Transport of the Proinflammatory Chemokines C-C Motif Chemokine Ligand 2 (MCP-1) and C-C Motif Chemokine Ligand 5 (RANTES) across the Intact Mouse Blood-Brain Barrier Is Inhibited by Heparin and Eprodisate and Increased with Systemic Inflammation.

One important function of the vascular blood-brain barrier (BBB) is to facilitate neuroimmune communication. The BBB fulfills this function, in part, through its ability to transport cytokines and chemokines. C-C motif chemokine receptor 2 (CCL2) (MCP-1) and C-C motif chemokine receptor 5 (CCL5) (RANTES) are proinflammatory chemokines that mediate neuroimmune responses to acute insults and aspects of brain injury and neurodegenerative diseases; however, a blood-to-brain transport system has not been evaluated for either chemokine in vivo. Therefore, we determined whether CCL2 and CCL5 in blood can cross the intact BBB and enter the brain. Using CD-1 mice, we found that 125I-labeled CCL2 and CCL5 crossed the BBB and entered the brain parenchyma. We next aimed to identify the mechanisms of 125I-CCL2 and 125I-CCL5 transport in an in situ brain perfusion model. We found that both heparin and eprodisate inhibited brain uptake of 125I-CCL2 and 125I-CCL5 in situ, whereas antagonists of their receptors, CCR2 or CCR5, respectively, did not, suggesting that heparan sulfates at the endothelial surface mediate BBB transport. Finally, we showed that CCL2 and CCL5 transport across the BBB increased following a single injection of 0.3 mg/kg lipopolysaccharide. These data demonstrate that CCL2 and CCL5 in the brain can derive, in part, from the circulation, especially during systemic inflammation. Further, binding to the BBB-associated heparan sulfate is a mechanism by which both chemokines can cross the intact BBB, highlighting a novel therapeutic target for treating neuroinflammation. SIGNIFICANCE STATEMENT: Our work demonstrates that C-C motif chemokine ligand 2 (CCL2) and C-C motif chemokine ligand 5 (CCL5) can cross the intact blood-brain barrier and that transport is robustly increased during inflammation. These data suggest that circulating CCL2 and CCL5 can contribute to brain levels of each chemokine. We further show that the transport of both chemokines is inhibited by heparin and eprodisate, suggesting that CCL2/CCL5-heparan sulfate interactions could be therapeutically targeted to limit accumulation of these chemokines in the brain.

High-mobility group box 1 (HMGB1) crosses the BBB bidirectionally.

High-mobility group box 1 (HMGB1) is a ubiquitous protein that regulates transcription in the nucleus, and is an endogenous damage-associated molecular pattern molecule that activates the innate immune system. HMGB1 activates the TLR4 and RAGE recepto, inducing downstream signals reminiscent of cytokines that have been found to cross the blood-brain barrier (BBB). Blood HMGB1 increases in stroke, sepsis, senescence, alcohol binge drinking and other conditions. Here, we examined the ability of HMGB1 radioactively labeled with iodine (I-HMGB1) to cross the BBB. We found that I-HMGB1 readily entered into mouse brain from the circulation with a unidirectional influx rate of 0.654 µl/g-min. All brain regions tested took up I-HMGB1; uptake was greatest by the olfactory bulb and least in the striatum. Transport was not reliably inhibited by unlabeled HMGB1 nor by inhibitors of TLR4, TLR2, RAGE, or CXCR4. Uptake was enhanced by co-injection of wheatgerm agglutinin, suggestive of involvement of absorptive transcytosis as a mechanism of transport. Induction of inflammation/neuroinflammation with lipopolysaccharide is known to increase blood HMGB1; we report here that brain transport is also increased by LPS-induced inflammation. Finally, we found that I-HMGB1 was also transported in the brain-to-blood direction, with both unlabeled HMGB1 or lipopolysaccharide increasing the transport rate. These results show that HMGB1 can bidirectionally cross the BBB and that those transport rates are enhanced by inflammation. Such transport provides a mechanism by which HMGB1 levels would impact neuroimmune signaling in both the brain and periphery.

Timing matters: Sex differences in inflammatory and behavioral outcomes following repetitive blast mild traumatic brain injury.

BACKGROUND: Repetitive blast-related mild traumatic brain injury (mTBI) caused by exposure to high explosives is increasingly common among warfighters as well as civilians. While women have been serving in military positions with increased risk of blast exposure since 2016, there are few published reports examining sex as a biological variable in models of blast mTBI, greatly limiting diagnosis and treatment capabilities. As such, here we examined outcomes of repetitive blast trauma in female and male mice in relation to potential behavioral, inflammatory, microbiome, and vascular dysfunction at multiple timepoints. METHODS: In this study we utilized a well-established blast overpressure model to induce repetitive (3x) blast-mTBI in both female and male mice. Acutely following repetitive exposure, we measured serum and brain cytokine levels, blood-brain barrier (BBB) disruption, fecal microbial abundance, and locomotion and anxiety-like behavior in the open field assay. At the one-month timepoint, in female and male mice we assessed behavioral correlates of mTBI and PTSD-related symptoms commonly reported by Veterans with a history of blast-mTBI using the elevated zero maze, acoustic startle, and conditioned odorant aversion paradigms. RESULTS: Repetitive blast exposure resulted in both similar (e.g., increased IL-6), and disparate (e.g., IL-10 increase only in females) patterns of acute serum and brain cytokine as well as gut microbiome changes in female and male mice. Acute BBB disruption following repetitive blast exposure was apparent in both sexes. While female and male blast mice both exhibited acute locomotor and anxiety-like deficits in the open field assay, only male mice exhibited adverse behavioral outcomes that lasted at least one-month. DISCUSSION: Representing a novel survey of potential sex differences following repetitive blast trauma, our results demonstrate unique similar yet divergent patterns of blast-induced dysfunction in female vs. male mice and highlight novel targets for future diagnosis and therapeutic development.

Blood-brain barrier penetration of non-replicating SARS-CoV-2 and S1 variants of concern induce neuroinflammation which is accentuated in a mouse model of Alzheimer's disease.

COVID-19 and especially Long COVID are associated with severe CNS symptoms and may place persons at risk to develop long-term cognitive impairments. Here, we show that two non-infective models of SARS-CoV-2 can cross the blood-brain barrier (BBB) and induce neuroinflammation, a major mechanism underpinning CNS and cognitive impairments, even in the absence of productive infection. The viral models cross the BBB by the mechanism of adsorptive transcytosis with the sugar N-acetylglucosamine being key. The delta and omicron variants cross the BB B faster than the other variants of concern, with peripheral tissue uptake rates also differing for the variants. Neuroinflammation induced by icv injection of S1 protein was greatly enhanced in young and especially in aged SAMP8 mice, a model of Alzheimer's disease, whereas sex and obesity had little effect.

The Effects of Viruses on Insulin Sensitivity and Blood-Brain Barrier Function.

In this review manuscript, we discuss the effects of select common viruses on insulin sensitivity and blood-brain barrier (BBB) function and the potential overlapping and distinct mechanisms involved in these effects. More specifically, we discuss the effects of human immunodeficiency virus (HIV), herpes, hepatitis, influenza, respiratory syncytial virus (RSV), and SARS-CoV-2 viruses on insulin sensitivity and BBB function and the proposed underlying mechanisms. These viruses differ in their ability to be transported across the BBB, disrupt the BBB, and/or alter the function of the BBB. For RSV and SARS-CoV-2, diabetes increases the risk of infection with the virus, in addition to viral infection increasing the risk for development of diabetes. For HIV and hepatitis C and E, enhanced TNF-a levels play a role in the detrimental effects. The winter of 2022-2023 has been labeled as a tridemic as influenza, RSV, and COVID-19 are all of concern during this flu season. There is an ongoing discussion about whether combined viral exposures of influenza, RSV, and COVID-19 have additive, synergistic, or interference effects. Therefore, increased efforts are warranted to determine how combined viral exposures affect insulin sensitivity and BBB function.

Ultrastructural Remodeling of the Blood-Brain Barrier and Neurovascular Unit by Lipopolysaccharide-Induced Neuroinflammation.

The blood-brain barrier (BBB) is an interface primarily comprised of brain endothelial cells (BECs), separating the central nervous system (CNS) from the systemic circulation while carefully regulating the transport of molecules and inflammatory cells, and maintaining the required steady-state environment. Inflammation modulates many BBB functions, but the ultrastructural cytoarchitectural changes of the BBB with inflammation are understudied. Inflammation was induced in male 8-10-week-old CD-1 mice with intraperitoneal lipopolysaccharide (LPS), using a regimen (3 mg/kg at 0, 6, and 24 h) that caused robust BBB disruption but had minimal lethality at the study timepoint of 28 h. Perfusion-fixed brains were collected and the frontal cortical layer III regions were analyzed using a transmission electron microscopy (TEM). The LPS-treated mice had pronounced ultrastructural remodeling changes in BECs that included plasma membrane ruffling, increased numbers of extracellular microvesicles, small exosome formation, aberrant BEC mitochondria, increased BEC transcytosis, while tight junctions appeared to be unaltered. Aberrant pericytes were contracted with rounded nuclei and a loss of their elongated cytoplasmic processes. Surveilling microglial cells were attracted to the neurovascular unit (NVU) of BECs, and astrocyte detachment and separation were associated with the formation of a perivascular space and pericapillary edema. The LPS treatment resulted in numerous ultrastructural aberrant remodeling changes to the neurovascular unit's BECs, microglia, pericytes, and astrocytes. In summary, a disturbance of the NVU morphology is a consequence of LPS treatment.

Effects of Ozone on Sickness and Depressive-like Behavioral and Biochemical Phenotypes and Their Regulation by Serum Amyloid A in Mice.

Ozone (O3) is an air pollutant that primarily damages the lungs, but growing evidence supports the idea that O3 also harms the brain; acute exposure to O3 has been linked to central nervous system (CNS) symptoms such as depressed mood and sickness behaviors. However, the mechanisms by which O3 inhalation causes neurobehavioral changes are limited. One hypothesis is that factors in the circulation bridge communication between the lungs and brain following O3 exposure. In this study, our goals were to characterize neurobehavioral endpoints of O3 exposure as they relate to markers of systemic and pulmonary inflammation, with a particular focus on serum amyloid A (SAA) and kynurenine as candidate mediators of O3 behavioral effects. We evaluated O3-induced dose-, time- and sex-dependent changes in pulmonary inflammation, circulating SAA and kynurenine and its metabolic enzymes, and sickness and depressive-like behaviors in Balb/c and CD-1 mice. We found that 3 parts per million (ppm) O3, but not 2 or 1 ppm O3, increased circulating SAA and lung inflammation, which were resolved by 48 h and was worse in females. We also found that indoleamine 2,3-dioxygenase (Ido1) mRNA expression was increased in the brain and spleen 24 h after 3 ppm O3 and that kynurenine was increased in blood. Sickness and depressive-like behaviors were observed at all O3 doses (1-3 ppm), suggesting that behavioral responses to O3 can occur independently of increased SAA or neutrophils in the lungs. Using SAA knockout mice, we found that SAA did not contribute to O3-induced pulmonary damage or inflammation, systemic increases in kynurenine post-O3, or depressive-like behavior but did contribute to weight loss. Together, these findings indicate that acute O3 exposure induces transient symptoms of sickness and depressive-like behaviors that may occur in the presence or absence of overt pulmonary neutrophilia and systemic increases of SAA. SAA does not appear to contribute to pulmonary inflammation induced by O3, although it may contribute to other aspects of sickness behavior, as reflected by a modest effect on weight loss.

Viable human brain microvessels for the study of aging and neurodegenerative diseases.

The brain microvasculature is altered in normal aging and in the presence of disease processes, such as neurodegeneration or ischemia, but there are few methods for studying living tissues. We now report that viable microvessels (MV) are readily isolated from brain tissue of subjects enrolled in studies of neurodegenerative diseases who undergo rapid autopsy (performed with <12 h postmortem interval - PMI). We find that these MV retain their morphology and cellular components and are fairly uniform in size. Sufficient MV (~3-5000) are obtained from 3 to 4 g of tissue to allow for studies of cellular composition as well as extracellular matrix (ECM). Using live/dead assays, these MV are viable for up to 5 days in tissue culture media (2D) designed to support endothelial cells and up to 11 days post-isolation in a 3-dimensional (3D) matrix (Low Growth Factor Matrigel™). Assays that measure the reducing potential of live cells \demonstrated that the majority of the MV maintain high levels of metabolic activity for a similar number of days as the live/dead assays. Functional cellular components (such as tight junctions and transporter proteins) and ECM of MV in tissue culture media, and to a lesser extent in 3D matrices, were readily visualized using immunofluorescence techniques. MV in tissue culture media are lysed and protein content analyzed, but MV in 3D matrix first require removal of the supporting matrix, which can confound the analysis of MV ECM. Finally, MV can be preserved in cryoprotective media, whereby over 50% retain their baseline viability upon thawing. In summary, we find that MV isolated from human brains undergoing rapid autopsy are viable in standard tissue culture for up to 5 days and the timeframe for experiments can be extended up to 11 days by use of a supportive 3D matrix. Viable human MV allow for temporal and spatial analysis of relevant cellular and ECM components that have implications for microvascular function in neurodegenerative diseases, vascular brain injury, and neurotrauma.

Decoding perineuronal net glycan sulfation patterns in the Alzheimer's disease brain.

The extracellular matrix (ECM) of the brain comprises unique glycan "sulfation codes" that influence neurological function. Perineuronal nets (PNNs) are chondroitin sulfate-glycosaminoglycan (CS-GAG) containing matrices that enmesh neural networks involved in memory and cognition, and loss of PNN matrices is reported in patients with neurocognitive and neuropsychiatric disorders including Alzheimer's disease (AD). Using liquid chromatography tandem mass spectrometry (LC-MS/MS), we show that patients with a clinical diagnosis of AD-related dementia undergo a re-coding of their PNN-associated CS-GAGs that correlates to Braak stage progression, hyperphosphorylated tau (p-tau) accumulation, and cognitive impairment. As these CS-GAG sulfation changes are detectable prior to the regional onset of classical AD pathology, they may contribute to the initiation and/or progression of the underlying degenerative processes and implicate the brain matrix sulfation code as a key player in the development of AD clinicopathology.

Characteristics of Exosomes and the Vascular Landscape Regulate Exosome Sequestration by Peripheral Tissues and Brain.

Exosomes mediate intercellular communication, shuttling messages between cells and tissues. We explored whether exosome tissue sequestration is determined by the exosomes or the tissues using ten radiolabeled exosomes from human or murine, cancerous or noncancerous cell lines. We measured sequestration of these exosomes by the liver, kidney, spleen, and lung after intravenous injection into male CD-1 mice. Except for kidney sequestration of three exosomes, all exosomes were incorporated by all tissues, but sequestration levels varied greatly among exosomes and tissues. Species of origin (mouse vs. human) or source (cancerous vs. noncancerous cells) did not influence tissue sequestration. Sequestration of J774A.1 exosomes by liver involved the mannose-6 phosphate (M6P) receptor. Wheatgerm agglutinin (WGA) or lipopolysaccharide (LPS) treatments enhanced sequestration of exosomes by brain and lung but inhibited sequestration by liver and spleen. Response to LPS was not predictive of response to WGA. Path and heat map analyses included our published results for brain and found distinct clusters among the exosomes and the tissues. In conclusion, we found no evidence for a universal binding site controlling exosome-tissue interactions. Instead, sequestration of exosomes by tissues is differentially regulated by both exosomes and tissues and may be stimulated or inhibited by WGA and inflammation.

Amyloid Beta Pathology Exacerbates Weight Loss and Brain Cytokine Responses following Low-Dose Lipopolysaccharide in Aged Female Tg2576 Mice.

Systemic inflammation has been implicated in the progression of Alzheimer's disease (AD); however, less is understood about how existing AD pathology contributes to adverse outcomes following acute inflammatory insults. In the present study, our goal was to determine how AD-associated amyloid beta (Aß) pathology influences the acute neuroinflammatory and behavioral responses to a moderate systemic inflammatory insult. We treated 16-18-month-old female Tg2576 (Tg) mice, which overproduce human Aß and develop plaques, and age-matched wild-type (WT) littermate mice with an intraperitoneal injection of 0.33 mg/kg lipopolysaccharide (LPS) or saline. Mice were then evaluated over the next 28 h for sickness/depressive-like behaviors (food intake, weight loss, locomotion, and sucrose preference), systemic inflammation (serum amyloid A, SAA), blood-brain barrier (BBB) disruption, astrogliosis (glial fibrillary acidic protein/GFAP), Aß, and cytokine levels in the brain. We found that LPS caused a larger reduction in body weight in Tg vs. WT mice, but that other behavioral responses to LPS did not differ by genotype. BBB disruption was not apparent in either genotype following LPS. Concentrations of the systemic inflammatory marker, SAA, in the blood and brain were significantly increased with LPS but did not significantly differ by genotype. GFAP was increased in Tg mice vs. WT but was not significantly affected by LPS in either genotype. Finally, LPS-induced increases of eight cytokines (IL-1ß, IL-6, IL-12 (p40), IL-10, IL-17A, MIP-1α/CCL3, MIP-1ß/CCL4, and RANTES/CCL5) were found to be significantly higher in Tg mice vs. WT. In summary, our data show that Aß pathology exacerbates the neuroinflammatory response to LPS and identifies cytokines that are selectively regulated by Aß. The association of worse neuroinflammation with greater weight loss in Tg mice suggests that Aß pathology could contribute to poor outcomes following a systemic inflammatory insult.

Interactions of Lipids, Lipoproteins, and Apolipoproteins with the Blood-Brain Barrier.

Lipids and lipoproteins are a diverse group of substances and their interactions with the blood-brain barrier (BBB) is similarly diverse. Some lipoproteins such as high density lipoprotein (HDL), apolipoprotein (apo) A-I, apoJ, some free fatty acids, and triglycerides cross the BBB whereas others such as apoE do not. Some forms of cholesterol can cross the BBB and others do not. Lipids can have effects on BBB preservation and function: HDL may protect the BBB during multiple sclerosis, cholesterol can disrupt the BBB, and triglycerides inhibit the transport of leptin across the BBB and the activation of the hypothalamic leptin receptor. ApoE is associated with many effects on the BBB, with the specific isoform apoE4 having detrimental effects. In summary, the diverse ways in which lipids, lipoproteins, and apolipoproteins interact with the BBB is important in both health and disease.

Neuroimmune Axes of the Blood-Brain Barriers and Blood-Brain Interfaces: Bases for Physiological Regulation, Disease States, and Pharmacological Interventions.

Central nervous system (CNS) barriers predominantly mediate the immune-privileged status of the brain, and are also important regulators of neuroimmune communication. It is increasingly appreciated that communication between the brain and immune system contributes to physiologic processes, adaptive responses, and disease states. In this review, we discuss the highly specialized features of brain barriers that regulate neuroimmune communication in health and disease. In section I, we discuss the concept of immune privilege, provide working definitions of brain barriers, and outline the historical work that contributed to the understanding of CNS barrier functions. In section II, we discuss the unique anatomic, cellular, and molecular characteristics of the vascular blood-brain barrier (BBB), blood-cerebrospinal fluid barrier, and tanycytic barriers that confer their functions as neuroimmune interfaces. In section III, we consider BBB-mediated neuroimmune functions and interactions categorized as five neuroimmune axes: disruption, responses to immune stimuli, uptake and transport of immunoactive substances, immune cell trafficking, and secretions of immunoactive substances. In section IV, we discuss neuroimmune functions of CNS barriers in physiologic and disease states, as well as pharmacological interventions for CNS diseases. Throughout this review, we highlight many recent advances that have contributed to the modern understanding of CNS barriers and their interface functions.

Deficient Leptin Cellular Signaling Plays a Key Role in Brain Ultrastructural Remodeling in Obesity and Type 2 Diabetes Mellitus.

The triad of obesity, metabolic syndrome (MetS), Type 2 diabetes mellitus (T2DM) and advancing age are currently global societal problems that are expected to grow over the coming decades. This triad is associated with multiple end-organ complications of diabetic vasculopathy (maco-microvessel disease), neuropathy, retinopathy, nephropathy, cardiomyopathy, cognopathy encephalopathy and/or late-onset Alzheimer's disease. Further, obesity, MetS, T2DM and their complications are associated with economical and individual family burdens. This review with original data focuses on the white adipose tissue-derived adipokine/hormone leptin and how its deficient signaling is associated with brain remodeling in hyperphagic, obese, or hyperglycemic female mice. Specifically, the ultrastructural remodeling of the capillary neurovascular unit, brain endothelial cells (BECs) and their endothelial glycocalyx (ecGCx), the blood-brain barrier (BBB), the ventricular ependymal cells, choroid plexus, blood-cerebrospinal fluid barrier (BCSFB), and tanycytes are examined in female mice with impaired leptin signaling from either dysfunction of the leptin receptor (DIO and db/db models) or the novel leptin deficiency (BTBR ob/ob model).

Interactions of SARS-CoV-2 with the Blood-Brain Barrier.

Emerging data indicate that neurological complications occur as a consequence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The blood-brain barrier (BBB) is a critical interface that regulates entry of circulating molecules into the CNS, and is regulated by signals that arise from the brain and blood compartments. In this review, we discuss mechanisms by which SARS-CoV-2 interactions with the BBB may contribute to neurological dysfunction associated with coronavirus disease of 2019 (COVID-19), which is caused by SARS-CoV-2. We consider aspects of peripheral disease, such as hypoxia and systemic inflammatory response syndrome/cytokine storm, as well as CNS infection and mechanisms of viral entry into the brain. We also discuss the contribution of risk factors for developing severe COVID-19 to BBB dysfunction that could increase viral entry or otherwise damage the brain.

ApoE and cerebral insulin: Trafficking, receptors, and resistance.

Central nervous system (CNS) insulin resistance is associated with Alzheimer's disease (AD). In addition, the apolipoprotein E4 (apoE4) isoform is a risk factor for AD. The connection between these two factors in relation to AD is being actively explored. We summarize this literature with a focus on the transport of insulin and apoE across the blood-brain barrier (BBB) and into the CNS, the impact of apoE and insulin on the BBB, and the interactions between apoE, insulin, and the insulin receptor once present in the CNS. We highlight how CNS insulin resistance is apparent in AD and potential ways to overcome this resistance by repurposing currently approved drugs, with apoE genotype taken into consideration as the treatment response following most interventions is apoE isoform-dependent. This review is part of a special issue focusing on apoE in AD and neurodegeneration.

Pericytes Suppress Brain Metastasis from Lung Cancer In Vitro.

Metastasis of lung cancer to the brain is associated with poor outcomes and limited therapeutic options. The blood-brain barrier (BBB) plays a major role in brain metastasis. However, little is known about the role of pericytes in brain metastasis formation. This study aimed to reveal the interaction between pericytes and cancer cells. We established in vitro BBB models with rat primary cultured BBB-related cells (endothelial cells, astrocytes, and pericytes) and investigated the relationship between BBB-related cells and metastatic cancer cell lines. We observed a significant decrease in transendothelial electrical resistance with metastatic cancer cells in monolayer and coculture models with astrocytes. In contrast, the coculture model with pericytes showed inhibition of the decrease in transendothelial electrical resistance with metastatic cancer cells. In addition, the expression of tight junction protein was preserved only in the coculture model with pericytes. The conditioned medium of pericytes with metastatic cancer cells suppressed the proliferation of the cancer cells significantly. This study revealed that brain pericytes are the major regulators of the resistance of the BBB to lung cancer metastasis to the brain. Pericytes exert an anti-metastatic effect and thus have potential for the preventive treatment of brain metastasis.

Leucine competes with kynurenine for blood-to-brain transport and prevents lipopolysaccharide-induced depression-like behavior in mice.

Inflammation activates indoleamine 2,3-dioxygenase (IDO) which metabolizes tryptophan into kynurenine. Circulating kynurenine is transported into the brain by the large amino transporter LAT1 at the level of the blood-brain barrier. We hypothesized that administration of leucine that has a high affinity for LAT1 should prevent the entry of kynurenine into the brain and attenuate the formation of neurotoxic kynurenine metabolites. To test whether leucine could prevent inflammation-induced depression-like behavior, mice were treated with lipopolysaccharide (LPS, 0.83 mg/kg IP) or saline and treated with L-leucine (50 mg/kg, IP) or vehicle administered before and 6 h after LPS. Depression-like behavior was measured by increased duration of immobility in the forced swim test and decreased sucrose preference. Leucine decreased brain kynurenine levels, blocked LPS-induced depression-like behavior and had antidepressant-like effects in control mice. Leucine had no effect of its own on sickness behavior and neuroinflammation. To confirm that leucine acts by interfering with the transport of kynurenine into the brain, mice were injected with L-leucine (300 mg/kg, IP) immediately before kynurenine (33 mg/kg IP) and brain kynurenine and depression-like behavior were measured 3 h later. Leucine did prevent the entry of exogenous kynurenine into the brain and abrogated depression-like behavior measured by increased duration of immobility in the forced swim test. Additional experiments using an in vitro model of the blood-brain barrier confirmed that kynurenine competes with leucine at the level of the amino acid transporter LAT1 for brain uptake. These experiments also revealed that efflux was the dominant direction of kynurenine transport and was largely independent of LAT1 and leucine, which explains why leucine could block brain uptake of kynurenine without affecting brain clearance. These findings demonstrate that leucine has antidepressant properties vis-à-vis inflammation-induced depression and one mechanism for this is by blocking the ability of kynurenine to enter the brain.

Comparison of the rate of dedifferentiation with increasing passages among cell sources for an in vitro model of the blood-brain barrier.

Cell culture-based blood-brain barrier (BBB) models are useful experimental tools for developing central nervous system drugs. Several endothelial cell sources exist for BBB models, including primary cultured brain endothelial cells and immortalized cell lines. Among them, primary cell-based models are considered suitable for the functional analysis of the BBB; however, little is known about the utility of low-passage brain endothelial cells for this purpose. In this study, we investigated the effect of passage on brain endothelial cells from human, mouse and rat brain tissue as BBB models. We established in vitro BBB models using primary brain endothelial cells (Passage 1-Passage 4) from humans, mice, and rats. To analyze the effect of cell type on BBB function, we evaluated transendothelial electrical resistance (TEER) and performed immunofluorescence staining of tight junction proteins. Among the brain endothelial cell models, TEER was highest in the Passage 1 (P1) cell-based BBB model. There was no adequate increase in TEER in other low-passage cultures (P2-P4). A confluent, non-overlapping, uniform monolayer of cells in all P1 cell-based models was visible on immunostaining of tight junction proteins, whereas it was weak or undetectable in more passaged cultures. Increasing passages cultured of brain endothelial cells did not exhibit restrictive BBB function regardless of the cell source and despite culturing with pericytes and astrocytes. Among the tested culture models, only the lowest cultured cell-based models are suitable for functional analysis of the BBB.

Transport of Extracellular Vesicles across the Blood-Brain Barrier: Brain Pharmacokinetics and Effects of Inflammation.

Extracellular vesicles can cross the blood-brain barrier (BBB), but little is known about passage. Here, we used multiple-time regression analysis to examine the ability of 10 exosome populations derived from mouse, human, cancerous, and non-cancerous cell lines to cross the BBB. All crossed the BBB, but rates varied over 10-fold. Lipopolysaccharide (LPS), an activator of the innate immune system, enhanced uptake independently of BBB disruption for six exosomes and decreased uptake for one. Wheatgerm agglutinin (WGA) modulated transport of five exosome populations, suggesting passage by adsorptive transcytosis. Mannose 6-phosphate inhibited uptake of J774A.1, demonstrating that its BBB transporter is the mannose 6-phosphate receptor. Uptake rates, patterns, and effects of LPS or WGA were not predicted by exosome source (mouse vs. human) or cancer status of the cell lines. The cell surface proteins CD46, AVß6, AVß3, and ICAM-1 were variably expressed but not predictive of transport rate nor responses to LPS or WGA. A brain-to-blood efflux mechanism variably affected CNS retention and explains how CNS-derived exosomes enter blood. In summary, all exosomes tested here readily crossed the BBB, but at varying rates and by a variety of vesicular-mediated mechanisms involving specific transporters, adsorptive transcytosis, and a brain-to-blood efflux system.