Brain Barrier Damage Research Articles

Brain Immune Cell Infiltration and Serum Metabolomic Characteristics Reveal that Lauric Acid Promotes Immune Cell Infiltration in Brain and Streptococcus suis Meningitis in Mice.

Although naturally Streptococcus suis serotype 2 (SS2) causes meningitis resulting in death or sequela of neurological symptoms in pigs and humans, severely threatening public health in the world, it has been difficult to build up and confirm experimental meningitis mouse models with obvious neurological syndrome for about two decades, which strongly hampers the in-depth study on the control measures and mechanisms of SS2-induced meningitis. In this study, a typical meningitis mouse model of SS2 was successfully established, as confirmed by the behavioral indicators of balance beam test, suspension test, and gait analysis. With bacteria gathering in the brain, distinguishable unique features including meningeal thickening, vacuolization of the Nissl body, brain barrier damage, glial cell activation, and more infiltration of T cells, macrophages, and DCs are observed in SS2 meningitis mice with typical neurological signs. Some meningitis mice were also accompanied by identical nephritis, ophthalmia, and cochlearitis. Investigation of the metabolic features demonstrated the downregulated cholic acid and upregulated 2-hydroxyvaleric acid, tetrahydrocortisone, nicotinic acid, and lauric acid in blood serum of mice and piglets with meningitis. And feeding trials show that lauric acid can promote meningitis by promoting the infiltration of immune cells into brain. These findings demonstrated that infection of ICR (improved castle road) mice with SS2 was able to induce typical meningitis accompanied by immune cell infiltration and lauric acid upregulation. These data provide a basis for the deep study of SS2 meningitis.

Fe3O4 Nanozymes Improve Neuroblast Differentiation and Blood-Brain Barrier Integrity of the Hippocampal Dentate Gyrus in D-Galactose-Induced Aged Mice.

Aging is a process associated with blood–brain barrier (BBB) damage and the reduction in neurogenesis, and is the greatest known risk factor for neurodegenerative disorders. However, the effects of Fe3O4 nanozymes on neurogenesis have rarely been studied. This study examined the effects of Fe3O4 nanozymes on neuronal differentiation in the dentate gyrus (DG) and BBB integrity of D-galactose-induced aged mice. Long-term treatment with Fe3O4 nanozymes (10 μg/mL diluted in ddH2O daily) markedly increased the doublecortin (DCX) immunoreactivity and decreased BBB injury induced by D-galactose treatment. In addition, the decreases in the levels of antioxidant proteins including superoxide dismutase (SOD) and catalase as well as autophagy-related proteins such as Becin-1, LC3II/I, and Atg7 induced by D-galactose treatment were significantly ameliorated by Fe3O4 nanozymes in the DG of the mouse hippocampus. Furthermore, Fe3O4 nanozyme treatment showed an inhibitory effect against apoptosis in the hippocampus. In conclusion, Fe3O4 nanozymes can relieve neuroblast damage and promote neuroblast differentiation in the hippocampal DG by regulating oxidative stress, apoptosis, and autophagy.

Abstract WP252: Tamoxifen Treatment Worsens Astrocyte Activation And Mitochondrial Stress From Oxygen Glucose Deprivation

Introduction: Tamoxifen (TAM), a selective estrogen receptor modifier, is a first-line treatment option for hormone receptor positive breast cancer. TAM inhibits the downstream estrogen receptor signaling in tumor cells. Stroke risk is increased in women with low estrogen levels, such as early-onset menopause, and doubles in women after the age of 55, which corresponds with the average age of menopause. Women with breast cancer who were treated with tamoxifen had an increased risk for ischemic stroke (82%). Since stroke severity is associated with increased blood brain barrier (BBB) damage, we investigated the effects of tamoxifen on activation and mitochondrial stress in astrocytes, which is a cellular component of the BBB. Methods: Cultured female human astrocytes were treated with tamoxifen (1uM), 17ß-estradiol (10nM), or vehicle (DMSO) for 24 hours. Cells were separated into two groups: normoxic (21% O2, 25mM glucose) or oxygen and glucose deprivation (OGD) conditions 6 hours before analysis. ROS production was measured using the Cellular ROS Assay Kit (Red) and aquaporin-4 levels were visualized via immunocytochemistry. Mitochondrial function was assessed by using a Seahorse XFe96 Analyzer. Results: Tamoxifen treatment increased Aquaporin-4 protein levels (p <0.0001) in cultured astrocytes after OGD compared to both vehicle (DMSO) and 17ß-estradiol treatment. While there was no significant effect, tamoxifen treated cells showed a trend toward (p =0.0732) increased ROS production after OGD compared to vehicle. Additionally, tamoxifen reduced oxygen consumption rate (p =0.0197) and basal respiration under normoxic conditions in astrocytes when compared with DMSO or 17ß-estradiol treatment. Conclusions: Tamoxifen treatment altered the expression of a water channel protein and decreased the ability to respond to oxidative stress in astrocytes. Aquaporin-4, when localized to the astrocytic end feet, facilitates edema after stroke, and ischemic stroke by definition puts brain tissue under oxidative stress. Our findings suggest tamoxifen may increase edema, which is known to impair stroke recovery. These data suggest a mechanism by which breast cancer treatment may increase the risk for severe ischemic stroke. Supported by RFAG042189 to FS

Abstract TP105: The Neuroprotective Efficacy of Regional Cerebral Hypothermia Induced via a New Intrajugular Cooling Method in Experimental Ischemic Stroke

Background: Therapeutic hypothermia as a neuroprotective strategy after stroke has been well established in animal models, but its application in humans has been limited by logistical challenges. To provide a solution to these challenges, this study was designed to determine the hypothermic and neuroprotective efficacy of infusing cold saline directly into the internal jugular (IJ) vein, and compare the effects of IJ regional cerebral hypothermia to those achieved by intra-carotid (IC) artery regional hypothermia in an ischemic stroke model. Methods: Middle cerebral artery occlusion was established in rats using an intraluminal filament. Regional cerebral hypothermia was then achieved by infusion of 6 ml of 0 °C isotonic saline by micro-catheter into the right IJ or right IC for 30 minutes immediately after the initiation of reperfusion. Infarct sizes, neurological deficits, edema volume, blood brain barrier (BBB) damage, BBB associated molecules (MMP-9 and AQP-4), and apoptosis associated proteins (Bcl-2 and cleaved Caspase-3) were measured. Results: Both IJ and IC infusion led to a significant, rapid reduction in brain temperatures while having a minimal effect on rectal temperatures. Infarct sizes were significantly reduced at 24 hours after reperfusion by IC and IJ treatments. Neurologic deficits were also significantly improved by IC and IJ infusion compared to control models over a 28-day observation period. Both IC and IJ significantly reduced Evans Blue leakage and edema volume compared with control groups. MMP-9 and AQP-4 expression were also reduced, Bcl-2 increased, and cleaved Caspase-3 decreased in the IC and IJ groups. Conclusion: Compared to IC infusion, IJ infusion conferred a similar degree of regional cerebral hypothermia and neuroprotection following ischemic stroke. Given the ease of establishing vascular access via the internal jugular vein and the powerful neuroprotection that hypothermia provides, IJ regional brain cooling could be utilized as a promising hypothermia-induction modality due to its safe, quick, and effective features.

Increased 14-3-3β and γ protein isoform expressions in parasitic eosinophilic meningitis caused by Angiostrongylus cantonensis infection in mice

The 14-3-3 proteins are cerebrospinal fluid (CSF) markers of neuronal damage during infectious meningitis and Creutzfeldt-Jakob disease. Little is known about dynamic changes in the individual isoforms in response to parasitic eosinophilic meningitis. The purposes of this study were to determine the 14-3-3 protein isoform patterns, examine the kinetics and correlate the severity of blood brain barrier (BBB) damage with the expressions of these markers in mice with eosinophilic meningitis.Mice were orally infected with 50 A. cantonensis L3 via an oro-gastric tube and sacrificed every week for 3 consecutive weeks after infection. The Evans blue method and BBB junctional protein expressions were used to measure changes in the BBB. Hematoxylin and eosin staining was used to analyze pathological changes in the mice brains following 1–3 weeks of infection with A. cantonensis. The levels of 14-3-3 protein isoforms in serum/CSF and brain homogenates were analyzed by Western blot, and immunohistochemistry (IHC) was used to explore the different isoform distributions of 14-3-3 proteins and changes in BBB junctional proteins in the mice brain meninges. Dexamethasone was injected intraperitoneally from the seventh day post infection (dpi) until the end of the study (21 dpi) to study the changes in BBB junctional proteins. The amounts of Evans blue, tight junction and 14-3-3 protein isoforms in the different groups of mice were compared using the nonparametric Kruskal-Wallis test.There were significant increases in 14-3-3 protein isoforms β and γ in the CSF in the second and third weeks after infection compared to the controls and first week of infection, which were correlated with the severity of BBB damage in brain histology, and Evans blue extravasation. Using IHC to assess the distribution of 14-3-3 protein isoforms and changes in BBB junctional proteins in the mice brain meninges, the expressions of isoforms β, γ, ε, and θ and junctional proteins occludin and claudin-5 in the brain meninges increased over a 3-week period after infection compared to the controls and 1 week after infection. The administration of dexamethasone decreased the expressions of BBB junctional proteins occludin and claudin-5 in the mice brain meninges.Our findings support that 14-3-3 proteins β and γ can potentially be used as a CSF marker of neuronal damage in parasitic eosinophilic meningitis caused by A. cantonensis.

Prostaglandin E2 EP4 Receptor Activation Attenuates Neuroinflammation and Early Brain Injury Induced by Subarachnoid Hemorrhage in Rats

Activation of E prostanoid 4 receptor (EP4) shows neuroprotective effects in multiple central nervous system (CNS) lesions, but the roles of EP4 receptor in subarachnoid hemorrhage (SAH) are not explored. This study was designed to research the effects of EP4 modulation on early brain injury (EBI) after experimental SAH in rats. We found that the administration of EP4 selective agonist AE1-329 significantly improved neurological dysfunction, blood brain barrier (BBB) damage and brain edema at 24 h after SAH. Furthermore, AE1-329 obviously reduced the number of activated microglia and the mRNA and protein levels of pro-inflammatory cytokines, and increased Ser1177 phosphorylated endothelial nitric oxide synthase (Ser1177 p-eNOS). Moreover, AE1-329 significantly reduced the number of TUNEL-positive cells and active caspase-3 in cortex after SAH. The EP4 selective antagonist AE3-208 was also administrated and the opposite effects were achieved. Our results indicate that activation of EP4 protects brain from EBI through downregulating neuroinflammation reaction after SAH.

Abstract 49: Endothelial-targeted Overexpression of Heat Shock Protein 27 Ameliorates Rapid Blood Brain Barrier Impairment and Improves Long Term Outcomes After Ischemia and Reperfusion

Introduction: Blood brain barrier (BBB) damage resulting from ischemia/reperfusion (I/R) disrupts the neurovascular unit and leads to poor patient outcomes. We recently discovered that caveolin-1-independent subtle structural aberrations of brain microvascular endothelial cells (BMECs), such as abnormal actin polymerization, stress fiber formation and subsequent junctional protein (JP) disassembly, are a novel mechanism for rapid BBB breach after I/R. Hypothesis: Heat shock protein 27 (HSP27) reduces BBB breakdown after I/R by inhibiting actin polymerization and JP disassembly in BMECs. Methods: Neuron- or EC-specific HSP27-overexpressing mice were subjected to 1 h MCAO and reperfusion. BBB damage, tissue histology, and neurobehavioral performance were assessed up to 35 d after I/R. I/R-induced BBB damage was also simulated in BMEC cultures, where gene manipulations were achieved using lentiviral vectors. Cell-permeable TAT-HSP27 protein was injected i.v. into mice after I/R to rapidly elevate HSP27 in BMECs. Results: Targeted overexpression of HSP27 within ECs—but not neurons—was sufficient to reduce early (1-3 h) and late (24 h) BBB damage after I/R (p<0.01). Mechanistically, HSP27 suppressed I/R-induced actin polymerization, stress fibers, and JP disassembly in BMECs, but independent of its anti-cell death properties. Intracerebral infiltration of neutrophils and macrophages was attenuated in EC-HSP27 mice by 35% and 60%, respectively (n=6, p<0.05) at 48 h after I/R. Infarct size was reduced by 35% at 72 h, and sensorimotor functions (p<0.01, cylinder and corner tests) were improved in EC-HSP27 mice up to 21 d. Post-I/R injection of TAT-HSP27 markedly reduced BBB damage and elicited sustained (up to 35 d) protection against neurological deficits. Conclusions: HSP27 protects against BBB disruption after I/R by inhibiting actin polymerization and JP disassembly in BMECs. HSP27 may be a therapy for ischemic stroke in conjunction with reperfusion.

Abstract TP108: Blood Occludin Level Indicates the Extent of Early Blood Brain Barrier Damage in Ischemic Stroke

Background and Purpose: Fear of symptomatic intracerebral hemorrhage (ICH) has been the primary reason for withholding tPA thrombolysis from acute ischemic stroke patients. Early blood brain barrier (BBB) damage is appreciated to be closely associated with post-thrombolysis ICH, while it remains a technical challenge for rapid assessment of BBB damage before tPA administration. Our recent data showed that cerebral ischemia induced rapid degradation of tight junction protein occludin in ischemic cerebromicrovessels. This study further investigates whether the cleaved occludin is released into the blood stream and how blood occludin levels correlate to the extent of ischemic BBB damage. Methods: Male Sprague Dawley rats were subjected to 1.5, 3, 4.5, 12 and 24 hours of middle cerebral artery occlusion (MCAO), followed by 5-min reperfusion. Blood samples were taken before and after MCAO. Blood occludin was assessed by ELISA. BBB permeability was measured by Evans blue dye leakage. Occludin cleavage was identified on immunoblots. Results: MCAO induced Evans blue dye leakage and blood occludin increase in a duration-dependent manner. Blood occludin increase concurrently occurred with the loss of occludin from ischemic cerebral microvessels. Western blot analysis identified two cleaved occludin fragments (31- and 55- kDa) in the blood. Lastly, blood occludin levels remained significantly higher than its basal level within the first 24 hours after MCAO onset. Conclusions: Our results indicate that blood occludin levels correlate well with the extents of BBB damage and thus may serve as a potential biomarker for evaluating the risk of hemorrhagic transformation before tPA administration.

Visual impairment in diabetic patients with and without established blood‐retinal barrier leakage and relation with the status of the blood‐brain barrier

AbstractPurpose To assess visual impairment in diabetic patients with and without established blood‐retinal barrier leakage and relation with the status of the blood‐brain barrier.Methods We assessed 37 diabetic patients (29 type2; mean Hb1Ac 7.93[1.44]) using four different approaches: a two‐alternative forced‐choice multi‐psychophysical channel test for speed discrimination and achromatic contrast as well as chromatic L, M and S cone contrast discrimination; multifocal electrorretinography(mfERG) and brain barrier integrity assessment using a permeability measure Ktrans for dynamic contrast enhanced MRI (Cramer et. al, 2013) in 4 regions of interest(ROIs). We selected two diabetic groups with and without measured retinal barrier leakage. Spearman’s rank were used for correlations and Mann‐Whitney U test for comparison between groups for a set p value threshold of 0.05.Results We found that presence of retinal leakage was not associated with changes in blood brain barrier permeability at any of the studied ROIs. Interestingly we found, in particular in diabetic type 2 patients, significant negative correlations between the amplitude and/or implicit time of N1 and P1 components of the multifocal electrorretinogram and brain leakage, for the most external stimulation rings. Psychophysical performance was impaired in particular for S but was not correlated with barrier status.Conclusion Our findings suggest that retina and brain blood barriers are patho physiologically unrelated in diabetes. We found a direct correlation between neurophysiological measures and brain barrier damage, suggesting that both may have a direct common cause.

Erythropoietin pretreatment suppresses seizures and prevents the increase in inflammatory mediators during pentylenetetrazole-induced generalized seizures

Erythropoietin (EPO) suppresses epileptic seizures, but the mechanism is unclear. The search for novel targets in the therapy of epilepsy has focused recently on brain inflammation since brain inflammation and the associated blood–brain barrier (BBB) damage appears to be an integral part of epilepsy pathophysiology. We examined the effects of EPO on proinflammatory mediators in brain and serum in PTZ-induced generalized seizure model. The inflammation markers (IL-1β, TNF-α, IL-6, IL-10), BBB and neuron damage markers (S100B, Neuron specific enolase; NSE, respectively) in serum and brain of Sprague–Dawley male rats were examined with the ELISA method. Nitric oxide synthase (NOS) isoforms were investigated immunohistochemically in hippocampus. EPO treatment 4 h and 24 h before PTZ administration had diverse effects. EPO treatment 4 h before PTZ administration elongated the seizure latency, decreased the inflammation and damage markers in serum and brain significantly, whereas EPO treatment 24 h before PTZ administration lowered inflammation and damage markers to control levels and decreased the seizure stage. PTZ-induced seizures increased inducible NOS (iNOS) activity and decreased endothelial NOS (eNOS) activity in hippocampus. Both EPO pretreatments reversed these effects. These findings, i.e., decreased iNOS activity and increased eNOS activity by EPO suggest the first time that the favorable effect of EPO pretreatment on inflammatory mediators triggered by PTZ-induced seizures. This can provide further insight into epilepsy treatment and new prophylactic strategies against epilepsy risk.

Systemic inflammatory challenges compromise survival after experimental stroke via augmenting brain inflammation, blood- brain barrier damage and brain oedema independently of infarct size

BackgroundSystemic inflammation impairs outcome in stroke patients and experimental animals via mechanisms which are poorly understood. Circulating inflammatory mediators can activate cerebrovascular endothelium or glial cells in the brain and impact on ischaemic brain injury. One of the most serious early clinical complications of cerebral ischaemia is brain oedema, which compromises survival in the first 24-48 h. It is not understood whether systemic inflammatory challenges impair outcome after stroke by increasing brain injury only or whether they have direct effects on brain oedema, cerebrovascular inflammation and blood-brain barrier damage.MethodsWe used two different systemic inflammatory stimuli, acute endotoxin treatment and anaphylaxis to study mechanisms of brain injury after middle cerebral artery occlusion (MCAo). Ischaemic brain injury, blood-brain barrier damage and oedema were analysed by histological techniques. Systemic cytokine responses and inflammatory changes in the brain were analysed by cytometric bead array, immunofluorescence, in situ hibridization and quantitative real-time PCR.ResultsSystemic inflammatory challenges profoundly impaired survival in the first 24 h after experimental stroke in mice, independently of an increase in infarct size. Systemic lipopolysaccharide (LPS) dose-dependently increased mortality (50-100%) minutes to hours after cerebral ischaemia. Acute anaphylactic challenge in ovalbumin-sensitised mice affected stroke more seriously when induced via intraperitoneal administration compared to intravenous. Both LPS and anaphylaxis induced inflammatory changes in the blood and in the brain prior to experimental stroke. Plasma cytokine levels were significantly higher after LPS, while increased IL-10 levels were seen after anaphylaxis. After MCAo, both LPS and anaphylaxis increased microglial interleukin-1α (IL-1α) expression and blood-brain barrier breakdown. LPS caused marked granulocyte recruitment throughout the ipsilateral hemisphere. To investigate whether reduction of ischaemic damage can improve outcome in systemic inflammation, controlled hypothermia was performed. Hypothermia reduced infarct size in all treatment groups and moderately improved survival, but failed to reduce excess oedema formation after anaphylaxis and LPS-induced neuroinflammation.ConclusionsOur results suggest that systemic inflammatory conditions induce cerebrovascular inflammation via diverse mechanisms. Increased brain inflammation, blood-brain barrier injury and brain oedema formation can be major contributors to impaired outcome in mice after experimental stroke with systemic inflammatory stimuli, independently of infarct size.

Effects of Normobaric Hyperoxia in a Rat Model of Transient Focal Cerebral Ischemia and Reperfusion

3 Background: The role of therapeutic oxygen in treatment of acute stroke is controversial. Oxygen improves cellular aerobic metabolism and can salvage ischemic tissue. However, oxygen free radicals can increase blood brain barrier (BBB) damage, and oxygen can induce vasoconstriction, which could worsen stroke outcome. We studied the effects of normobaric oxygen in cerebral ischemia and reperfusion. Methods: Rats were subjected to normobaric hyperoxia (FiO2 100%) or normoxia (FiO2 30%) during two hour filament occlusion and one hour reperfusion of the middle cerebral artery. Twenty-four hour infarct volumes, regional cerebral blood flow (rCBF) using laser Doppler flowmetry, and severity of BBB damage (assessed by quantifying Evan’s Blue dye (EB) leakage after one hour of reperfusion) were compared between groups. Results: Physiological parameters were similar in hyperoxic and control groups, except for paO2, which was expectedly higher in the hyperoxic group (pO2 484 mm Hg) as compared to controls (pO2 118 mm Hg). Mean rCBF dropped to 25% after onset of ischemia and recovered to 75–90% after arterial unocclusion, indicating successful reperfusion. Mean total (right hemispheric) infarct volume was 65 mm 3 in the hyperoxia group and 209 mm 3 in controls, p 3 in hyperoxic rats and 129 mm 3 in controls (p 3 in hyperoxic rats and 80 mm 3 in controls (p Conclusion: Total and cortical infarct volumes can be significantly reduced with normobaric hyperoxia during transient cerebral ischemia and reperfusion. Hyperoxia does not decrease blood flow to ischemic brain, and its benefit in reducing infarct volume may outweigh any potential damage from BBB damage.

Neuropeptide Levels after Pentylenetetrazol Kindling in the Rat.

Levels of several neuropeptides were measured in the frontal cortex, dorsal hippocampus, striatum, and amygdala/pyriform cortex in rats kindled for 5 weeks by daily injection of pentylenetetrazol (30 mg/kg, i.p.). Significantly increased concentrations (by 30 - 140%) were found in all examined brain areas for neuropeptide Y, somatostatin (except hippocampus) and neurokinin-like immunoreactivity 10 days after the last kindling session. Similar but less pronounced changes were also found 24 h after the last seizure. The increase in total neurokinin-like immunoreactivity was due to a marked increase in neurokinin B as revealed by HPLC analysis. Increases in peptide levels, however, were restricted to fully kindled animals. At the same time no changes in levels of substance P, vasoactive intestinal polypeptide and calcitonin gene-regulated peptide were observed. Cholecystokinin octapeptide was enhanced only in the hippocampus (by 46%). The increases in neuropeptide Y, somatostatin, and neurokinin-like immunoreactivity subsided after 3 months. A markedly decreased seizure threshold was observed 10 days and 2 months after the final kindling session. No nerve cell degeneration was observed in kindled rats 24 h or 10 days after the last pentylenetetrazol injection. Some animals (2 of 4), however, exhibited signs of blood - brain barrier damage when examined 24 h after the last kindling session which may reflect the preceding convulsions. No such changes were detected after 10 days. The increases in peptide levels may suggest increased activity of respective neurons which, at least to some degree, may be associated with gamma-aminobutyric acid. The changes in peptide levels may be more closely related to the kindling procedure itself than to the decreased seizure threshold of the animals.