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Amyloid-beta protein (Abeta) is well recognized as having a significant role in the pathogenesis of Alzheimer's disease (AD). The reason for the presence of Abeta and its physiological role in non-disease states is not clear. In these studies, low doses of Abeta enhanced memory retention in two memory tasks and enhanced acetylecholine production in the hippocampus in vivo. We then tested whether endogenous Abeta has a role in learning and memory in young, cognitively intact mice by blocking endogenous Abeta in healthy 2-month-old CD-1 mice. Blocking Abeta with antibody to Abeta or DFFVG (which blocks Abeta binding) or decreasing Abeta expression with antisense directed at the Abeta precursor, AbetaPP, all resulted in impaired learning in T-maze foot-shock avoidance. Finally, Abeta 1-42 facilitated induction and maintenance of long term potentiation in hippocampal slices, whereas antibodies to Abeta inhibited hippocampal LTP. In conclusion, these results indicate that in normal healthy young animals the presence of Abeta is important for learning and memory. [ABSTRACT FROM AUTHOR]

Decreased clearance is the main reason amyloid-beta protein (Abeta) is increased in the brains of patients with Alzheimer's disease (AD). The neurovascular hypothesis states that this decreased clearance is caused by impairment of low density lipoprotein receptor related protein-1 (LRP-1), the major brain-to-blood transporter of Abeta at the blood-brain barrier (BBB). As deletion of the LRP-1 gene is a lethal mutation, we tested the neurovascular hypothesis by developing a cocktail of phosphorothioate antisenses directed against LRP-1 mRNA. We found these antisenses in comparison to random antisense selectively decreased LRP-1 expression, reduced BBB clearance of Abeta42, increased brain levels of Abeta42, and impaired learning ability and recognition memory in mice. These results support dysfunction of LRP-1 at the BBB as a mechanism by which brain levels of Abeta could increase and AD would be promoted. [ABSTRACT FROM AUTHOR]

Acute consumption of ethanol results in reversible changes in learning and memory whereas chronic ethanol consumption of six or more months produces permanent deficits and neural damage in rodents. The goal of the current paper was determine whether shorter durations of chronic ethanol ingestion in mice would produce long-term deficits in learning and memory after the cessation of ethanol. We first examined the effects of four and eight weeks of 20% ethanol followed by a three week withdrawal period on learning and memory in mice. We determined that three weeks after eight, but not four, weeks of 20% ethanol consumption resulted in deficits in learning and long-term memory (seven days) in T-maze footshock avoidance and Greek Cross brightness discrimination, step-down passive avoidance and shuttlebox active avoidance. Short-term memory (1 hr) was not affected. The deficit was not related to changes in thiamine status, caloric intake, or nonmnemonic factors, such as, activity or footshock sensitivity. Lastly, we examined if the mice recovered after longer durations of withdrawal. After eight weeks of ethanol, we compared mice after three and 12 weeks of withdrawal. Mice that had been off ethanol for both three and 12 weeks were impaired in T-maze footshock avoidance compared to the controls. The current results indicate that a duration of ethanol consumption as short as eight weeks produces deficits in learning and memory that are present 12 weeks after withdrawal. [ABSTRACT FROM AUTHOR]

Substances cross the blood-brain barrier (BBB) by a variety of mechanisms. These include transmembrane diffusion, saturable transporters, adsorptive endocytosis, and the extracellular pathways. Here, we focus on the chief characteristics of two mechanisms especially important in drug delivery: transmembrane diffusion and transporters. Transmembrane diffusion is non-saturable and depends, on first analysis, on the physicochemical characteristics of the substance. However, brain-to-blood efflux systems, enzymatic activity, plasma protein binding, and cerebral blood flow can greatly alter the amount of the substance crossing the BBB. Transport systems increase uptake of ligands by roughly 10-fold and are modified by physiological events and disease states. Most drugs in clinical use to date are small, lipid soluble molecules that cross the BBB by transmembrane diffusion. However, many drug delivery strategies in development target peptides, regulatory proteins, oligonucleotides, glycoproteins, and enzymes for which transporters have been described in recent years. We discuss two examples of drug delivery for newly discovered transporters: that for phosphorothioate oligonucleotides and for enzymes. [ABSTRACT FROM AUTHOR]

Background: Animal-assisted therapy (AAT) is claimed to have a variety of benefits, but almost all published results are anecdotal. We characterized the resident population in long-term care facilities desiring AAT and determined whether AAT can objectively improve loneliness.Methods: Of 62 residents, 45 met inclusion criteria for the study. These 45 residents were administered the Demographic and Pet History Questionnaire (DPHQ) and Version 3 of the UCLA Loneliness Scale (UCLA-LS). They were then randomized into three groups (no AAT; AAT once/week; AAT three times/week; n = 15/group) and retested with the UCLA-LS near the end of the 6-week study.Results: Use of the DPHQ showed residents volunteering for the study had a strong life-history of emotional intimacy with pets and wished that they currently had a pet. AAT was shown by analysis of covariance followed by pairwise comparison to have significantly reduced loneliness scores in comparison with the no AAT group.Conclusions: The desire for AAT strongly correlates with previous pet ownership. AAT reduces loneliness in residents of long-term care facilities. [ABSTRACT FROM AUTHOR]

Angelman syndrome (AS) is a rare genetic neurodevelopmental disorder with profoundly debilitating symptoms with no FDA-approved cure or therapeutic. Brain-derived neurotrophic factor (BDNF), and its receptor tropomyosin receptor kinase B (TrkB), have a well-established role as regulators of synaptic plasticity, dendritic outgrowth and spine formation. Previously, we reported that the association of postsynaptic density protein 95 (PSD-95) with TrkB is critical for intact BDNF signaling in the AS mouse model, as illustrated by attenuated PLCγ and PI3K signaling and intact MAPK pathway signaling. These data suggest that drugs tailored to enhance the TrkB-PSD-95 interaction may provide a novel approach for the treatment of AS and a variety of neurodevelopmental disorders (NDDs). To evaluate this critical interaction, we synthesized a class of high-affinity PSD-95 ligands that bind specifically to the PDZ3 domain of PSD-95, denoted as Syn3 peptidomimetic ligands. We evaluated Syn3 and its analog D-Syn3 (engineered using dextrorotary (D)-amino acids) in vivo using the Ube3a exon 2 deletion mouse model of AS. Following systemic administration of Syn3 and D-Syn3, we demonstrate improvement in the seizure domain of AS. Learning and memory using the novel object recognition assay also illustrated improved cognition following Syn3 and D-Syn3, along with restored long-term potentiation. A pharmacokinetic analysis of D-Syn3 demonstrates that it crosses the blood-brain barrier (BBB), and the brain influx rate is in the range of CNS therapeutics. Finally, D-Syn3 treated mice showed a partial rescue in motor learning. Neither Syn3 nor D-Syn3 improved gross exploratory locomotion deficits, nor gait impairments that have been well documented in the AS rodent models. These findings highlight the need for further investigation of this compound class as a potential therapeutic for AS and other genetic NDDs., (© 2024. The Author(s), under exclusive licence to American College of Neuropsychopharmacology.)

Brain microvascular dysfunction is an important feature of Alzheimer's disease (AD). To better understand the brain microvascular molecular signatures of AD, we processed and analyzed isolated human brain microvessels by data-independent acquisition liquid chromatography with tandem mass spectrometry (DIA LC-MS/MS) to generate a quantitative dataset at the peptide and protein level. Brain microvessels were isolated from parietal cortex grey matter using protocols that preserve viability for downstream functional studies. Our cohort included 23 subjects with clinical and neuropathologic concordance for Alzheimer's disease, and 21 age-matched controls. In our analysis, we identified 168 proteins whose abundance was significantly increased, and no proteins that were significantly decreased in AD. The most highly increased proteins included amyloid beta, tau, midkine, SPARC related modular calcium binding 1 (SMOC1), and fatty acid binding protein 7 (FABP7). Additionally, Gene Ontology (GO) enrichment analysis identified the enrichment of increased proteins involved in cellular detoxification and antioxidative responses. A systematic evaluation of protein functions using the UniProt database identified groupings into common functional themes including the regulation of cellular proliferation, cellular differentiation and survival, inflammation, extracellular matrix, cell stress responses, metabolism, coagulation and heme breakdown, protein degradation, cytoskeleton, subcellular trafficking, cell motility, and cell signaling. This suggests that AD brain microvessels exist in a stressed state of increased energy demand, and mount a compensatory response to ongoing oxidative and cellular damage that is associated with AD. We also used public RNAseq databases to identify cell-type enriched genes that were detected at the protein level and found no changes in abundance of these proteins between control and AD groups, indicating that changes in cellular composition of the isolated microvessels were minimal between AD and no-AD groups. Using public data, we additionally found that under half of the proteins that were significantly increased in AD microvessels had concordant changes in brain microvascular mRNA, implying substantial discordance between gene and protein levels. Together, our results offer novel insights into the molecular underpinnings of brain microvascular dysfunction in AD., (© 2024. The Author(s).)

The blood-brain barrier (BBB) plays central roles in the maintenance and health of the brain. Its mechanisms to safeguard the brain against xenobiotics and endogenous toxins also make the BBB the primary obstacle to the development of drugs for the central nervous system (CNS). Here, we review classic examples of the intersection of clinical medicine, drug delivery, and the BBB. We highlight the role of lipid solubility (heroin), saturable brain-to-blood (efflux: opiates) and blood-to-brain (influx: nutrients, vitamins, and minerals) transport systems, and adsorptive transcytosis (viruses and incretin receptor agonists). We examine how the disruption of the BBB that occurs in certain diseases (tumors) can also be modulated (osmotic agents and microbubbles) and used to deliver treatments, and the role of extracellular pathways in gaining access to the CNS (albumin and antibodies). In summary, this review provides a historical perspective of the key role of the BBB in delivery of drugs to the brain in health and disease., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

There are genetic and environmental risk factors that contribute to the development of cognitive decline in Alzheimer's disease (AD). Some of these include the genetic predisposition of the apolipoprotein E4 genotype, consuming a high-fat diet (HFD), and the female sex. Brain insulin receptor resistance and deficiency have also been shown to be associated with AD and cognitive impairment. Intranasal (INL) insulin enhances cognition in AD, but the response varies due to genotype, diet, and sex. We investigated here the combination of these risk factors in a humanized mouse model, expressing E3 or E4, following a HFD in males and females on cognitive performance and the brain distribution of insulin following INL delivery. The HFD had a negative effect on survival in male mice only, requiring sex to be collapsed. We found many genotype, diet, and genotype x diet effects in anxiety-related tasks. We further found beneficial effects of INL insulin in our memory tests, with the most important findings showing a beneficial effect of INL insulin in mice on a HFD. We found insulin distribution throughout the brain after INL delivery was largely unaffected by diet and genotype, indicating these susceptible groups can still receive adequate levels of insulin following INL delivery. Our findings support the involvement of brain insulin signaling in cognition and highlight continuing efforts investigating mechanisms resulting from treatment with INL insulin., (© 2024. The Author(s).)

Targeting brain insulin resistance (BIR) has become an attractive alternative to traditional therapeutic treatments for Alzheimer's disease (AD). Incretin receptor agonists (IRAs), targeting either or both of the glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) receptors, have proven to reverse BIR and improve cognition in mouse models of AD. We previously showed that many, but not all, IRAs can cross the blood-brain barrier (BBB) after intravenous (IV) delivery. Here we determined if widespread brain uptake of IRAs could be achieved by circumventing the BBB using intranasal (IN) delivery, which has the added advantage of minimizing adverse gastrointestinal effects of systemically delivered IRAs. Of the 5 radiolabeled IRAs tested (exenatide, dulaglutide, semaglutide, DA4-JC, and DA5-CH) in CD-1 mice, exenatide, dulaglutide, and DA4-JC were successfully distributed throughout the brain following IN delivery. We observed significant sex differences in uptake for DA4-JC. Dulaglutide and DA4-JC exhibited high uptake by the hippocampus and multiple neocortical areas. We further tested and found the presence of AD-associated Aβ pathology minimally affected uptake of dulaglutide and DA4-JC. Of the 5 tested IRAs, dulaglutide and DA4-JC are best capable of accessing brain regions most vulnerable in AD (neocortex and hippocampus) after IN administration. Future studies will need to be performed to determine if IN IRA delivery can reduce BIR in AD or animal models of that disorder., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Introduction: Brain insulin resistance and deficiency is a consistent feature of Alzheimer's disease (AD). Insulin resistance can be mediated by the surface expression of the insulin receptor (IR). Cleavage of the IR generates the soluble IR (sIR)., Methods: We measured the levels of sIR present in cerebrospinal fluid (CSF) from individuals along the AD diagnostic spectrum from two cohorts: Seattle ( n  = 58) and the Consortium for the Early Identification of Alzheimer's Disease-Quebec (CIMA-Q; n  = 61). We further investigated the brain cellular contribution for sIR using human cell lines., Results: CSF sIR levels were not statistically different in AD. CSF sIR and amyloid beta (Aβ)42 and Aβ40 levels significantly correlated as well as CSF sIR and cognition in the CIMA-Q cohort. Human neurons expressing the amyloid precursor protein "Swedish" mutation generated significantly greater sIR and human astrocytes were also able to release sIR in response to both an inflammatory and insulin stimulus., Discussion: These data support further investigation into the generation and role of sIR in AD., Highlights: Cerebrospinal fluid (CSF) soluble insulin receptor (sIR) levels positively correlate with amyloid beta (Aβ)42 and Aβ40.CSF sIR levels negatively correlate with cognitive performance (Montreal Cognitive Assessment score).CSF sIR levels in humans remain similar across Alzheimer's disease diagnostic groups.Neurons derived from humans with the "Swedish" mutation in which Aβ42 is increased generate increased levels of sIR.Human astrocytes can also produce sIR and generation is stimulated by tumor necrosis factor α and insulin., Competing Interests: The authors declare no conflicts of interest. Author disclosures are available in the supporting information., (© 2024 The Author(s). Alzheimer's & Dementia: Diagnosis, Assessment & Disease Monitoring published by Wiley Periodicals LLC on behalf of Alzheimer's Association.)

Alzheimer's disease (AD) involves a complex pathological process that evolves over years, and its etiology is understood as a classic example of gene-environment interaction. The notion that exposure to microbial organisms may play some role in AD pathology has been proposed and debated for decades. New evidence from model organisms and -omic studies, as well as epidemiological data from the recent COVID-19 pandemic and widespread use of vaccines, offers new insights into the "germ hypothesis" of AD. To review new evidence and identify key research questions, the Duke/University of North Carolina (Duke/UNC) Alzheimer's Disease Research Center hosted a virtual symposium and workshop: "New Approaches for Understanding the Potential Role of Microbes in Alzheimer's disease." Discussion centered around the antimicrobial protection hypothesis of amyloid accumulation, and other mechanisms by which microbes could influence AD pathology including immune cell activation, changes in blood-brain barrier, or direct neurotoxicity. This summary of proceedings reviews the content presented in the symposium and provides a summary of major topics and key questions discussed in the workshop., Competing Interests: This symposium was supported by funding from Dr. Leslie Norins and Mrs. Rainey Norins and the Benter Foundation and further supported by the NIH (P30AG072958; R38AG065762 to HEW). The work presented by symposium speakers receives grant funding. Monica M. Diaz is supported by the NIH (K23MH131466), the Alzheimer's Association (AARGD-22-924896) and the American Academy of Neurology. Kenneth E. Schmader received support from the Duke Pepper Older Americans Independence Center (NIA P30AG028716). Manolis Kellis has received NIH support from the following grants: AG054012|AD-complexity, AG058002|AD-dissection, AG062377|AD-endosomes, AG081017|ADpathogens, NS129032|VCID, NS110453|ADRD, NS115064|Vascular, AG062335|AD-Psychosis, AG074003|SexAD., (Published by Elsevier Inc.)

The blood-brain barrier (BBB) predominantly regulates insulin transport into and levels within the brain. The BBB is also an important site of insulin binding and mediator of insulin receptor (INSR) signaling. The insulin transporter is separate from the INSR, highlighting the important, unique role of each protein in this structure. After a brief introduction on the structure of insulin and the INSR, we discuss the importance of insulin interactions at the BBB, the properties of the insulin transporter and the role of the BBB insulin transporter in various physiological conditions. We go on to further describe insulin BBB signaling and the impact not only within brain endothelial cells but also the cascade into other cell types within the brain. We close with future considerations to advance our knowledge about the importance of insulin at the BBB., (Copyright © 2024. Published by Elsevier Inc.)

Background: A number of peptide incretin receptor agonists (IRAs) show promise as therapeutics for Alzheimer's disease (AD) and Parkinson's disease (PD). Transport across the blood-brain barrier (BBB) is one way for IRAs to act directly within the brain. To determine which IRAs are high priority candidates for treating these disorders, we have studied their brain uptake pharmacokinetics., Methods: We quantitatively measure the ability of four IRAs to cross the BBB. We injected adult male CD-1 mice intravenously with 125 I- or 14 C-labeled albiglutide, dulaglutide, DA5-CH, or tirzepatide and used multiple-time regression analyses to measure brain kinetics up to 1 hour. For those IRAs failing to enter the brain 1 h after intravenous injection, we also investigated their ability to enter over a longer time frame (i.e., 6 h)., Results: Albiglutide and dulaglutide had the fastest brain uptake rates within 1 hour. DA5-CH appears to enter the brain rapidly, reaching equilibrium quickly. Tirzepatide does not appear to cross the BBB within 1 h after iv injection but like albumin, did so slowly over 6 h, presumably via the extracellular pathways., Conclusions: We find that IRAs can cross the BBB by two separate processes; one that is fast and one that is slow. Three of the four IRAs investigated here have fast rates of transport and should be taken into consideration for testing as AD and PD therapeutics as they would have the ability to act quickly and directly on the brain as a whole.

There is a paucity of information regarding efficacious pharmacological neuroprotective strategies to attenuate or reduce brain injury in neonates. Lipopolysaccharide (LPS) disrupts blood-brain barrier (BBB) function in adult rodents and increases inflammation in adults and neonates. Human blood-derived Inter-alpha Inhibitor Proteins (IAIPs) are neuroprotective, improve neonatal survival after LPS, and attenuate LPS-induced disruption of the BBB in adult male mice. We hypothesized that LPS also disrupts the function of the BBB in neonatal mice and that IAIPs attenuate the LPS-induced BBB disruption in male and female neonatal mice. IAIPs were administered to neonatal mice after LPS and BBB permeability quantified with intravenous 14 C-sucrose and 99m Tc-albumin. Although repeated high doses (3 mg/kg) of LPS in neonates resulted in high mortality rates and a robust increase in BBB permeability, repeated lower doses (1 mg/kg) of LPS resulted in lower mortality rates and disruption of the BBB in both male and female neonates. IAIP treatment attenuated disruption of the BBB similarly to sucrose and albumin after exposure to low-dose LPS in neonatal mice. Exposure to low-dose LPS elevated IAIP concentrations in blood, but it did not appear to increase the systemic levels of Pre-alpha inhibitor (PaI), one of the family members of the IAIPs that contains heavy chain 3. We conclude that IAIPs attenuate LPS-related disruption of the BBB in both male and female neonatal mice., Competing Interests: Declaration of Competing Interest Joseph Qiu and Yow-Pin Lim are employed by ProThera Biologics and Yow-Pin Lim has a significant financial stake in the company. All other authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The field of peptides exploded in the 1970's and has continued to be a major area of discovery. Among the early discoveries was that peptides administered peripherally could affect brain functions. This led Kastin to propose that peptides could cross the blood-brain barrier (BBB). Although initially very controversial, Kastin, I, and others demonstrated not only that peptides can cross the BBB, but elucidated many fundamental characteristics of that passage. That work was in large part the basis of the 2022 Viktor Mutt Lectureship. Here, we review some of the early work with current updates on topics related to the penetration of peptides across the BBB. We briefly review mechanisms by which peripherally administered peptides can affect brain function without crossing the BBB, and then review the major mechanisms by which peptides and their analogs have been show to cross the BBB: transmembrane diffusion, saturable transport, and adsorptive transcytosis. Saturable transport systems are adaptable to physiologic changes and can be altered by disease states. In particular, the transport across the BBB of insulin and of pituitary adenylate cyclase activating polypeptide (PACAP) illustrate many of the concepts regarding peptide transport across the BBB., (Published by Elsevier Inc.)

Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Research on the neuroprotective effect of pituitary adenylate cyclase-activating polypeptide (PACAP) and its use as a therapeutic agent has grown over the past 30 years. Both in vitro and in vivo experiments have shown that PACAP exerts a strong neuroprotective effect in many central and peripheral neuronal diseases. Various delivery routes have been employed from intravenous (IV) injections to intracerebroventricular (ICV) administration, leading either to systemic or topical delivery of the peptide. Over the last decade, a growing interest in the use of intranasal (IN) administration of PACAP and other therapeutic agents has emerged as an alternative delivery route to target the brain. The aim of this review is to summarize the findings on the neuroprotective effect of PACAP and to discuss how the IN administration of PACAP could contribute to target the effects of this pleiotropic peptide.

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The blood-brain barrier (BBB) is a critical physiochemical interface that regulates communication between the brain and blood. It is comprised of brain endothelial cells which regulate the BBB's barrier and interface properties and is surrounded by supportive brain cell types including pericytes and astrocytes. Recent reports have suggested that the BBB undergoes dysfunction during normative aging and in disease. In this review, we consider the effect of cellular senescence, one of the nine hallmarks of aging, on the BBB. We first characterize known normative age-related changes at the BBB, and then evaluate changes in neurodegenerative diseases, with an emphasis on if/how cellular senescence is influencing these changes. We then discuss what insight has been gained from in vitro and in vivo studies of cellular senescence at the BBB. Finally, we evaluate mechanisms by which cellular senescence in peripheral pathologies can indirectly or directly affect BBB function.

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Inc.)

Background: Insulin transport across the blood-brain barrier (BBB) is a highly regulated, saturable process, known to be affected by many peripheral substrates including insulin itself and triglycerides. This is in contrast to insulin leakage into peripheral tissues. Whether the central nervous system (CNS) can control the rate of insulin uptake by brain remains to be determined. Insulin BBB interactions are impaired in Alzheimer's disease (AD) and CNS insulin resistance is widely prevalent in AD. Therefore, if CNS insulin controls the rate of insulin transport across the BBB, then the defective transport of insulin seen in AD could be one manifestation of the resistance to CNS insulin observed in AD., Methods: We investigated whether enhancing CNS insulin levels or induction of CNS insulin resistance using an inhibitor of the insulin receptor altered the blood-to-brain transport of radioactively labeled insulin in young, healthy mice., Results: We found that insulin injected directly into the brain decreased insulin transport across the BBB for whole brain and the olfactory bulb in male mice, whereas insulin receptor blockade decreased transport in female mice for whole brain and hypothalamus. Intranasal insulin, currently being investigated as a treatment in AD patients, decreased transport across the BBB of the hypothalamus., Conclusions: These results suggest CNS insulin can control the rate of insulin brain uptake, connecting CNS insulin resistance to the rate of insulin transport across the BBB., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Brain glucose hypometabolism is an early sign of Alzheimer's disease (AD), and interventions which offset this deficit, such as ketogenic diets, show promise as AD therapeutics. Conversely, high-fat feeding may exacerbate AD risk. We analyzed the metabolomic profile of cerebrospinal fluid (CSF) in a pilot study of older adults who underwent saline and triglyceride (TG) infusions. Older adults (12 cognitively normal (CN), age 65.3 ± 8.1, and 9 with cognitive impairment (CI), age 70.9 ± 8.6) underwent a 5 h TG or saline infusion on different days using a random crossover design; CSF was collected at the end of infusion. Aqueous metabolites were measured using a targeted mass spectroscopy (MS) platform focusing on 215 metabolites from over 35 different metabolic pathways. Data were analyzed using MetaboAnalyst 4.0 and SAS. Of the 215 targeted metabolites, 99 were detectable in CSF. Only one metabolite significantly differed by treatment: the ketone body 3-hydroxybutyrate (HBA). Post hoc analyses showed that HBA levels were associated with age and markers of metabolic syndrome and demonstrated different correlation patterns for the two treatments. When analyzed by cognitive diagnosis group, TG-induced increases in HBA were over 3 times higher for those with cognitive impairment (change score CN +9.8 uM ± 8.3, CI +32.4 ± 7.4, p = 0.0191). Interestingly, individuals with cognitive impairment had higher HBA levels after TG infusion than those with normal cognition. These results suggest that interventions that increase plasma ketones may lead to higher brain ketones in groups at risk for AD and should be confirmed in larger intervention studies.

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

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.

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.

Ozone (O 3 ) is an air pollutant that primarily damages the lungs, but growing evidence supports the idea that O 3 also harms the brain; acute exposure to O 3 has been linked to central nervous system (CNS) symptoms such as depressed mood and sickness behaviors. However, the mechanisms by which O 3 inhalation causes neurobehavioral changes are limited. One hypothesis is that factors in the circulation bridge communication between the lungs and brain following O 3 exposure. In this study, our goals were to characterize neurobehavioral endpoints of O 3 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 O 3 behavioral effects. We evaluated O 3 -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) O 3 , but not 2 or 1 ppm O 3 , 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 O 3 and that kynurenine was increased in blood. Sickness and depressive-like behaviors were observed at all O 3 doses (1-3 ppm), suggesting that behavioral responses to O 3 can occur independently of increased SAA or neutrophils in the lungs. Using SAA knockout mice, we found that SAA did not contribute to O 3 -induced pulmonary damage or inflammation, systemic increases in kynurenine post-O 3 , or depressive-like behavior but did contribute to weight loss. Together, these findings indicate that acute O 3 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 O 3 , although it may contribute to other aspects of sickness behavior, as reflected by a modest effect on weight loss.

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 125 I-labeled CCL2 and CCL5 crossed the BBB and entered the brain parenchyma. We next aimed to identify the mechanisms of 125 I-CCL2 and 125 I-CCL5 transport in an in situ brain perfusion model. We found that both heparin and eprodisate inhibited brain uptake of 125 I-CCL2 and 125 I-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., (U.S. Government work not protected by U.S. copyright.)

Brain endothelial cells (BEC) of the vascular blood-brain barrier (BBB) interact with many different cell types in the brain, including microglia, the brain's resident immune cells. Physical associations of microglia with the BBB and the importance of these interactions in health and disease are an emerging area of study and likely involved in neuroimmune communication. In this mini-review, we consider how microglia and the BBB are intrinsically linked in the developing brain, discuss possible mechanisms that attract microglia to the vasculature in healthy physiological conditions, and examine the known microglial-vascular associated changes in systemic infection and various disease states. Our findings shed light on the complexities of microglial-vascular interactions and highlight the contributions of microglia to the functions of the neurovascular unit., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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.

Objectives: Cyclodextrins are increasingly used therapeutically. For example, 2-hydroxypropyl-ß-cyclodextrin (kleptose) is used for the treatment of Niemann-Pick disease. Kleptose crosses the blood-brain barrier poorly, in part because of a central nervous system (CNS)-to-blood (efflux) transporter, and so is administered by the intrathecal (IT) route in the treatment of Niemann-Pick disease., Methods: Here, we evaluated the uptake and distribution of kleptose by the brain and spinal cord after intranasal (IN) or IT delivery., Key Findings: Kleptose distributed to all regions of the brain and spinal cord after IN administration, with only 3.27% of the administered dose entering the bloodstream. Intrathecal delivery produced levels in the whole brain about 40 times higher than intranasal administration and about 20 times higher than previously found after intravenous administration. About 70-90% of the IT dose rapidly entered the bloodstream, in part because of the previously described efflux transporter. The uptake by CNS after IN and IT was both non-saturable. The uptake by the olfactory bulb, hypothalamus and pons-medulla was favoured by all routes of administration., Conclusions: Kleptose was taken up by all regions of the CNS after either IN or IT administration, but IN administration resulted in only a fraction of the uptake of the IT route., (Published by Oxford University Press on behalf of the Royal Pharmaceutical Society 2022.)

The concept of insulin resistance has been around since a few decades after the discovery of insulin itself. To allude to the classic Charles Dicken's novel published 62 years before the discovery of insulin, in some ways, this is the best of times, as the concept of insulin resistance has expanded to include the brain, with the realization that insulin has a life beyond the regulation of glucose. In other ways, it is the worst of times as insulin resistance is implicated in devastating diseases, including diabetes mellitus, obesity, and Alzheimer's disease (AD) that affect the brain. Peripheral insulin resistance affects nearly a quarter of the United States population in adults over age 20. More recently, it has been implicated in AD, with the degree of brain insulin resistance correlating with cognitive decline. This has led to the investigation of brain or central nervous system (CNS) insulin resistance and the question of the relation between CNS and peripheral insulin resistance. While both may involve dysregulated insulin signaling, the two conditions are not identical and not always interlinked. In this review, we compare and contrast the similarities and differences between peripheral and CNS insulin resistance. We also discuss how an apolipoprotein involved in insulin signaling and related to AD, apolipoprotein E (apoE), has distinct pools in the periphery and CNS and can indirectly affect each system. As these systems are both separated but also linked via the blood-brain barrier (BBB), we discuss the role of the BBB in mediating some of the connections between insulin resistance in the brain and in the peripheral tissues.

Ozone is a widespread air toxicant. Although its primary target organ is the lungs, emerging evidence suggests that ozone also has harmful effects on the brain. The vascular blood-brain barrier (BBB), an endothelial interface that regulates passage of substances between the brain and peripheral tissues, is a likely mediator of ozone's adverse effects on the brain. Ozone can cause BBB disruption, a pathological state in which the BBB becomes leaky, resulting in the unregulated entry of circulating substances into the brain. BBB disruption can be detected using many methods, which each have their strengths and limitations. Recent data suggest that BBB disruption can occur in mice following ozone exposures, albeit at a low level. Therefore, robust and highly sensitive assays for BBB disruption are needed. Assays commonly used to detect BBB disruption, however, can be time consuming, lack sensitivity, and can be vulnerable to artifacts that are typically not addressed in the experimental design. Radiochemical assays are among the most sensitive and specific for detecting subtle disruptions of the BBB and require minimal sample processing for detection. Radiochemical assays can also be multiplexed to include radiotracer conjugates of large and small molecular weights, and the uptake of each of them can provide information about the severity and mechanism of BBB disruption. Here, we describe a protocol to use two of these radiotracer conjugates, 14 C-sucrose and 99m Tc- albumin, to measure BBB disruption following an acute exposure to ozone in mice. We provide the steps to expose mice acutely to ozone, to label albumin with 99m Tc-pertechnetate, and to measure BBB disruption by evaluating permeability to 99m Tc-albumin and 14 C-sucrose after ozone exposure. These methods can be adapted to different ozone exposure paradigms and to different rodent species/strains, allowing for the sensitive and rapid assessment of BBB disruption that is detectable in whole brains or in brain regions. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Ozone exposures in mice Basic Protocol 2: Measurement of blood-brain barrier disruption by evaluating permeability to 14 C-sucrose and 99m Tc-albumin Support Protocol: Labeling of bovine serum albumin with 99m Tc., (© 2022 Wiley Periodicals LLC.)

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., (© 2021 the Alzheimer's Association.)

Disruption of the blood-brain barrier (BBB) can occur through different mechanisms and pathways. As these pathways result in increased permeability to different classes of substances, it is likely that the neurological insults that occur will also differ for these pathways. The major categories of BBB disruption are paracellular (between cells) and transcellular (across cells) with a subcategory of transcellular leakage involving vesicles (transcytotic). Older literature, as well as more recent studies, highlights the importance of the transcellular pathways in BBB disruption. Of the various transcytotic mechanisms that are thought to be active at the BBB, some are linked to receptor-mediated transcytosis, whereas others are likely involved in BBB disruption. For most capillary beds, transcytotic mechanisms are less clearly linked to permeability than are membrane spanning canaliculi and fenestrations. Disruption pathways share cellular mechanisms to some degree as exemplified by transcytotic caveolar and transcellular canaliculi formations. The discovery of some of the cellular components involved in transcellular mechanisms of BBB disruption and the ability to measure them are adding greatly to our classic knowledge, which is largely based on ultrastructural studies. Future work will likely address the conditions and diseases under which the various pathways of disruption are active, the different impacts that they have, and the cellular biology that underlies the different pathways to disruption.

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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Exercise has multiple beneficial effects including improving peripheral insulin sensitivity, improving central function such as memory, and restoring a dysregulated blood-brain barrier (BBB). Central nervous system (CNS) insulin resistance is a common feature of cognitive impairment, including Alzheimer's disease. Delivery of insulin to the brain can improve memory. Endogenous insulin must cross the BBB to directly act within the CNS and this transport system can be affected by various physiological states and serum factors. Therefore, the current study sought to investigate whether exercise could enhance insulin BBB transport as a mechanism for the underlying benefits of exercise on cognition. We investigated radioactive insulin BBB pharmacokinetics following an acute bout of exercise in young, male and female CD-1 mice. In addition, we investigated changes in serum levels of substrates that are known to affect insulin BBB transport. Finally, we measured the basal level of a downstream protein involved in insulin receptor signaling in various brain regions as well as muscle. We found insulin BBB transport in males was greater following exercise, and in males and females to both enhance the level of insulin vascular binding and alter CNS insulin receptor signaling, independent of changes in serum factors known to alter insulin BBB transport. NEW & NOTEWORTHY Central nervous system (CNS) insulin and exercise are beneficial for cognition. CNS insulin resistance is present in Alzheimer's disease. CNS insulin levels are regulated by transport across the blood-brain barrier (BBB). We show that exercise can enhance insulin BBB transport and binding of insulin to the brain's vasculature in mice. There were no changes in serum factors known to alter insulin BBB pharmacokinetics. We conclude exercise could impact cognition through regulation of insulin BBB transport.

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 (Aβ) 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 Aβ 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), Aβ, 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-1β, IL-6, IL-12 (p40), IL-10, IL-17A, MIP-1α/CCL3, MIP-1β/CCL4, and RANTES/CCL5) were found to be significantly higher in Tg mice vs. WT. In summary, our data show that Aβ pathology exacerbates the neuroinflammatory response to LPS and identifies cytokines that are selectively regulated by Aβ. The association of worse neuroinflammation with greater weight loss in Tg mice suggests that Aβ pathology could contribute to poor outcomes following a systemic inflammatory insult.