Engineered Wnt7a ligands rescue blood-brain barrier and cognitive deficits in a COVID-19 mouse model.

Respiratory infection with SARS-CoV-2 causes systemic vascular inflammation and cognitive impairment. We sought to identify the underlying mechanisms mediating cerebrovascular dysfunction and inflammation following mild respiratory SARS-CoV-2 infection. To this end, we performed unbiased transcriptional analysis to identify brain endothelial cell signalling pathways dysregulated by mouse adapted SARS-CoV-2 MA10 in aged immunocompetent C57Bl/6 mice in vivo. This analysis revealed significant suppression of Wnt/ß-catenin signalling, a critical regulator of blood-brain barrier (BBB) integrity. We therefore hypothesized that enhancing cerebrovascular Wnt/ß-catenin activity would offer protection against BBB permeability, neuroinflammation, and neurological signs in acute infection. Indeed, we found that delivery of cerebrovascular-targeted, engineered Wnt7a ligands protected BBB integrity, reduced T-cell infiltration of the brain, and reduced microglial activation in SARS-CoV-2 infection. Importantly, this strategy also mitigated SARS-CoV-2 induced deficits in the novel object recognition assay for learning and memory and the pole descent task for bradykinesia. These observations suggest that enhancement of Wnt/ß-catenin signalling or its downstream effectors could be potential interventional strategies for restoring cognitive health following viral infections.

Endothelial Cell APOE3 Regulates Neurovascular, Neuronal, and Behavioral Function.

BACKGROUND: Specialized brain endothelial cells and human APOE3 are independently important for neurovascular function, yet whether APOE3 expression by endothelial cells contributes to brain function is currently unknown. In the present study, we determined whether the loss of endothelial cell APOE3 impacts brain vascular and neural function. METHODS: We developed APOE3fl/fl/Cdh5(PAC)-CreERT2+/- (APOE3Cre+/-) and APOE3fl/fl/Cdh5(PAC)-CreERT2-/- (APOE3Cre-/-, control) mice and induced endothelial cell APOE3 knockdown with tamoxifen at ≈4 to 5 weeks of age. Neurovascular and neuronal function were evaluated by biochemistry, immunohistochemistry, behavioral testing, and electrophysiology at 9 months of age. RESULTS: We found that the loss of endothelial APOE3 expression was sufficient to cause neurovascular dysfunction including higher permeability and lower vessel coverage in tandem with deficits in spatial memory and fear memory extinction and a disruption of cortical excitatory/inhibitory balance. CONCLUSIONS: Our data collectively support the novel concept that endothelial APOE3 plays a critical role in the regulation of the neurovasculature, neural circuit function, and behavior.

In Vivo Proton Exchange Rate (kex ) MRI for the Characterization of Multiple Sclerosis Lesions in Patients.

BACKGROUND: Currently available radiological methods do not completely capture the diversity of multiple sclerosis (MS) lesion subtypes. This lack of information hampers the understanding of disease progression and potential treatment stratification. For example, inflammation persists in some lesions after gadolinium (Gd) enhancement resolves. Novel metabolic and molecular imaging methods may improve the current assessments of MS pathophysiology. PURPOSE: To compare the in vivo proton exchange rate (kex ) MRI with Gd-enhanced MRI for characterizing MS lesions. STUDY TYPE: Retrospective. SUBJECTS: Sixteen consecutively diagnosed relapsing-remitting multiple sclerosis (RRMS) patients. FIELD STRENGTH/SEQUENCE: 3.0T MRI with T2 -weighted imaging, postcontrast T1 -weighted imaging, and single-slice chemical exchange saturation transfer imaging. ASSESSMENT: MS lesions in white matter were assessed for Gd enhancement and kex elevation compared to normal-appearing white matter (NAWM). STATISTICAL TESTS: Student's t-test was used for analyzing the difference of kex values between lesions and NAWM, with statistical significance set at 0.05. RESULTS: Of all 153 MS lesions, 78 (51%) lesions were Gd-enhancing and 75 (49%) were Gd-negative. Without exception, all 78 Gd-enhancing lesions showed significantly elevated kex values compared to NAWM (924 ± 130 s-1 vs. 735 ± 61 s-1 , P < 0.05). Of 75 Gd-negative lesions, 18 lesions (24%) showed no kex elevation (762 ± 29 s-1 vs. 755 ± 28 s-1 , P = 0.47) and 57 (76%) showed significant kex elevation (950 ± 124 s-1 vs. 759 ± 48 s-1 , P < 0.05) compared to NAWM. MS lesions with kex elevation appeared nodular (118, 87.4%), ring-like (15, 11.1%), or irregular-shaped (2, 1.5%). DATA CONCLUSION: For Gd-enhancing lesions, kex MRI is highly consistent with Gd-enhanced images by showing 100% of elevated kex . For all Gd-negative lesions, the discrepancy on kex MRI may further differentiate active slowly expanding lesions or chronic inactive lesions, supporting kex as an imaging biomarker for tissue oxidative stress and inflammation. Level of Evidence 2 Technical Efficacy Stage 3 J. MAGN. RESON. IMAGING 2021;53:408-415.

Liver kinase B1 depletion from astrocytes worsens disease in a mouse model of multiple sclerosis.

Liver kinase B1 (LKB1) is a ubiquitously expressed kinase involved in the regulation of cell metabolism, growth, and inflammatory activation. We previously reported that a single nucleotide polymorphism in the gene encoding LKB1 is a risk factor for multiple sclerosis (MS). Since astrocyte activation and metabolic function have important roles in regulating neuroinflammation and neuropathology, we examined the serine/threonine kinase LKB1 in astrocytes in a chronic experimental autoimmune encephalomyelitis mouse model of MS. To reduce LKB1, a heterozygous astrocyte-selective conditional knockout (het-cKO) model was used. While disease incidence was similar, disease severity was worsened in het-cKO mice. RNAseq analysis identified Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriched in het-cKO mice relating to mitochondrial function, confirmed by alterations in mitochondrial complex proteins and reductions in mRNAs related to astrocyte metabolism. Enriched pathways included major histocompatibility class II genes, confirmed by increases in MHCII protein in spinal cord and cerebellum of het-cKO mice. We observed increased numbers of CD4+ Th17 cells and increased neuronal damage in spinal cords of het-cKO mice, associated with reduced expression of choline acetyltransferase, accumulation of immunoglobulin-γ, and reduced expression of factors involved in motor neuron survival. In vitro, LKB1-deficient astrocytes showed reduced metabolic function and increased inflammatory activation. These data suggest that metabolic dysfunction in astrocytes, in this case due to LKB1 deficiency, can exacerbate demyelinating disease by loss of metabolic support and increase in the inflammatory environment.

Endothelial Wnt/ß-catenin signaling reduces immune cell infiltration in multiple sclerosis.

Disruption of the blood-brain barrier (BBB) is a defining and early feature of multiple sclerosis (MS) that directly damages the central nervous system (CNS), promotes immune cell infiltration, and influences clinical outcomes. There is an urgent need for new therapies to protect and restore BBB function, either by strengthening endothelial tight junctions or suppressing endothelial vesicular transcytosis. Although wingless integrated MMTV (Wnt)/ß-catenin signaling plays an essential role in BBB formation and maintenance in healthy CNS, its role in BBB repair in neurologic diseases such as MS remains unclear. Using a Wnt/ß-catenin reporter mouse and several downstream targets, we demonstrate that the Wnt/ß-catenin pathway is up-regulated in CNS endothelial cells in both human MS and the mouse model experimental autoimmune encephalomyelitis (EAE). Increased Wnt/ß-catenin activity in CNS blood vessels during EAE progression correlates with up-regulation of neuronal Wnt3 expression, as well as breakdown of endothelial cell junctions. Genetic inhibition of the Wnt/ß-catenin pathway in CNS endothelium before disease onset exacerbates the clinical presentation of EAE, CD4+ T-cell infiltration into the CNS, and demyelination by increasing expression of vascular cell adhesion molecule-1 and the transcytosis protein Caveolin-1 and promoting endothelial transcytosis. However, Wnt signaling attenuation does not affect the progressive degradation of tight junction proteins or paracellular BBB leakage. These results suggest that reactivation of Wnt/ß-catenin signaling in CNS vessels during EAE/MS partially restores functional BBB integrity and limits immune cell infiltration into the CNS.

Vascular dysfunction-The disregarded partner of Alzheimer's disease.

Increasing evidence recognizes Alzheimer's disease (AD) as a multifactorial and heterogeneous disease with multiple contributors to its pathophysiology, including vascular dysfunction. The recently updated AD Research Framework put forth by the National Institute on Aging-Alzheimer's Association describes a biomarker-based pathologic definition of AD focused on amyloid, tau, and neuronal injury. In response to this article, here we first discussed evidence that vascular dysfunction is an important early event in AD pathophysiology. Next, we examined various imaging sequences that could be easily implemented to evaluate different types of vascular dysfunction associated with, and/or contributing to, AD pathophysiology, including changes in blood-brain barrier integrity and cerebral blood flow. Vascular imaging biomarkers of small vessel disease of the brain, which is responsible for >50% of dementia worldwide, including AD, are already established, well characterized, and easy to recognize. We suggest that these vascular biomarkers should be incorporated into the AD Research Framework to gain a better understanding of AD pathophysiology and aid in treatment efforts.

Pannexin1 Channels Are Required for Chemokine-Mediated Migration of CD4+ T Lymphocytes: Role in Inflammation and Experimental Autoimmune Encephalomyelitis.

Pannexin1 (Panx1) channels are large high conductance channels found in all vertebrates that can be activated under several physiological and pathological conditions. Our published data indicate that HIV infection results in the extended opening of Panx1 channels (5-60 min), allowing for the secretion of ATP through the channel pore with subsequent activation of purinergic receptors, which facilitates HIV entry and replication. In this article, we demonstrate that chemokines, which bind CCR5 and CXCR4, especially SDF-1α/CXCL12, result in a transient opening (peak at 5 min) of Panx1 channels found on CD4(+) T lymphocytes, which induces ATP secretion, focal adhesion kinase phosphorylation, cell polarization, and subsequent migration. Increased migration of immune cells is key for the pathogenesis of several inflammatory diseases including multiple sclerosis (MS). In this study, we show that genetic deletion of Panx1 reduces the number of the CD4(+) T lymphocytes migrating into the spinal cord of mice subjected to experimental autoimmune encephalomyelitis, an animal model of MS. Our results indicate that opening of Panx1 channels in response to chemokines is required for CD4(+) T lymphocyte migration, and we propose that targeting Panx1 channels could provide new potential therapeutic approaches to decrease the devastating effects of MS and other inflammatory diseases.

Loss of Allograft Inflammatory Factor-1 Ameliorates Experimental Autoimmune Encephalomyelitis by Limiting Encephalitogenic CD4 T-Cell Expansion.

Experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis (MS), is mediated by myelin-specific autoreactive T cells that cause inflammation and demyelination in the central nervous system (CNS), with significant contributions from activated microglia and macrophages. The molecular bases for expansion and activation of these cells, plus trafficking to the CNS for peripheral cells, are not fully understood. Allograft inflammatory factor-1 (Aif-1) (also known as ionized Ca(2+) binding adapter-1 [Iba-1]) is induced in leukocytes in MS and EAE; here we provide the first assessment of Aif-1 function in this setting. After myelin oligodendrocyte glycoprotein peptide (MOG35-55) immunization, Aif-1-deficient mice were less likely than controls to develop EAE and had less CNS leukocyte infiltration and demyelination; their spinal cords contained fewer CD4 T cells and microglia and more CD8 T cells. These mice also showed significantly less splenic CD4 T-cell expansion and activation, plus decreased proinflammatory cytokine expression. These findings identify Aif-1 as a potent molecule that promotes expansion and activation of CD4 T cells, plus elaboration of a proinflammatory cytokine milieu, in MOG35-55-induced EAE and as a potential therapeutic target in MS.

Caveolin-1 mediates blood-brain barrier permeability, neuroinflammation, and cognitive impairment in SARS-CoV-2 infection.

Blood-brain barrier (BBB) permeability can cause neuroinflammation and cognitive impairment. Caveolin-1 (Cav-1) critically regulates BBB permeability, but its influence on the BBB and consequent neurological outcomes in respiratory viral infections is unknown. We used Cav-1-deficient mice with genetically encoded fluorescent endothelial tight junctions to determine how Cav-1 influences BBB permeability, neuroinflammation, and cognitive impairment following respiratory infection with mouse adapted (MA10) SARS-CoV-2 as a model for COVID-19. We found that SARS-CoV-2 infection increased brain endothelial Cav-1 and increased transcellular BBB permeability to albumin, decreased paracellular BBB Claudin-5 tight junctions, and caused T lymphocyte infiltration in the hippocampus, a region important for learning and memory. Concordantly, we observed learning and memory deficits in SARS-CoV-2 infected mice. Importantly, genetic deficiency in Cav-1 attenuated transcellular BBB permeability and paracellular BBB tight junction losses, T lymphocyte infiltration, and gliosis induced by SARS-CoV-2 infection. Moreover, Cav-1 KO mice were protected from the learning and memory deficits caused by SARS-CoV-2 infection. These results establish the contribution of Cav-1 to BBB permeability and behavioral dysfunction induced by SARS-CoV-2 neuroinflammation.

Caveolin-1 mediates neuroinflammation and cognitive impairment in SARS-CoV-2 infection.

Leukocyte infiltration of the CNS can contribute to neuroinflammation and cognitive impairment. Brain endothelial cells regulate adhesion, activation, and diapedesis of T cells across the blood-brain barrier (BBB) in inflammatory diseases. The integral membrane protein Caveolin-1 (Cav-1) critically regulates BBB permeability, but its influence on T cell CNS infiltration in respiratory viral infections is unknown. In this study, we sought to determine the role of Cav-1 at the BBB in neuroinflammation in a COVID-19 mouse model. We used mice genetically deficient in Cav-1 to test the role of this protein in T cell infiltration and cognitive impairment. We found that SARS-CoV-2 infection upregulated brain endothelial Cav-1. Moreover, SARS-CoV-2 infection increased brain endothelial cell vascular cell adhesion molecule-1 (VCAM-1) and CD3+ T cell infiltration of the hippocampus, a region important for short term learning and memory. Concordantly, we observed learning and memory deficits. Importantly, genetic deficiency in Cav-1 attenuated brain endothelial VCAM-1 expression and T cell infiltration in the hippocampus of mice with SARS-CoV-2 infection. Moreover, Cav-1 KO mice were protected from the learning and memory deficits caused by SARS-CoV-2 infection. These results indicate the importance of BBB permeability in COVID-19 neuroinflammation and suggest potential therapeutic value of targeting Cav-1 to improve disease outcomes.

Schistosomiasis-associated pulmonary hypertension unveils disrupted murine gut-lung microbiome and reduced endoprotective Caveolin-1/BMPR2 expression.

Schistosomiasis-associated Pulmonary Arterial Hypertension (Sch-PAH) is a life-threatening complication of chronic S. mansoni infection that can lead to heart failure and death. During PAH, the expansion of apoptosis-resistant endothelial cells (ECs) has been extensively reported; however, therapeutic approaches to prevent the progression or reversal of this pathological phenotype remain clinically challenging. Previously, we showed that depletion of the anti-apoptotic protein Caveolin-1 (Cav-1) by shedding extracellular vesicles contributes to shifting endoprotective bone morphogenetic protein receptor 2 (BMPR2) towards transforming growth factor beta (TGF-ß)-mediated survival of an abnormal EC phenotype. However, the mechanism underlying the reduced endoprotection in PAH remains unclear. Interestingly, recent findings indicate that, similar to the gut, healthy human lungs are populated by diverse microbiota, and their composition depends significantly on intrinsic and extrinsic host factors, including infection. Despite the current knowledge that the disruption of the gut microbiome contributes to the development of PAH, the role of the lung microbiome remains unclear. Thus, using a preclinical animal model of Sch-PAH, we tested whether S. mansoni infection alters the gut-lung microbiome composition and causes EC injury, initiating the expansion of an abnormal EC phenotype observed in PAH. Indeed, in vivo stimulation with S. mansoni eggs significantly altered the gut-lung microbiome profile, in addition to promoting injury to the lung vasculature, characterized by increased apoptotic markers and loss of endoprotective expression of lung Cav-1 and BMPR2. Moreover, S. mansoni egg stimulus induced severe pulmonary vascular remodeling, leading to elevated right ventricular systolic pressure and hypertrophy, characteristic of PAH. In vitro, exposure to the immunodominant S. mansoni egg antigen p40 activated TLR4/CD14-mediated transient phosphorylation of Cav-1 at Tyr14 in human lung microvascular EC (HMVEC-L), culminating in a mild reduction of Cav-1 expression, but failed to promote death and shedding of extracellular vesicles observed in vivo. Altogether, these data suggest that disruption of the host-associated gut-lung microbiota may be essential for the emergence and expansion of the abnormal lung endothelial phenotype observed in PAH, in addition to S. mansoni eggs and antigens.

Matrix proteins plug a hole: How pericytes suppress blood brain barrier transcytosis.

How is the brain so efficient at excluding proteins, drugs, and immune cells from the blood? In this issue of Neuron, Ayloo et al. (2022) find that an extracellular matrix protein secreted by CNS pericytes shuts down endocytic transport in blood brain barrier endothelial cells.

Combining in vivo proton exchange rate (k ex) MRI with quantitative susceptibility mapping to further stratify the gadolinium-negative multiple sclerosis lesions.

Background: Conventional gadolinium (Gd)-enhanced MRI is currently used for stratifying the lesion activity of multiple sclerosis (MS) despite limited correlation with disability and disease activity. The stratification of MS lesion activity needs further improvement to better support clinics. Purpose: To investigate if the novel proton exchange rate (k ex ) MRI combined with quantitative susceptibility mapping (QSM) may help to further stratify non-enhanced (Gd-negative) MS lesions. Materials and methods: From December 2017 to December 2020, clinically diagnosed relapsing-remitting MS patients who underwent MRI were consecutively enrolled in this IRB-approved retrospective study. The customized MRI protocol covered conventional T2-weighted, T2-fluid-attenuated-inversion-recovery, pre- and post-contrast T1-weighted imaging, and quantitative sequences, including k ex MRI based on direct-saturation removed omega plots and QSM. Each MS lesion was evaluated based on its Gd-enhancement as well as its susceptibility and k ex elevation compared to the normal appearing white matter. The difference and correlation concerning lesion characteristics and imaging contrasts were analyzed using the Mann-Whitney U test or Kruskal-Wallis test, and Spearman rank analysis with p < 0.05 considered significant. Results: A total of 322 MS lesions from 30 patients were identified with 153 Gd-enhanced and 169 non-enhanced lesions. We found that the k ex elevation of all lesions significantly correlated with their susceptibility elevation (r = 0.30, p < 0.001). Within the 153 MS lesions with Gd-enhancement, ring-enhanced lesions showed higher k ex elevation than the nodular-enhanced ones' (p < 0.001). Similarly, lesions with ring-hyperintensity in QSM also had higher k ex elevation than the lesions with nodular-QSM-hyperintensity (p < 0.001). Of the 169 Gd-negative lesions, three radiological patterns were recognized according to lesion manifestations on the k ex map and QSM images: Pattern I (k ex + and QSM+, n = 114, 67.5%), Pattern II (only k ex + or QSM+, n = 47, 27.8%) and Pattern III (k ex - and QSM-, n = 8, 4.7%). Compared to Pattern II and III, Pattern I had higher k ex (p < 0.001) and susceptibility (p < 0.05) elevation. The percentage of Pattern I of each subject was negatively correlated with the disease duration (r = -0.45, p = 0.015). Conclusion: As a potential imaging biomarker for inflammation due to oxidative stress, in vivo k ex MRI combined with QSM is promising in extending the clinical classification of MS lesions beyond conventional Gd-enhanced MRI.

Autocrine Effects of Brain Endothelial Cell-Produced Human Apolipoprotein E on Metabolism and Inflammation in vitro.

Reports of APOE4-associated neurovascular dysfunction during aging and in neurodegenerative disorders has led to ongoing research to identify underlying mechanisms. In this study, we focused on whether the APOE genotype of brain endothelial cells modulates their own phenotype. We utilized a modified primary mouse brain endothelial cell isolation protocol that enabled us to perform experiments without subculture. Through initial characterization we found, that compared to APOE3, APOE4 brain endothelial cells produce less apolipoprotein E (apoE) and have altered metabolic and inflammatory gene expression profiles. Further analysis revealed APOE4 brain endothelial cultures have higher preference for oxidative phosphorylation over glycolysis and, accordingly, higher markers of mitochondrial activity. Mitochondrial activity generates reactive oxygen species, and, with APOE4, there were higher mitochondrial superoxide levels, lower levels of antioxidants related to heme and glutathione and higher markers/outcomes of oxidative damage to proteins and lipids. In parallel, or resulting from reactive oxygen species, there was greater inflammation in APOE4 brain endothelial cells including higher chemokine levels and immune cell adhesion under basal conditions and after low-dose lipopolysaccharide (LPS) treatment. In addition, paracellular permeability was higher in APOE4 brain endothelial cells in basal conditions and after high-dose LPS treatment. Finally, we found that a nuclear receptor Rev-Erb agonist, SR9009, improved functional metabolic markers, lowered inflammation and modulated paracellular permeability at baseline and following LPS treatment in APOE4 brain endothelial cells. Together, our data suggest that autocrine signaling of apoE in brain endothelial cells represents a novel cellular mechanism for how APOE regulates neurovascular function.

Deletion of astrocyte connexins 43 and 30 leads to a dysmyelinating phenotype and hippocampal CA1 vacuolation.

Astrocytes are coupled via gap junctions (GJs) comprising connexin 43 (Cx43) (Gja1) and Cx30 (Gjb6), which facilitate intercellular exchange of ions. Astrocyte connexins also form heterotypic GJs with oligodendrocytic somata and lamellae. Loss of oligodendrocyte gap junctions results in oligodendrocyte and myelin pathology. However, whether loss of astrocyte GJs affects oligodendrocytes and myelin is not known. To address this question, mice with astrocyte-targeted deletion of Cx43 and global loss of Cx30 [double knock-out (dKO)] were studied using Western blotting, immunohistochemistry, electron microscopy, and functional assays. Commencing around postnatal day 23 and persisting into old age, we found widespread pathology of white matter tracts comprising vacuolated oligodendrocytes and intramyelinic edema. In contrast, gray matter pathology was restricted to the CA1 region of the hippocampus, and consisted of edematous astrocytes. No differences were observed in synaptic density or total NeuN(+) cells in the hippocampus, or olig2(+) cells in the corpus callosum. However, in dKO mice, fewer CC1-positive mature oligodendrocytes were detected, and Western blotting indicated reduced myelin basic protein. Pathology was not noted in mice expressing a single allele of either Cx43 or Cx30. When compared with single connexin knock-outs, dKO mice were impaired in sensorimotor (rotarod, balance beam assays) and spatial memory tasks (object recognition assays). We conclude that loss of astrocytic GJs can result in white matter pathology that has functional consequences.

Neuroprotection and remyelination after autoimmune demyelination in mice that inducibly overexpress CXCL1.

In rodents, the chemokine CXCL1 both induces the proliferation and inhibits the migration of oligodendrocyte precursor cells. We previously reported that in multiple sclerosis, the same chemokine is expressed by hypertrophic astrocytes, which associate with oligodendrocytes that express the receptor CXCR2. To investigate whether chemokines influence repair after autoimmune demyelination, we generated GFAP-rtTA x beta-Gal-TRE-CXCL1 double-transgenic (Tg) mice that inducibly overexpress CXCL1 under the control of the astrocyte-specific gene, glial fibrillary acidic protein. Experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, was induced in these animals (and controls) by the subcutaneous injection of myelin oligodendrocyte glycoprotein, and after disease onset, CXCL1 production was initiated by the intraperitoneal injection of doxycycline. Double-Tg animals displayed a milder course of disease compared with both single (CXCL1 or glial fibrillary acidic protein)-Tg and wild-type controls. Pathologies were similar in all groups during the acute stage of disease. During the chronic disease phase, both inflammation and demyelination were diminished in double-Tg mice and Wallerian degeneration was markedly decreased. Remyelination was strikingly more prominent in double-Tg mice, together with an apparent increased number of oligodendrocytes. Moreover, cell proliferation, indicated by BrdU incorporation within the central nervous system, was more widespread in the white matter of double-Tg animals. These findings suggest a neuroprotective role for CXCL1 during the course of autoimmune demyelination.

Laminectomy and Spinal Cord Window Implantation in the Mouse.

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 - a tight junction protein.

Caveolin1 Is Required for Th1 Cell Infiltration, but Not Tight Junction Remodeling, at the Blood-Brain Barrier in Autoimmune Neuroinflammation.

Lymphocytes cross vascular boundaries via either disrupted tight junctions (TJs) or caveolae to induce tissue inflammation. In the CNS, Th17 lymphocytes cross the blood-brain barrier (BBB) before Th1 cells; yet this differential crossing is poorly understood. We have used intravital two-photon imaging of the spinal cord in wild-type and caveolae-deficient mice with fluorescently labeled endothelial tight junctions to determine how tight junction remodeling and caveolae regulate CNS entry of lymphocytes during the experimental autoimmune encephalomyelitis (EAE) model for multiple sclerosis. We find that dynamic tight junction remodeling occurs early in EAE but does not depend upon caveolar transport. Moreover, Th1, but not Th17, lymphocytes are significantly reduced in the inflamed CNS of mice lacking caveolae. Therefore, tight junction remodeling facilitates Th17 migration across the BBB, whereas caveolae promote Th1 entry into the CNS. Moreover, therapies that target both tight junction degradation and caveolar transcytosis may limit lymphocyte infiltration during inflammation.

Huntington's Disease iPSC-Derived Brain Microvascular Endothelial Cells Reveal WNT-Mediated Angiogenic and Blood-Brain Barrier Deficits.

Brain microvascular endothelial cells (BMECs) are an essential component of the blood-brain barrier (BBB) that shields the brain against toxins and immune cells. While BBB dysfunction exists in neurological disorders, including Huntington's disease (HD), it is not known if BMECs themselves are functionally compromised to promote BBB dysfunction. Further, the underlying mechanisms of BBB dysfunction remain elusive given limitations with mouse models and post-mortem tissue to identify primary deficits. We undertook a transcriptome and functional analysis of human induced pluripotent stem cell (iPSC)-derived BMECs (iBMEC) from HD patients or unaffected controls. We demonstrate that HD iBMECs have intrinsic abnormalities in angiogenesis and barrier properties, as well as in signaling pathways governing these processes. Thus, our findings provide an iPSC-derived BBB model for a neurodegenerative disease and demonstrate autonomous neurovascular deficits that may underlie HD pathology with implications for therapeutics and drug delivery.

Stepwise recruitment of transcellular and paracellular pathways underlies blood-brain barrier breakdown in stroke.

Brain endothelial cells form a paracellular and transcellular barrier to many blood-borne solutes via tight junctions (TJs) and scarce endocytotic vesicles. The blood-brain barrier (BBB) plays a pivotal role in the healthy and diseased CNS. BBB damage after ischemic stroke contributes to increased mortality, yet the contributions of paracellular and transcellular mechanisms to this process in vivo are unknown. We have created a transgenic mouse strain whose endothelial TJs are labeled with eGFP and have imaged dynamic TJ changes and fluorescent tracer leakage across the BBB in vivo, using two-photon microscopy in the t-MCAO stroke model. Although barrier function is impaired as early as 6 hr after stroke, TJs display profound structural defects only after 2 days. Conversely, the number of endothelial caveolae and transcytosis rate increase as early as 6 hr after stroke. Therefore, stepwise impairment of transcellular followed by paracellular barrier mechanisms accounts for the BBB deficits in stroke.