 0.11, p = 0.200). In contrast to the effects on Aß, the minor allele of this same variant seemed to enhance the association with blood-brain barrier dysfunction (ß = 7.0e-4, p = 0.009), suggesting that elevated sTREM2 may carry a much different interpretation in carriers vs. non-carriers of this allele. When evaluating the risk variant (rs6591561) across datasets, we did not observe a statistically significant interaction against any outcome in VMAP and observed opposing directions of associations in ADNI and ROS/MAP on Aß levels. Together, our results suggest that the protective effect of rs1582763 may act by decoupling the associations between sTREM2 and amyloid abundance, providing important mechanistic insight into sTREM2 changes and highlighting the need to incorporate genetic context into the analysis of sTREM2 levels, particularly if leveraged as a clinical biomarker of disease in the future.

Obesity-induced blood-brain barrier dysfunction: phenotypes and mechanisms.

Obesity, a burgeoning global health issue, is increasingly recognized for its detrimental effects on the central nervous system, particularly concerning the integrity of the blood-brain barrier (BBB). This manuscript delves into the intricate relationship between obesity and BBB dysfunction, elucidating the underlying phenotypes and molecular mechanisms. We commence with an overview of the BBB's critical role in maintaining cerebral homeostasis and the pathological alterations induced by obesity. By employing a comprehensive literature review, we examine the structural and functional modifications of the BBB in the context of obesity, including increased permeability, altered transport mechanisms, and inflammatory responses. The manuscript highlights how obesity-induced systemic inflammation and metabolic dysregulation contribute to BBB disruption, thereby predisposing individuals to various neurological disorders. We further explore the potential pathways, such as oxidative stress and endothelial cell dysfunction, that mediate these changes. Our discussion culminates in the summary of current findings and the identification of knowledge gaps, paving the way for future research directions. This review underscores the significance of understanding BBB dysfunction in obesity, not only for its implications in neurodegenerative diseases but also for developing targeted therapeutic strategies to mitigate these effects.

The Temporal Relationship between Blood-Brain Barrier Integrity and Microglial Response following Neonatal Hypoxia Ischemia.

Blood-brain barrier (BBB) dysfunction and neuroinflammation are key mechanisms of brain injury. We performed a time-course study following neonatal hypoxia-ischemia (HI) to characterize these events. HI brain injury was induced in postnatal day 10 rats by single carotid artery ligation followed by hypoxia (8% oxygen, 90 min). At 6, 12, 24, and 72 h (h) post-HI, brains were collected to assess neuropathology and BBB dysfunction. A significant breakdown of the BBB was observed in the HI injury group compared to the sham group from 6 h in the cortex and hippocampus (p < 0.001), including a significant increase in albumin extravasation (p < 0.0033) and decrease in basal lamina integrity and tight-junction proteins. There was a decrease in resting microglia (p < 0.0001) transitioning to an intermediate state from as early as 6 h post-HI, with the intermediate microglia peaking at 12 h (p < 0.0001), which significantly correlated to the peak of microbleeds. Neonatal HI insult leads to significant brain injury over the first 72 h that is mediated by BBB disruption within 6 h and a transitioning state of the resident microglia. Key BBB events coincide with the appearance of the intermediate microglial state and this relationship warrants further research and may be a key target for therapeutic intervention.

Activation of Wnt signaling mitigates blood-brain barrier disruption by inhibiting vesicular transcytosis after traumatic brain injury in mice.

Elevated transport of Caveolin-1 (CAV-1) vesicles within vascular endothelial cells constitutes a significant secondary pathogenic event contributing to the compromise of the blood-brain barrier (BBB) post-traumatic brain injury (TBI). While Wnt/ß-catenin signaling is recognized for its critical involvement in angiogenesis and the maintenance of BBB integrity, its influence on vascular endothelial transcytosis in the aftermath of TBI is not well-defined. This study aims to elucidate the impact of Wnt/ß-catenin signaling on cerebrovascular vesicular transcytosis following TBI. In this experiment, adult male wild-type (WT) C57BL/6 mice underwent various interventions. TBI was induced utilizing the controlled cortical impact technique. Post-TBI, mice were administered either an inhibitor or an agonist of Wnt signaling via intraperitoneal injection. Recombinant adeno-associated virus (rAAV) was administered intracerebroventricularly to modulate the expression of the CAV-1 inhibitory protein, Major facilitator superfamily domain-containing 2a (Mfsd2a). This research utilized Evans blue assay, Western blot analysis, immunofluorescence, transmission electron microscopy, and neurobehavioral assessments. Post-TBI observations revealed substantial increases in macromolecule (Evans blue and albumin) leakage, CAV-1 transport vesicle count, astrocyte end-feet edema, and augmented aquaporin-4 (AQP4) expression, culminating in BBB disruption. The findings indicate that Wnt signaling pathway inhibition escalates CAV-1 transport vesicle activity and aggravates BBB compromise. Conversely, activating this pathway could alleviate BBB damage by curtailing CAV-1 vesicle presence. Post-TBI, there is a diminution in Mfsd2a expression, which is directly influenced by the modulation of WNT signals. Employing a viral approach to regulate Mfsd2a, we established that its down-regulation undermines the protective benefits derived from reducing CAV-1 transport vesicles through WNT signal enhancement. Moreover, we verified that the WNT signaling agonist LiCl notably ameliorates neurological deficits following TBI in mice. Collectively, our data imply that Wnt/ß-catenin signaling presents a potential therapeutic target for safeguarding against BBB damage and enhancing neurological function after TBI.

Activation of NLRP3 inflammasome in a rat model of cerebral small vessel disease.

Cerebral small vessel disease (CSVD) is increasingly being recognized as a leading contributor to cognitive impairment in the elderly. However, there is a lack of effective preventative or therapeutic options for CSVD. In this exploratory study, we investigated the interplay between neuroinflammation and CSVD pathogenesis as well as the cognitive performance, focusing on NLRP3 signaling as a new therapeutic target. Spontaneously hypertensive stroke-prone (SHRSP) rats served as a CSVD model. We found that SHRSP rats showed decline in learning and memory abilities using morris water maze test. Activated NLRP3 signaling and an increased expression of the downstream pro-inflammatory factors, including IL (interleukin)-6 and tumor necrosis factor α were determined. We also observed a remarkable increase in the production of pyroptosis executive protein gasdermin D, and elevated astrocytic and microglial activation. In addition, we identify several neuropathological hallmarks of CSVD, including blood-brain barrier breakdown, white matter damage, and endothelial dysfunction. These results were in correlation with the activation of NLRP3 inflammasome. Thus, our findings reveal that the NLRP3-mediated inflammatory pathway could play a central role in the pathogenesis of CSVD, presenting a novel target for potential CSVD treatment.

Regulation of CNS pathology by Serpina3n/SERPINA3: The knowns and the puzzles.

Neuroinflammation, blood-brain barrier (BBB) dysfunction, neuron and glia injury/death and myelin damage are common central nervous system (CNS) pathologies observed in various neurological diseases and injuries. Serine protease inhibitor (Serpin) clade A member 3n (Serpina3n), and its human orthologue SERPINA3, is an acute-phase inflammatory glycoprotein secreted primarily by the liver into the bloodstream in response to systemic inflammation. Clinically, SERPINA3 is dysregulated in brain cells, cerebrospinal fluid and plasma in various neurological conditions. Although it has been widely accepted that Serpina3n/SERPINA3 is a reliable biomarker of reactive astrocytes in diseased CNS, recent data have challenged this well-cited concept, suggesting instead that oligodendrocytes and neurons are the primary sources of Serpina3n/SERPINA3. The debate continues regarding whether Serpina3n/SERPINA3 induction represents a pathogenic or a protective mechanism. Here, we propose possible interpretations for previously controversial data and present perspectives regarding the potential role of Serpina3n/SERPINA3 in CNS pathologies, including demyelinating disorders where oligodendrocytes are the primary targets. We hypothesise that the 'good' or 'bad' aspects of Serpina3n/SERPINA3 depend on its cellular sources, its subcellular distribution (or mis-localisation) and/or disease/injury types. Furthermore, circulating Serpina3n/SERPINA3 may cross the BBB to impact CNS pathologies. Cell-specific genetic tools are critically important to tease out the potential roles of cell type-dependent Serpina3n in CNS diseases/injuries.

The Microglial Transcriptome of Age-Associated Deep Subcortical White Matter Lesions Suggests a Neuroprotective Response to Blood-Brain Barrier Dysfunction.

Age-associated deep-subcortical white matter lesions (DSCLs) are an independent risk factor for dementia, displaying high levels of CD68+ microglia. This study aimed to characterize the transcriptomic profile of microglia in DSCLs and surrounding radiologically normal-appearing white matter (NAWM) compared to non-lesional control white matter. CD68+ microglia were isolated from white matter groups (n = 4 cases per group) from the Cognitive Function and Ageing Study neuropathology cohort using immuno-laser capture microdissection. Microarray gene expression profiling, but not RNA-sequencing, was found to be compatible with immuno-LCM-ed post-mortem material in the CFAS cohort and identified significantly differentially expressed genes (DEGs). Functional grouping and pathway analysis were assessed using the Database for Annotation Visualization and Integrated Discovery (DAVID) software, and immunohistochemistry was performed to validate gene expression changes at the protein level. Transcriptomic profiling of microglia in DSCLs compared to non-lesional control white matter identified 181 significant DEGs (93 upregulated and 88 downregulated). Functional clustering analysis in DAVID revealed dysregulation of haptoglobin-haemoglobin binding (Enrichment score 2.5, p = 0.017), confirmed using CD163 immunostaining, suggesting a neuroprotective microglial response to blood-brain barrier dysfunction in DSCLs. In NAWM versus control white matter, microglia exhibited 347 DEGs (209 upregulated, 138 downregulated), with significant dysregulation of protein de-ubiquitination (Enrichment score 5.14, p < 0.001), implying an inability to maintain protein homeostasis in NAWM that may contribute to lesion spread. These findings enhance understanding of microglial transcriptomic changes in ageing white matter pathology, highlighting a neuroprotective adaptation in DSCLs microglia and a potentially lesion-promoting phenotype in NAWM microglia.

Ibrutinib disrupts blood-tumor barrier integrity and prolongs survival in rodent glioma model.

In malignant glioma, cytotoxic drugs are often inhibited from accessing the tumor site due to the blood-tumor barrier (BTB). Ibrutinib, FDA-approved lymphoma agent, inhibits Bruton tyrosine kinase (BTK) and has previously been shown to independently impair aortic endothelial adhesion and increase rodent glioma model survival in combination with cytotoxic therapy. Yet additional research is required to understand ibrutinib's effect on BTB function. In this study, we detail baseline BTK expression in glioma cells and its surrounding vasculature, then measure endothelial junctional expression/function changes with varied ibrutinib doses in vitro. Rat glioma cells and rodent glioma models were treated with ibrutinib alone (1-10 µM and 25 mg/kg) and in combination with doxil (10-100 µM and 3 mg/kg) to assess additive effects on viability, drug concentrations, tumor volume, endothelial junctional expression and survival. We found that ibrutinib, in a dose-dependent manner, decreased brain endothelial cell-cell adhesion over 24 h, without affecting endothelial cell viability (p < 0.005). Expression of tight junction gene and protein expression was decreased maximally 4 h after administration, along with inhibition of efflux transporter, ABCB1, activity. We demonstrated an additive effect of ibrutinib with doxil on rat glioma cells, as seen by a significant reduction in cell viability (p < 0.001) and increased CNS doxil concentration in the brain (56 ng/mL doxil alone vs. 74.6 ng/mL combination, p < 0.05). Finally, Ibrutinib, combined with doxil, prolonged median survival in rodent glioma models (27 vs. 16 days, p < 0.0001) with brain imaging showing a - 53% versus - 75% volume change with doxil alone versus combination therapy (p < 0.05). These findings indicate ibrutinib's ability to increase brain endothelial permeability via junctional disruption and efflux inhibition, to increase BTB drug entry and prolong rodent glioma model survival. Our results motivate the need to identify other BTB modifiers, all with the intent of improving survival and reducing systemic toxicities.

Brain endothelial GSDMD activation mediates inflammatory BBB breakdown.

The blood-brain barrier (BBB) protects the central nervous system from infections or harmful substances1; its impairment can lead to or exacerbate various diseases of the central nervous system2-4. However, the mechanisms of BBB disruption during infection and inflammatory conditions5,6 remain poorly defined. Here we find that activation of the pore-forming protein GSDMD by the cytosolic lipopolysaccharide (LPS) sensor caspase-11 (refs. 7-9), but not by TLR4-induced cytokines, mediates BBB breakdown in response to circulating LPS or during LPS-induced sepsis. Mice deficient in the LBP-CD14 LPS transfer and internalization pathway10-12 resist BBB disruption. Single-cell RNA-sequencing analysis reveals that brain endothelial cells (bECs), which express high levels of GSDMD, have a prominent response to circulating LPS. LPS acting on bECs primes Casp11 and Cd14 expression and induces GSDMD-mediated plasma membrane permeabilization and pyroptosis in vitro and in mice. Electron microscopy shows that this features ultrastructural changes in the disrupted BBB, including pyroptotic endothelia, abnormal appearance of tight junctions and vasculature detachment from the basement membrane. Comprehensive mouse genetic analyses, combined with a bEC-targeting adeno-associated virus system, establish that GSDMD activation in bECs underlies BBB disruption by LPS. Delivery of active GSDMD into bECs bypasses LPS stimulation and opens the BBB. In CASP4-humanized mice, Gram-negative Klebsiella pneumoniae infection disrupts the BBB; this is blocked by expression of a GSDMD-neutralizing nanobody in bECs. Our findings outline a mechanism for inflammatory BBB breakdown, and suggest potential therapies for diseases of the central nervous system associated with BBB impairment.

Strategies to Improve Drug Delivery Across the Blood-Brain Barrier for Glioblastoma.

PURPOSE OF REVIEW: Glioblastoma remains resistant to most conventional treatments. Despite scientific advances in the past three decades, there has been a dearth of effective new treatments. New approaches to drug delivery and clinical trial design are needed. RECENT FINDINGS: We discuss how the blood-brain barrier and tumor microenvironment pose challenges for development of effective therapies for glioblastoma. Next, we discuss treatments in development that aim to overcome these barriers, including novel drug designs such as nanoparticles and antibody-drug conjugates, novel methods of drug delivery, including convection-enhanced and intra-arterial delivery, and novel methods to enhance drug penetration, such as blood-brain barrier disruption by focused ultrasound and laser interstitial thermal therapy. Lastly, we address future opportunities, positing combination therapy as the best strategy for effective treatment, neoadjuvant and window-of-opportunity approaches to simultaneously enhance therapeutic effectiveness with interrogation of on-treatment biologic endpoints, and adaptive platform and basket trials as imperative for future trial design. New approaches to GBM treatment should account for the blood-brain barrier and immunosuppression by improving drug delivery, combining treatments, and integrating novel clinical trial designs.