Lipopolysaccharide, VE-cadherin, HMGB1, and HIF-1α levels are elevated in the systemic circulation in chronic migraine patients with medication overuse headache: evidence of leaky gut and inflammation.

OBJECTIVE: Medication overuse headache (MOH) was recently shown to be associated with leaky gut in rodents. We aimed to investigate whether chronic migraine (CM) patients with MOH have elevated lipopolysaccharide levels and inflammatory molecules in blood circulation. MATERIALS AND METHODS: The study included women participants (40 CM patients with NSAID overuse headache, 35 episodic migraine (EM) patients, and 20 healthy non-headache sufferers). Migraine duration, monthly migraine headache days, MigSCog, HADS-D, HADS-A, and HIT-6 scores were recorded. Serum samples were collected to measure circulating LPS, LPS binding protein (LBP), tight junction protein occludin, adherens junction protein vascular endothelial cadherin (VE-cadherin), CGRP, HMGB1, HIF-1α, IL-6, and IL-17 levels. RESULTS: Serum LPS, VE-Cadherin, CGRP, HIF-1α, and IL-6 levels were significantly higher in the CM + MOH group compared to the EM group and healthy controls while serum LBP and HMGB1 were higher in the CM + MOH group compared to healthy controls. IL-17 and occludin levels were comparable between the three groups. Serum HMGB1 levels in EM patients were higher compared to the control group. Mig-SCog and HIT-6 scores were higher in the CM + MOH group compared to EM patients. HADS-A and HADS-D scores were significantly higher in the CM + MOH group compared to EM patients and healthy controls, and they were also higher in EM patients compared to healthy subjects. LPS levels were correlated with VE-cadherin and occludin levels. The number of monthly migraine headache days was positively correlated with serum LPS, HIF-1α, VE-cadherin, and IL-6 levels, HADS-A, HADS-D, HIT-6, and MigSCog scores. CONCLUSION: We have evidence for the first time that CM + MOH is associated with elevated serum LPS and LBP levels suggestive of LPS leak into the systemic circulation. Higher levels of nociceptive and/or pro-inflammatory molecules such as HMGB1, HIF-1α, IL-6, and CGRP may play a role in trigeminal sensitization and neurobiology of MOH. Intestinal hyperpermeability and consequent inflammatory response should be considered as a potential contributory factor in patients with MOH.

Serum claudin-5, claudin-11, occludin, vinculin, paxillin, and beta-catenin levels in preschool children with autism spectrum disorder.

AIM: Increased intestinal and blood-brain barriers (BBB) permeability has been suggested to have a role in autism spectrum disorder (ASD). Claudin-5, claudin-11, occludin, ß-catenin, vinculin, and paxillin are crucial components of these barriers. This study assessed concentrations of these molecules in preschool children with ASD. METHODS: A total of 80 children with ASD and 40 controls aged 18-60 months were enrolled in this study. Serum levels of biochemical variables were determined using commercial enzyme-linked immunosorbent assay kits. RESULTS: Serum claudin-11, occludin, and ß-catenin levels were significantly higher in the ASD group than in the control group. However, no significant difference for serum claudin-5, vinculin, and paxillin levels was detected between the groups. CONCLUSION: These findings suggest that claudin-11, occludin, and ß-catenin may be involved in the pathogenesis of ASD. These proteins may affect the brain by causing dysregulation in intestinal or blood-brain barrier permeability or with other unknown mechanisms.

Higher serum occludin after successful reperfusion Is associated with early neurological deterioration.

AIMS: Early neurological deterioration (END) is an important factor that affects prognosis in patients with acute ischemic stroke. We explored the relationship between serum occludin levels after successful reperfusion and END in patients treated with endovascular thrombectomy (EVT). METHODS: We prospectively enrolled 120 stroke patients who underwent EVT with successful reperfusion. Enzyme-linked immunosorbent assay was used to detect the serum occludin levels on admission and within 1 h after successful reperfusion. Receiver operating characteristic curves (ROC) and regression analysis were used to compare the relationship between serum occludin and END after thrombectomy. RESULTS: Among the 120 patients, 36 (30%) experienced END. The END group had higher serum occludin levels than the non-END group after successful reperfusion [4.31 (3.71-5.38) vs 6.32 (5.88-6.99), p < 0.001]. The ROC curve showed that postoperative serum occludin levels had a significant prediction value for END (AUC: 0.86, p < 0.001). Regression analysis showed that serum occludin was an independent risk factor for END in EVT patients (adjusted odds ratio: 4.46, 95% confidence interval: 1.92-10.32; p < 0.001). CONCLUSIONS: The higher serum occludin levels were strongly related to END after successful reperfusion. Serum occludin may be an independent risk factor for END in EVT patients.

Function and Biomarkers of the Blood-Brain Barrier in a Neonatal Germinal Matrix Haemorrhage Model.

Germinal matrix haemorrhage (GMH), caused by rupturing blood vessels in the germinal matrix, is a prevalent driver of preterm brain injuries and death. Our group recently developed a model simulating GMH using intrastriatal injections of collagenase in 5-day-old rats, which corresponds to the brain development of human preterm infants. This study aimed to define changes to the blood-brain barrier (BBB) and to evaluate BBB proteins as biomarkers in this GMH model. Regional BBB functions were investigated using blood to brain 14C-sucrose uptake as well as using biotinylated BBB tracers. Blood plasma and cerebrospinal fluids were collected at various times after GMH and analysed with ELISA for OCLN and CLDN5. The immunoreactivity of BBB proteins was assessed in brain sections. Tracer experiments showed that GMH produced a defined region surrounding the hematoma where many vessels lost their integrity. This region expanded for at least 6 h following GMH, thereafter resolution of both hematoma and re-establishment of BBB function occurred. The sucrose experiment indicated that regions somewhat more distant to the hematoma also exhibited BBB dysfunction; however, BBB function was normalised within 5 days of GMH. This shows that GMH leads to a temporal dysfunction in the BBB that may be important in pathological processes as well as in connection to therapeutic interventions. We detected an increase of tight-junction proteins in both CSF and plasma after GMH making them potential biomarkers for GMH.

Circulating tight-junction proteins are potential biomarkers for blood-brain barrier function in a model of neonatal hypoxic/ischemic brain injury.

BACKGROUND: Neonatal encephalopathy often leads to lifelong disabilities with limited treatments currently available. The brain vasculature is an important factor in many neonatal neurological disorders but there is a lack of diagnostic tools to evaluate the brain vascular dysfunction of neonates in the clinical setting. Measurement of blood-brain barrier tight-junction (TJ) proteins have shown promise as biomarkers for brain injury in the adult. Here we tested the biomarker potential of tight-junctions in the context of neonatal brain injury. METHODS: The levels of TJ-proteins (occluding, claudin-5, and zonula occludens protein 1) in both blood plasma and cerebrospinal fluid (CSF) as well as blood-brain barrier function via 14C-sucrose (342 Da) and Evans blue extravasation were measured in a hypoxia/ischemia brain-injury model in neonatal rats. RESULTS: Time-dependent changes of occludin and claudin-5 levels could be measured in blood and CSF after hypoxia/ischemia with males generally having higher levels than females. The levels of claudin-5 in CSF correlated with the severity of the brain injury at 24 h post- hypoxia/ischemia. Simultaneously, we detected early increase in blood-brain barrier-permeability at 6 and 24 h after hypoxia/ischemia. CONCLUSIONS: Levels of circulating claudin-5 and occludin are increased after hypoxic/ischemic brain injuries and blood-brain barrier-impairment and have promise as early biomarkers for cerebral vascular dysfunction and as a tool for risk assessment of neonatal brain injuries.

Peripheral Blood Occludin Level as a Biomarker for Perioperative Cerebral Edema in Patients with Brain Tumors.

OBJECTIVE: Cerebral edema is a common complication of brain tumors in the perioperative period. However, there is currently no reliable and convenient method to evaluate the extent of brain edema. The objective is to explore the effectiveness of serum occludin on predicting the extent of perioperative brain edema and outcome in patients with brain tumors. METHODS: This prospective study enrolled 55 patients with brain tumors and 24 healthy controls in Sanbo Brain Hospital from June 2019 through November 2019. Serum occludin levels were measured preoperatively and on postoperative day 1. Peritumoral edema was assessed preoperatively using MRI. Pericavity brain edema on postoperative day 1 was evaluated using CT. RESULTS: Compared with healthy controls, the serum occludin level was higher in patients with brain tumors both preoperatively and postoperatively (P < 0.001). The serum occludin level correlated positively with the degree of brain edema preoperatively (r = 0.78, P < 0.001) and postoperatively (r = 0.59, P < 0.001). At an optimal cutoff of 3.015 ng/mL, the preoperative serum occludin level discriminated between mild and severe preoperative brain edema with a sensitivity of 90.48% and specificity of 84.62%. At an optimal cutoff value of 3.033 ng/mL, the postoperative serum occludin level distinguished between mild and severe postoperative brain edema with a sensitivity of 97.30% and specificity of 55.56%. CONCLUSIONS: The serum occludin level is associated with cerebral edema and could potentially be used as a biomarker for perioperative cerebral edema. This trial is registered with ChiCTR1900023742.

Normobaric Hyperoxia Reduces Blood Occludin Fragments in Rats and Patients With Acute Ischemic Stroke.

BACKGROUND AND PURPOSE: Damage of the blood-brain barrier (BBB) increases the incidence of neurovascular complications, especially for cerebral hemorrhage after tPA (tissue-type plasminogen activator) therapy. Currently, there is no effective method to evaluate the extent of BBB damage to guide tPA use. Herein, we investigated whether blood levels of tight junction proteins could serve as biomarker of BBB damages in acute ischemic stroke (AIS) in both rats and patients. We examined whether this biomarker could reflect the extent of BBB permeability during cerebral ischemia/reperfusion and the effects of normobaric hyperoxia (NBO) on BBB damage. METHODS: Rats were exposed to NBO (100% O2) or normoxia (21% O2) during middle cerebral artery occlusion. BBB permeability was determined. Occludin and claudin-5 in blood and cerebromicrovessels were measured. Patients with AIS were assigned to oxygen therapy or room air for 4 hours, and blood occludin and claudin-5 were measured at different time points after stroke. RESULTS: Cerebral ischemia/reperfusion resulted in the degradation of occludin and claudin-5 in microvessels, leading to increased BBB permeability in rats. In blood samples, occludin increased with 4-hour ischemia and remained elevated during reperfusion, correlating well with its loss from ischemic cerebral microvessels. NBO treatment both prevented occludin degradation in microvessels and reduced occludin levels in blood, leading to improved neurological functions in rats. In patients with AIS receiving intravenous tPA thrombolysis, the blood occludin was already elevated when patients arrived at hospital (within 4.5 hours since symptoms appeared) and remained at a high level for 72 hours. NBO significantly lowered the level of blood occludin and improved neurological functions in patients with AIS. CONCLUSIONS: Blood occludin may be a clinically viable biomarker for evaluating BBB damage during ischemia/reperfusion. NBO therapy has the potential to reduce blood occludin, protect BBB, and improve outcome in AIS patients with intravenous tPA thrombolysis. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT02974283.

Applying fluid biomarkers to Alzheimer's disease.

Alzheimer's disease (AD) is a common neurodegenerative disease that starts with a clinically silent phase of a decade or more during which brain pathologies accumulate predominantly in the medial temporal lobe but also elsewhere in the brain. Network dysfunction and clinical symptoms typically appear when senile plaque (amyloid-ß) and neurofibrillary tangle (tau) pathologies meet in the brain parenchyma, producing synapse and neuronal loss. For plaque and tangle pathologies, reliable fluid biomarkers have been developed. These require sampling of cerebrospinal fluid. Reliable blood tests for plaque and tangle pathologies are currently lacking, but blood tests for general neurodegeneration have recently been developed. In AD, plaques and tangles often coexist with other pathologies, including Lewy bodies, and to what extent these contribute to symptoms is currently unknown. There are also important differential diagnoses that may be possible to distinguish from AD with the aid of biomarkers. The scope of this review is fluid biomarkers for AD and related pathologies. The purpose is to provide the reader with an updated account of currently available fluid biomarkers for AD and clinically relevant differential diagnoses.

Blood Occludin Level as a Potential Biomarker for Early Blood Brain Barrier Damage Following Ischemic Stroke.

Concern about intracerebral hemorrhage (ICH) is the primary reason for withholding tPA therapy from patients with ischemic stroke. Early blood brain barrier (BBB) damage is the major risk factor for fatal post-thrombolysis ICH, but rapidly assessing BBB damage before tPA administration is highly challenging. We recently reported that ischemia induced rapid degradation of tight junction protein occludin in cerebromicrovessels. The present study investigates whether the cleaved occludin is released into the blood stream and how blood occludin levels correlate to the extent of BBB damage using a rat model of ischemic stroke. Cerebral ischemia induced a time-dependent increase of blood occludin with a sharp increase at 4.5-hour post-ischemia onset, which concurrently occurred with the loss of occludin from ischemic cerebral microvessels and a massive BBB leakage at 4.5-hour post-ischemia. Two major occludin fragments were identified in the blood during cerebral ischemia. Furthermore, blood occludin levels remained significantly higher than its basal level within the first 24 hours after ischemia onset. Our findings demonstrate that blood occludin levels correlate well with the extent of BBB damage and thus may serve as a clinically relevant biomarker for evaluating the risk of ICH before tPA administration.

Circulating tight junction proteins mirror blood-brain barrier integrity in leukaemia central nervous system metastasis.

The aim of this study was to evaluate the clinical significance of circulating tight junction (TJ) proteins as biomarkers reflecting of leukaemia central nervous system (CNS) metastasis. TJs [claudin5 (CLDN5), occludin (OCLN) and ZO-1] concentrations were measured in serum and cerebrospinal fluid (CSF) samples obtained from 45 leukaemia patients. Serum ZO-1 was significantly higher (p < 0.05), but CSF ZO-1 levels were not significantly higher in the CNS leukaemia (CNSL) compared to the non-CNSL. The CNSL patients also had a lower CLDN5/ZO1 ratio in both serum and CSF than in non-CNSL patients (p < 0.05). The TJ index was negatively associated with WBCCSF , ALBCSF and BBB values in leukaemia patients. Among all of the parameters studied, CLDN5CSF had the highest specificity in discriminating between CNSL and non-CNSL patients. Therefore, analysing serum and CSF levels of CLDN5, OCLN and the CLDN5/ZO1 ratio is valuable in evaluating the potential of leukaemia CNS metastasis. Copyright © 2016 John Wiley & Sons, Ltd.

Thymol and Carvacrol Affect Hybrid Tilapia through the Combination of Direct Stimulation and an Intestinal Microbiota-Mediated Effect: Insights from a Germ-Free Zebrafish Model.

BACKGROUND: Essential oils (EOs) are commonly used as animal feed additives. Information is lacking on the mechanisms driving the beneficial effects of EOs in animals, especially the role played by the intestinal microbiota of the host. OBJECTIVE: The purpose of this study was to clarify the relative contribution of direct effects of EOs on the physiology and immune system of tilapia and indirect effects mediated by the intestinal microbiota by using a germ-free zebrafish model. METHODS: Juvenile hybrid tilapia were fed a control diet or 1 of 4 treatment diets containing 60-800 mg Next Enhance 150 (NE) (an EO product containing equal levels of thymol and carvacrol)/kg for 6 wk. The key humoral and cellular innate immune parameters were evaluated after the feeding period. In another experiment, the gut microbiota of tilapia fed a control or an NE diet (200 mg/kg) for 2 wk were transferred to 3-d postfertilization (dpf) germ-free (GF) zebrafish, and the expression of genes involved in innate immunity and tight junctions was evaluated in zebrafish at 6 dpf. Lastly, NE was directly applied to 3-dpf GF zebrafish at 3 doses ranging from 0.2 to 20 mg/L, and the direct effect of NE on zebrafish was evaluated after 1 and 3 d. RESULTS: NE supplementation at 200 mg/kg enhanced phagocytosis activity of head kidney macrophages (×1.36) (P < 0.05) and plasma lysozyme activity (×1.69) of tilapia compared with the control (P < 0.001), indicating an immunostimulatory effect. Compared with those colonized with control microbiota, GF zebrafish colonized with NE microbiota showed attenuated induction of immune response marker genes serum amyloid a (Saa; ×0.62), interleukin 1ß (Il1ß; ×0.29), and interleukin 8 (Il8; ×0.62) (P < 0.05). NE treatment of GF zebrafish at 2 and 20 mg/L for 1 d upregulated the expression of Il1ß (×2.44) and Claudin1 (×1.38), respectively (P < 0.05), whereas at day 3 the expression of Occludin2 was higher (×3.30) in the 0.2-mg NE/L group compared with the GF control (P < 0.05). CONCLUSION: NE may affect the immunity of tilapia through a combination of factors, i.e., primarily through a direct effect on host tissue (immune-stimulating) but also an indirect effect mediated by microbial changes (immune-relieving).

PROGNOSTIC VALUE OF PLASMA TIGHT-JUNCTION PROTEINS FOR SEPSIS IN EMERGENCY DEPARTMENT: AN OBSERVATIONAL STUDY.

BACKGROUND: Studies have illustrated that the breakdown of tight junction (TJ) contributed to an increase in vascular permeability in response to stimulation of inflammatory cytokines. Additionally, the release of TJ-associated proteins into the circulation was observed in many diseases. The present study was designed to investigate whether plasma levels of TJ-associated proteins could serve as predictors of severity and clinical outcome of sepsis. METHODS: In total, 51 septic patients were enrolled. The peripheral blood samples were collected for each patient on emergency department arrival. Plasma levels of occludin (OCLN), claudins (CLDN)-5, and zonula occludens (ZO)-1 and serum levels of procalcitonin (PCT) and lactate were measured. In addition, APACHE II score as well as SOFA score was calculated. The prognostic values of OCLN, CLDN-5, and ZO-1 were compared with the first 24-h maximum APACHE II score and SOFA score. RESULTS: The median levels of OCLN and ZO-1 were elevated with sepsis severity. The levels of plasma OCLN and ZO-1 were positively correlated with APACHE II score, SOFA score as well as lactate levels of the patients. The levels of ZO-1 revealed valuable diagnostic capacity to diagnose MODS, and the areas under the receiver-operating characteristic (AUC) curves of ZO-1 were similar to those of lactate levels, but better than those of PCT levels. The prognostic value for in-hospital mortality of ZO-1 was comparable to that of lactate levels, APACHE II score, and SOFA score, and superior to OCLN or PCT. CONCULSIONS: OCLN and ZO1 levels appear to be early prognostic markers in patients suffering from sepsis.

A New Panel of Blood Biomarkers for the Diagnosis of Mild Traumatic Brain Injury/Concussion in Adults.

No routine tests currently exist to objectively diagnose mild traumatic brain injury (mTBI)/concussion. Previously reported biomarkers for mTBI represented proteins released from damaged neurons or glia. However, low levels of these proteins, and/or the complexity of assays used for their detection, limits implementation of these biomarkers in routine practice. Here, we sought to identify proteins whose synthesis is altered post-mTBI and whose blood levels could be measured using standard immunoassays. Adult patients sustaining a concussion within the past 24 h were enrolled. Controls were uninjured subjects and patients with orthopedic injury (OI). Four candidate biomarkers were identified: copeptin; galectin 3 (LGALS3); matrix metalloproteinase 9 (MMP9); and occludin (OCLN). A 3.4-fold decrease (p<0.0001) in plasma concentration of copeptin was found in mTBI patients within 8 h after accident, compared to uninjured controls. Plasma levels of LGALS3, MMP9, and OCLN increased 3.6- to 4.5-fold (p<0.0001) within the same time frame postinjury. Levels of at least two biomarkers were altered beyond their respective cut-off values in 90% of mTBI patients, whereas in none of uninjured controls were levels of two biomarkers simultaneously changed. A positive correlation (r=0.681; p<0.001) between plasma levels of LGALS3 and OCLN was also found in mTBI patients, whereas in OI patients or uninjured subjects, these variables did not correlate. This panel of biomarkers discerns, with high accuracy, patients with isolated concussion from uninjured individuals within the first 8 h after accident. These biomarkers can also aid in diagnosing concussion in the presence of OI.

Serum tight-junction proteins predict hemorrhagic transformation in ischemic stroke patients.

OBJECTIVE: To evaluate the significance of circulating tight-junction (TJ) proteins as predictors of hemorrhagic transformation (HT) in ischemic stroke patients. METHODS: We examined 458 consecutive ischemic stroke patients, 7.2% of whom had clinically evident HT. None of the patients was treated with thrombolytic drugs. Serum levels of standard markers of blood-brain barrier (BBB) breakdown (S100B, neuron-specific enolase), TJ proteins (occludin [OCLN], claudin 5 [CLDN5], zonula occludens 1 [ZO1]), and molecules involved in BBB disintegration (matrix metalloproteinase 9 and vascular endothelial growth factor [VEGF]) were assessed upon admission to the emergency department. A clinical deterioration caused by HT (cdHT) was defined as an increase of ≥4 points in the NIH Stroke Scale score in combination with a visible HT on a CT scan performed immediately after the onset of new neurologic symptoms. RESULTS: Patients with cdHT had higher concentrations of OCLN, S100B, and the CLDN5/ZO1 ratio, and a lower level of VEGF than those without cdHT. CLDN5 levels also correlated with cdHT occurrence when estimated within 3 hours of stroke onset. We also demonstrated correlations between the levels of circulating TJ molecules and the level of S100B, which is a previously established marker of BBB disruption. CONCLUSIONS: Analyzing serum levels of TJ proteins, like CLDN5, OCLN, and CLDN5/ZO1 ratio, as well as S100B and VEGF, is an effective way to screen for clinical deterioration caused by HT in ischemic stroke patients, both within and after the IV thrombolysis time window.