Blood-brain Barrier Ultrastructure Research Articles

Protective Effect of Protocatechuic Aldehyde on Cerebral Ischemia/Reperfusion Injury in Rats through Blood-Brain Barrier Protection.

Cerebral ischemia can lead to destruction of the blood-brain barrier (BBB), the main cause of cerebral edema and cerebral infarction. BBB damage is also one of the key factors affecting the result of drug therapy. We studied the protective effect of 5-day pretreatment with protocatechuic aldehyde (PAL) at doses of 10 and 20 mg/kg on BBB function and structure after middle cerebral artery occlusion/reperfusion (MCAO/R) in rats. The infarct volume, behavioral neurological deficit score, and Evans blue content in the brain were estimated. We also evaluated the content of nitric oxide (NO) and activities of inducible and neuronal NO synthases. Expression of aquaporin-4 (AQP-4), occludin, claudin-5, and MMP-3 in the brain tissues was estimated by Western blotting. The BBB ultrastructure was analyzed under an electron microscope. We revealed that PAL at both used doses significantly reduced the neurological deficit score, brain infarct volume, and Evans blue extravasation. Electron microscopy showed that PAL significantly improved the ultrastructure of BBB and alleviated its injury. Pretreatment with PAL increased expression of occludin and claudin-5 and reduced expression of AQP-4 and MMP-3. At the same time, the release of NO and activities of NO synthases were notably inhibited. Our results suggest that PAL can be a promising compound to attenuate cerebral ischemia resulting from occlusion/reperfusion injury via BBB protection.

Impaired learning and memory in male mice induced by sodium arsenite was associated with MMP-2/MMP-9-mediated blood-brain barrier disruption and neuronal apoptosis

Arsenic is a widespread environmental contaminant known to accumulate in the brain, leading to cognitive impairment. However, the exact mechanisms by which arsenic causes cognitive deficits remain unclear. The present study aims to discover whether the destruction of the blood-brain barrier (BBB) mediated by matrix metalloproteinases 2 and matrix metalloproteinases 9 (MMP-2 and MMP-9) and subsequent neuronal apoptosis are involved in arsenic-induced cognitive impairment. Ninety male mice were given 0, 25, and 50 mg/L NaAsO2 in drinking water and 30 mg/kg doxycycline hyclate (DOX, an inhibitor of MMPs) gavage for 12 weeks to observe the alterations in learning and memory of mice, the morphology of hippocampal neurons, as well as the BBB permeability and ultrastructure, the localization and expression of tight junction proteins, MMP-2, and MMP-9. Our findings indicated that arsenic exposure induced learning and memory impairment in mice, accompanied by neuronal loss and apoptosis. Furthermore, arsenic exposure increased hematogenous IgG leakage into the brain, disrupted the tight junctions, reduced the expression of Claudin5, Occludin, and ZO1 in the endothelial cells, and increased the expression of MMP-2 and MMP-9 in the endothelial cells and astrocytes. Finally, DOX intervention preserved BBB integrity, alleviated hippocampal neuronal apoptosis, and improved cognitive impairment in mice caused by arsenic exposure. Our research demonstrates that cognitive disfunction in mice induced by arsenic exposure is associated with MMP-2 and MMP-9-mediated BBB destruction and neuronal apoptosis. The current investigation provides new insights into mechanisms of arsenic neurotoxicity and suggests that MMP-2 and MMP-9 may serve as potential therapeutic targets for treating arsenic-induced cognitive dysfunction in the future.

Microglia are not necessary for maintenance of blood-brain barrier properties in health, but PLX5622 alters brain endothelial cholesterol metabolism

Microglia, the resident immune cells of the central nervous system, are intimately involved in the brain's most basic processes, from pruning neural synapses during development to preventing excessive neuronal activity throughout life. Studies have reported both helpful and harmful roles for microglia at the blood-brain barrier (BBB) in the context of disease. However, less is known about microglia-endothelial cell interactions in the healthy brain. To investigate the role of microglia at a healthy BBB, we used the colony-stimulating factor 1 receptor (CSF1R) inhibitor PLX5622 to deplete microglia and analyzed the BBB ultrastructure, permeability, and transcriptome. Interestingly, we found that, despite their direct contact with endothelial cells, microglia are not necessary for the maintenance of BBB structure, function, or gene expression in the healthy brain. However, we found that PLX5622 treatment alters brain endothelial cholesterol metabolism. This effect was independent from microglial depletion, suggesting that PLX5622 has off-target effects on brain vasculature.

ASK1-K716R reduces neuroinflammation and white matter injury via preserving blood–brain barrier integrity after traumatic brain injury

BackgroundTraumatic brain injury (TBI) is a significant worldwide public health concern that necessitates attention. Apoptosis signal-regulating kinase 1 (ASK1), a key player in various central nervous system (CNS) diseases, has garnered interest for its potential neuroprotective effects against ischemic stroke and epilepsy when deleted. Nonetheless, the specific impact of ASK1 on TBI and its underlying mechanisms remain elusive. Notably, mutation of ATP-binding sites, such as lysine residues, can lead to catalytic inactivation of ASK1. To address these knowledge gaps, we generated transgenic mice harboring a site-specific mutant ASK1 Map3k5-e (K716R), enabling us to assess its effects and elucidate potential underlying mechanisms following TBI.MethodsWe employed the CRIPR/Cas9 system to generate a transgenic mouse model carrying the ASK1-K716R mutation, aming to investigate the functional implications of this specific mutant. The controlled cortical impact method was utilized to induce TBI. Expression and distribution of ASK1 were detected through Western blotting and immunofluorescence staining, respectively. The ASK1 kinase activity after TBI was detected by a specific ASK1 kinase activity kit. Cerebral microvessels were isolated by gradient centrifugation using dextran. Immunofluorescence staining was performed to evaluate blood–brain barrier (BBB) damage. BBB ultrastructure was visualized using transmission electron microscopy, while the expression levels of endothelial tight junction proteins and ASK1 signaling pathway proteins was detected by Western blotting. To investigate TBI-induced neuroinflammation, we conducted immunofluorescence staining, quantitative real-time polymerase chain reaction (qRT-PCR) and flow cytometry analyses. Additionally, immunofluorescence staining and electrophysiological compound action potentials were conducted to evaluate gray and white matter injury. Finally, sensorimotor function and cognitive function were assessed by a battery of behavioral tests.ResultsThe activity of ASK1-K716R was significantly decreased following TBI. Western blotting confirmed that ASK1-K716R effectively inhibited the phosphorylation of ASK1, JNKs, and p38 in response to TBI. Additionally, ASK1-K716R demonstrated a protective function in maintaining BBB integrity by suppressing ASK1/JNKs activity in endothelial cells, thereby reducing the degradation of tight junction proteins following TBI. Besides, ASK1-K716R effectively suppressed the infiltration of peripheral immune cells into the brain parenchyma, decreased the number of proinflammatory-like microglia/macrophages, increased the number of anti-inflammatory-like microglia/macrophages, and downregulated expression of several proinflammatory factors. Furthermore, ASK1-K716R attenuated white matter injury and improved the nerve conduction function of both myelinated and unmyelinated fibers after TBI. Finally, our findings demonstrated that ASK1-K716R exhibited favorable long-term functional and histological outcomes in the aftermath of TBI.ConclusionASK1-K716R preserves BBB integrity by inhibiting ASK1/JNKs pathway in endothelial cells, consequently reducing the degradation of tight junction proteins. Additionally, it alleviates early neuroinflammation by inhibiting the infiltration of peripheral immune cells into the brain parenchyma and modulating the polarization of microglia/macrophages. These beneficial effects of ASK1-K716R subsequently result in a reduction in white matter injury and promote the long-term recovery of neurological function following TBI.

DDEL-10. ULTRASOUND-ENHANCED DRUG DELIVERY IN HUMANS ALTERS ENDOTHELIAL PHENOTYPE AND PERTURBS THE ULTRASTRUCTURE OF THE BLOOD BRAIN BARRIER

Abstract The use of low-intensity pulsed ultrasound with concomitant injection of microbubbles (LIPU/MB) achieves local and reversible opening the blood-brain barrier (BBB), enhancing the delivery of systemically administered drugs to the brain parenchyma. Electron microscopic studies conducted in animals proposed that LIPU/MB enhances paracellular diffusion across the cerebral vasculature, by disrupting tight junction proteins between endothelial cells, as well as enhancement of caveolar transcytosis. The effects of LIPU/MB on the ultrastructure of the human BBB and cerebral endothelial phenotype have not been systematically investigated. Here we report a first in-human electron microscopic study, examining endothelial phenotype and BBB ultrastructure in the peri-tumoral brain of humans after undergoing LIPU/MB-enhanced drug delivery. Non-eloquent peritumoral brain was biopsied at different time points (4-63 minutes) following an intraoperative LIPU/MB procedure in three patients who underwent a surgical resection of recurrent glioblastoma through a phase I clinical trial [NCT04528680]. Transmission electron microscopy was used to examine cross sections of the microvasculature and associated components of the BBB. We observed a significant (P = 0.0397) time-dependent decrease in the frequency of endothelial caveolae immediately after sonication compared to non-sonicated control vessels, which resolved within 1 hour of treatment. We also observed a time-dependent increase in membrane-bound vacuoles in the endothelial cytoplasm following sonication (P = 0.0002, one-way ANOVA). Sonicated blood vessels occasionally showed swollen astrocytes, convoluted luminal protections, and lightening of otherwise dense tight junction proteins, not frequently observed in non-sonicated biopsies from the same patients. Our study shows that LIPU/MB changes the endothelial phenotype within the cerebral vasculature, and suggests the accumulation of vacuoles in endothelial cells after BBB opening.

Effects of electroacupuncture on the blood-brain-barrier and proinflammatory cytokine IL-1β and IL-18 in the hippocampus of rats with vascular dementia

To observe the effect of electroacupuncture (EA) on blood-brain barrier (BBB) permeability and proinflammatory cytokines interleukin-1β (IL-1β) and interleukin-18 (IL-18) in the hippocampus of vascular dementia (VD) rats, so as to explore the mechanism of EA on treatment of VD. SD male rats were randomly divided into sham operation, model, and EA groups, with 15 rats in each group. The VD rat model was established by permanently occlusion of the bilateral middle cerebral artery. Rats of the EA group received EA at "Baihui" (GV20), "Dazhui" (GV14), and bilateral "Shenshu"(BL23) for 30 min, 6 days a week for a total of 4 weeks. Morris water maze test was used to assess the cognitive function of rats. Evans blue staining was used to detect the BBB permeability, transmission electron microscopy and ELISA were used to detect the ultrastructure of BBB and the contents of hippocampal IL-1β and IL-18, respectively. Following modeling, compared with the sham operation group, the mean escape latency of model group was significantly prolonged (P<0.01), the times of crossing the platform were significantly decreased (P<0.01), the content of Evans blue, and the contents of IL-1β and IL-18 in hippocampus were increased (P<0.01). After the intervention, comparison between the model and EA groups showed that the average escape latency of rats in EA group was significantly shortened (P<0.01), the times of crossing the platform were increased (P<0.05), the content of Evans blue, and the contents of IL-1β and IL-18 in hippocampus were significantly decreased (P<0.01). The ultrastructure of BBB was moderately damaged in the model group, which was evidenced by blurred endothelial cell membrane structure, obviously dropsical astrocyte foot process, and decreased tight junctions. The ultrastructure of BBB was slightly damaged and astrocyte foot had no obvious edema in the EA group. EA can significantly improve the learning and memory ability of VD rats and improve the BBB permeability, which may be related to its effect in inhibiting the expression of IL-1β and IL-18 in the hippocampus.

Cuprizone feeding induces swollen astrocyte endfeet

The cuprizone model is a widely used model to study the pathogenesis of multiple sclerosis (MS). Due to the selective loss of mature oligodendrocytes and myelin, it is mainly being used to study demyelination and the mechanisms of remyelination, as well as the efficiency of compounds or therapeutics aiming at remyelination. Although early investigations using high dosages of cuprizone reported the occurrence of hydrocephalus, it has long been assumed that cuprizone feeding at lower dosages does not induce changes at the blood–brain barrier (BBB). Here, by analyzing BBB ultrastructure with high-resolution electron microscopy, we report changes at astrocytic endfeet surrounding vessels in the brain parenchyma. Particularly, edema formation around blood vessels and swollen astrocytic endfeet already occurred after feeding low dosages of cuprizone. These findings indicate changes in BBB function that will have an impact on the milieu of the central nervous system (CNS) in the cuprizone model and need to be considered when studying the mechanisms of de- and remyelination.

Storax Inhibits Caveolae-Mediated Transcytosis at Blood-Brain Barrier After Ischemic Stroke in Rats.

Background and Purpose: Blood-brain barrier (BBB) disruption following ischemic stroke (IS) contributes to hemorrhagic transformation, brain edema, increased neural dysfunction, secondary injury, and mortality. The prevailing view attributes the destruction of tight junction proteins (TJs) to the resulting BBB damage following IS. However, recent studies define a stepwise impairment of the transcellular barrier followed by the paracellular barrier which accounts for the BBB leakage in IS. The increased endothelial transcytosis that has been proven to be caveolae-mediated, preceding and independent of TJs disintegration. Emerging experimental investigations suggested Storax attenuates BBB damage after stroke. This study aimed to test our hypothesis that Storax inhibits caveolae-mediated transcytosis at BBB after ischemic stroke in rats. Methods: Male Wistar rats (250–300 g) were subjected to transient middle cerebral artery occlusion (t-MCAO). Brain water content and the cerebral infarction size were assessed by brain tissue drying-wet method and 2,3,5-triphenyltetrazolium chloride (TTC) staining. BBB permeability was detected by the leakage of Evans blue and Albumin-Alexa594. The ultrastructure of BBB was examined by transmission electron microscopy (TEM). Cav-1 and Mfsd2a were quantified by western blotting and immunofluorescence staining, AQP4, PDGFR-β, ZO-1 and Occludin were quantified by western blotting. Results: Storax treatment of 0.1 g/kg had no significant effects on brain lesions. Storax treatment of 0.2, 0.4, and 0.8 g/kg led to a significant decrease in infarction size, and the Storax 0.4, 0.8 g/kg groups displayed a significant reduction in brain water content. Storax treatment of 0.8 g/kg showed mild toxic reactions. Thus, 0.4 g/kg Storax was selected as the optimal dose for subsequent studies. Storax significantly inhibited the fluorescent albumin intensity in the brain parenchyma and the number of caveolae in ECs, alongside attenuating the ultrastructural disruption of BBB at 6 h after stroke. Meanwhile, Storax significantly increased the expression of Mfsd2a and PDGFR-β, and decrease the expression of Cav-1 and AQP4, corresponding to the significantly decreased Cav-1 positive cells and increased Mfsd2a positive cells. However, Storax has no significant effects on Evan blue leakage or the expression ZO-1, Occludin. Conclusion: Our experimental findings demonstrate Storax treatment inhibits caveolae-mediated transcytosis at BBB in the focal stroke model of rats. We also speculate that regulation of Cav-1, Mfsd2a, AQP4, and PDGFR-β expressions might be associated with its beneficial pharmacological effect, but remain to define and elucidate in future investigation.

Maresin 1 improves cognitive decline and ameliorates inflammation and blood-brain barrier damage in rats with chronic cerebral hypoperfusion

BackgroundChronic inflammation and blood–brain barrier destruction are interrelated pathological changes in chronic cerebral hypoperfusion (CCH) that promote vascular cognitive impairment (VCI). Therefore, we discussed the impact of the macrophage mediator in resolving inflammation 1 (Maresin 1) on the CCH-induced cognitive impairment and its underlying mechanisms. Methods66 rats were randomly divided into three groups: Sham (n = 22), 2VO (n = 22), and 2VO + MaR1 (n = 22). Rats in three groups received 2-Vessel Occlusion (2VO) or sham operation and received intrathecal delivery of PBS or MaR1. Hippocampal blood flow and Modified neurological severity scores (mNSS) were used to confirm models’ effect. Blood-brain barrier (BBB) damage was assessed by Evans blue (EB) leakage experiments and spectrophotometry, the BBB ultrastructure was observed with a transmission electron microscope (TEM), and the expression of zonula occluden-1 (ZO-1), claudin-5, and matrix metalloproteinases-9 (MMP-9) were detected with Enzyme-Linked Immunosorbent Assay (ELISA). Morris water maze (MWM) was used to assess cognitive function. Tumor necrosis factor (TNF-α), interleukin-1β (IL-1β), and nuclear factor-κB (NF-κB) expression were examined by Western blotting (WB) and ELISA. Immunofluorescence was used to detect microglia, astrocytes and oligodendrocytes. ResultsRats developed obvious cognitive impairment by CCH. BBB showed EB leakage, ultrastructural destruction, degradation of ZO-1, Claudin-5, and up-regulation of MMP-9. Inactivation of oligodendrocytes, activation of microglia and astrocyte and increased expression of NF-κB, TNF-α, and IL-1β has been detected. MaR1 administration significantly reverted these changes. ConclusionMaR1 can improve the CCH-induced cognitive impairment. Inflammatory resolution and BBB protection may be the mechanism of MaR1 to prevent CCH-induced cognitive impairment.

Neurocapillary-Modulation

ObjectivesWe consider two consequences of brain capillary ultrastructure in neuromodulation. First, blood-brain barrier (BBB) polarization as a consequence of current crossing between interstitial space and the blood. Second, interstitial current flow distortion around capillaries impacting neuronal stimulation. Materials and MethodsWe developed computational models of BBB ultrastructure morphologies to first assess electric field amplification at the BBB (principle 1) and neuron polarization amplification by the presence of capillaries (principle 2). We adapt neuron cable theory to develop an analytical solution for maximum BBB polarization sensitivity. ResultsElectrical current crosses between the brain parenchyma (interstitial space) and capillaries, producing BBB electric fields (EBBB) that are >400x of the average parenchyma electric field (ĒBRAIN), which in turn modulates transport across the BBB. Specifically, for a BBB space constant (λBBB) and wall thickness (dth-BBB), the analytical solution for maximal BBB electric field (EABBB) is given as: (ĒBRAIN × λBBB)/dth-BBB. Electrical current in the brain parenchyma is distorted around brain capillaries, amplifying neuronal polarization. Specifically, capillary ultrastructure produces ∼50% modulation of the ĒBRAIN over the ∼40 μm inter-capillary distance. The divergence of EBRAIN (Activating function) is thus ∼100 kV/m2 per unit ĒBRAIN. ConclusionsBBB stimulation by principle 1 suggests novel therapeutic strategies such as boosting metabolic capacity or interstitial fluid clearance. Whereas the spatial profile of EBRAIN is traditionally assumed to depend only on macroscopic anatomy, principle 2 suggests a central role for local capillary ultrastructure—which impact forms of neuromodulation including deep brain stimulation (DBS), spinal cord stimulation (SCS), transcranial magnetic stimulation (TMS), electroconvulsive therapy (ECT), and transcranial electrical stimulation (tES)/transcranial direct current stimulation (tDCS).

Synergic Neuroprotection Between Ligusticum Chuanxiong Hort and Borneol Against Ischemic Stroke by Neurogenesis via Modulating Reactive Astrogliosis and Maintaining the Blood–Brain Barrier

Background: Ligusticum chuanxiong Hort (LCH) is a famous ethnomedicine in Asia known for its excellent output on stroke treatment, and borneol usually acts as an assistant for its reducing permeability of the blood–brain barrier (BBB) after stroke. Although their synergy against brain ischemia was verified in previous studies, the potential mechanism is still unknown. Methods: The research aimed to explore the exact synergic mechanisms between LCH and borneol on neurogenesis within the areas of the dentate gyrus and subventricular zone. After treating middle cerebral artery occlusion rats with LCH (0.1 g/kg) and/or borneol (0.08 g/kg), the neurological severity score, brain infarct ratio, Nissl staining, Evans blue permeability, BBB ultrastructure, and expressions of von Willebrand factor and tight junction–associated proteins were measured. Co-localizations of Nestin+/BrdU+ and doublecortin+/BrdU+, and expressions of neuronal nuclei (NeuN) and glial fibrillary acidic protein (GFAP) were observed under a fluorescence microscope. Moreover, astrocyte polarization markers of complement component 3 and pentraxin 3, and relevant neurotrophins were also detected by immunoblotting. Results: Basically, LCH and borneol had different focuses, although both of them decreased infarct areas, and increased quantity of Nissl bodies and expression of brain-derived neurotrophic factor. LCH increased the neurological severity score, NeuN+ cells, and the ratios of Nestin+/BrdU+ and doublecortin+/BrdU+, and decreased GFAP+ cells and ciliary neurotrophic factor expression. Additionally, it regulated the expressions of complement component 3 and pentraxin 3 to transform astrocyte phenotypes. Borneol improved BBB ultrastructure and increased the expressions of von Willebrand factor, tight junction–associated proteins, vascular endothelial growth factor, and vascular endothelial growth factor receptor 2. Unexpectedly, their combined therapy showed more obvious regulations on the Nissl score, Evans blue permeability, doublecortin+/BrdU+, NeuN+ cells, brain-derived neurotrophic factor, and vascular endothelial growth factor than both of their monotherapies. Conclusions: The results indicated that LCH and borneol were complementary to each other in attenuating brain ischemia by and large. LCH mainly promoted neural stem cell proliferation, neurogenesis, and mature neuron preservation, which was probably related to the transformation of reactive astrocytes from A1 subtype to A2, while borneol preferred to maintain the integrity of the BBB, which provided neurogenesis with a homeostatic environment.

Damage to the Blood Brain Barrier Structure and Function from Nano Titanium Dioxide Exposure Involves the Destruction of Key Tight Junction Proteins in the Mouse Brain.

Numerous studies have proven that nano titanium dioxide (nano TiO₂) can accumulate in animal brains, where it damages the blood brain barrier (BBB); however, whether this process involves destruction of tight junction proteins in the mouse brain has not been adequately investigated. In this study, mice were exposed to nano TiO₂ for 30 consecutive days, and then we used transmission electron microscopy to observe the BBB ultrastructure and the Evans blue assay to evaluate the permeability of the BBB. Our data suggested that nano TiO₂ damaged the BBB ultrastructure and increased BBB permeability. Furthermore, we used immunofluorescence and Western blotting to examine the expression of key tight junction proteins, including Occludin, ZO-1, and Claudin-5 in the mouse brain. Our data showed that nano TiO₂ reduced Occludin, ZO-1 and Claudin-5 expression. Taken together, nano TiO₂-induced damage to the BBB structure and function may involve the destruction of key tight junction proteins.

Rac1/Wave2/Arp3 Pathway Mediates Rat Blood-Brain Barrier Dysfunction under Simulated Microgravity Based on Proteomics Strategy.

The blood-brain barrier (BBB) is critical to maintaining central nervous system (CNS) homeostasis. However, the effects of microgravity (MG) on the BBB remain unclear. This study aimed to investigate the influence of simulated MG (SMG) on the BBB and explore its potential mechanism using a proteomic approach. Rats were tail-suspended to simulate MG for 21 days. SMG could disrupt the BBB, including increased oxidative stress levels, proinflammatory cytokine levels, and permeability, damaged BBB ultrastructure, and downregulated tight junctions (TJs) and adherens junctions (AJs) protein expression in the rat brain. A total of 554 differentially expressed proteins (DEPs) induced by SMG were determined based on the label-free quantitative proteomic strategy. The bioinformatics analysis suggested that DEPs were mainly enriched in regulating the cell–cell junction and cell–extracellular matrix biological pathways. The inhibited Ras-related C3 botulinum toxin substrate 1 (Rac1)/Wiskott–Aldrich syndrome protein family verprolin-homologous protein 2 (Wave2)/actin-related protein 3 (Arp3) pathway and the decreased ratio of filamentous actin (F-actin) to globular actin contributed to BBB dysfunction induced by SMG. In the human brain microvascular endothelial cell (HBMECs), SMG increased the oxidative stress levels and proinflammatory cytokine levels, promoted apoptosis, and arrested the cell cycle phase. Expression of TJs and AJs proteins were downregulated and the distribution of F-actin was altered in SMG-treated HBMECs. The key role of the Rac1/Wave2/Arp3 pathway in BBB dysfunction was confirmed in HBMECs with a specific Rac1 agonist. This study demonstrated that SMG induced BBB dysfunction and revealed that Rac1/Wave2/Arp3 could be a potential signaling pathway responsible for BBB disruption under SMG. These results might shed a novel light on maintaining astronaut CNS homeostasis during space travel.

Blood-brain barrier permeability towards small and large tracers in a mouse model of osmotic demyelination syndrome

During osmotic demyelination syndrome (ODS), myelin and oligodendrocyte are lost according to specific patterns in centro- or extra-pontine regions. In both experimental model of ODS and human cases, brain lesions are locally correlated with the disruption of the blood brain-barrier (BBB). The initiation, the degree and the duration of blood-brain barrier (BBB) opening as well as its contribution to brain damages are still a matter of debate. Using a panel of intravascular tracers from low- to high- molecular weight (from 0.45 kDa 150 kDa), we have assessed the BBB permeability at different timings of ODS induced experimentally in mice. ODS was mimicked according to a protocol of rapid correction of a chronic hyponatremia. We demonstrated that BBB leakage towards smallest tracers Lucifer Yellow (0.45 kDa) and Texas Red-dextran (3 kDa) was delayed by 36 h compared to the first clues of oligodendrocyte loss (occurring 12 h post-correction of hyponatremia). At 48 h post-correction and concomitantly to myelin loss, BBB was massively disrupted as attested by accumulation of Evans Blue (69 kDa) and IgG (150 kDa) in brain parenchyma. Analysis of BBB ultrastructure verified that brain endothelial cells had minimal alterations during chronic hyponatremia and at 12 h post-correction of hyponatremia. However, brain endothelium yielded worsened alterations at 48 h, such as enlarged vesicular to tubular-like cytoplasmic profiles of pinocytosis and/or transcytosis, local basal laminae abnormalities and sub-endothelial cavities. The protein expressions of occludin and claudin-1, involved in inter-endothelial tight junctions, were also downregulated at 48 h post-correction of hyponatremia. Our results revealed that functional BBB opening occured late in pre-established ODS lesions, and therefore was not a primary event initiating oligodendrocyte damages in the mouse model of ODS.

Effect of 3-Aminobenzamide on the Ultrastructure of Astrocytes and Microvessels After Focal Cerebral Ischemia in Rats.

The disruption of blood–brain barrier (BBB) is a critical event in the formation of brain edema during early phases of ischemic brain injury. Poly(ADP-ribose) polymerase (PARP) activation, which contributes to BBB damage, has been reported in ischemia–reperfusion and traumatic brain injury. Here, we investigated the effect of 3-aminobenzamide (3-AB), a PARP-1 inhibitor, on the ultrastructure of BBB. Male Sprague Dawley rats were suffered from 90 minutes of middle cerebral artery occlusion, followed by 4.5 hours or 22.5 hours of reperfusion (R). The vehicle or 3-AB (10 mg/kg) was administered intraperitoneally (ip) 60 minutes after lacking of blood. Tissue Evans Blue (EB) levels, ultrastructures of astrocytes and microvessels, and areas of perivascular edema were examined in penumbra and core, at I 1.5 hours /R 4.5 hours and I 1.5 hours /R 22.5 hours, respectively. The severity of ultrastructural changes was graded with a scoring system in each group. We showed that 3-AB treatment significantly decreased tissue EB levels and ultrastructural scores, attenuated damages in astrocytes and microvessels, and reduced areas of perivascular edema. In conclusion, PARP inhibition may provide a novel therapeutic approach to ischemic brain injury.

Protection of blood-brain barrier as a potential mechanism for enriched environments to improve cognitive impairment caused by chronic cerebral hypoperfusion

BackgroundChronic cerebral hypoperfusion (CCH) is a common pathophysiological basis for Alzheimer’s Disease and vascular dementia in the early stages. It has been confirmed that blood-brain barrier (BBB) destruction is a key factor in CCH-related cognitive impairment. Here we explored the effects of an enriched environment (EE) intervention on CCH-induced BBB destruction and cognitive impairment, and the underlying mechanism. MethodsRats in the EE group were exposed to an EE, while the standard environment (SE) group was maintained in a standard cage with bedding but no other objects. On day 14, CCH was induced via permanent bilateral common carotid artery occlusion (2VO). Next, Evans blue (EB) leakage in the hippocampus was measured by chemical colorimetry to dynamically evaluate BBB permeability. On day 28, the BBB ultrastructure was observed using transmission electron microscopy. The expression levels of BBB integrity-related proteins, matrix metalloproteinases-2/-9 (MMP-2/-9), and the classical Wnt/β-catenin signaling pathway-related proteins were detected using western-blotting techniques. On day 43, cognitive function was assessed using the Morris water maze. ResultsAfter 2VO, CCH rats exposed to the SE developed obvious cognitive impairment and BBB destruction. BBB damage was manifested through increased EB leakage, ultrastructural destruction, degradation of BBB integrity-related proteins, and up-regulation of MMP-2/-9. These changes were significantly alleviated after the EE intervention. In addition, EEs activated the Wnt/β-catenin signaling pathway in the hippocampus of rats. ConclusionsThese results suggest that protection of the BBB may be a novel mechanism by which EEs ameliorate CCH-induced cognitive impairment, and this effect may be related to the activation of the Wnt/β-catenin pathway.

Effects of moxibustion on the structure and function of blood brain barrier in Alzheimer's disease model rats

To investigate the effects of moxibustion on the structure and function of blood-brain barrier in Alzheimer's disease (AD) model rats. Forty-eight SD rats were randomly divided into 4 groups: normal control group, sham operation group, model group, moxibustion group. Model group and moxibustion group rats were injected with aggregated Aβ25-35 by bilateral hippocampus. In the rat model, the sham-operated group was injected with the same amount of normal saline in the bilateral hippocampus, and the normal group was not treated. After successful modeling, the moxibustion treatment was given at 2～3 cm above the Baihui, Shenshu and Yintang points of the moxibustion group rats, each time for 10 min, once a day, continuous treatment for 21 d. The Morris water maze test was used to evaluate the learning and memory ability of rats in each group. The Evans blue method was used to detect the permeability of blood-brain barrier. The ultrastructure of blood-brain barrier was observed under electron microscope. The matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase 9 (MMP-9) positive cells in hippocampus were detected by immunohistochemistry. Compared with the sham operation group, the escape latency was significantly increased (P＜0.01), and the space exploration time was decreased (P＜0.01), the learning and memory function in model group was impaired seriously, the Evans blue content in the brain was increased significantly (P＜0.01), the perivascular edema became larger, and the blood-brain barrier structure function was impaired. At the same time, the positive expressions of MMP-2 and MMP-9 in hippocampus were increased significantly (P＜0.01). Compared with model group, the learning and memory ability in moxibustion group rats was enhanced (P＜ 0.05), the content of Evans blue in the brain was decreased (P＜0.05), the degree of perivascular edema was reduced, and the damage of blood-brain barrier was improved. Positive expressions of MMP-2 and MMP-9 in hippocampus were decreased (P＜0.05 or P＜ 0.01). Moxibustion can decrease the expressions of MMP-2 and MMP-9, and reduce the damage of the structure and function of blood-brain barrier, thereby improving the learning and memory ability of AD model rats, and exerting therapeutic effects.

Effect of quercetin pretreatment on permeability of blood-brain barrier in a rat model of global cerebral ischemia-reperfusion

Objective To evaluate the effect of quercetin pretreatment on the permeability of blood-brain barrier in a rat model of global cerebral ischemia-reperfusion (I/R). Methods Sixty-three clean-grade healthy male Sprague-Dawley rats, weighing 300-350 g, aged 4-5 months, were divided into 3 groups (n=21 each) using a random number table method: sham operation group (group S), group I/R and quercetin pretreatment group (group Q). Global cerebral I/R was induced by occlusion of bilateral common carotid arteries combined with hypotension (mean arterial pressure was maintained at 35-45 mmHg) in chloral hydrate-anesthetized rats.Quercetin 25 μmol/kg was injected intraperitoneally twice a day for 3 consecutive days starting from 3 days before establishment of the model in group Q, while the equal volume of normal saline was given instead at the corresponding time points in group S and group I/R, respectively.The animals were sacrificed at 24 h of reperfusion and brains were removed to determine the brain water content, Evans blue (EB) content and expression of occludin protein in cerebral cortex (by Western blot) and to observe the ultrastructure of blood-brain barrier. Results Compared with group S, the brain water content and EB content were significantly increased, the expression of occludin protein was down-regulated (P<0.05), and the injury to ultrastructure of blood-brain barrier was accentuated in I/R and Q groups.Compared with group I/R, the brain water content and EB content were significantly decreased, the expression of occludin protein was up-regulated (P<0.05), and the injury to ultrastructure of blood-brain barrier was significantly attenuated in group Q. Conclusion Quercetin pretreatment can decrease the permeability of blood-brain barrier and attenuate brain edema, and the mechanism may be related to up-regulated expression of occludin protein in a rat model of global cerebral I/R. Key words: Quercetin; Cerebral; Ischemia-reperfusion; Blood-brain barrier

Protective effects of 8-MOP on blood-brain barrier via the Nrf-2/HO-1 pathway in mice model of cerebral infarction.

To explore the effect of 8-MOP on the blood-brain barrier in mice model of cerebral infarction and the underlying mechanism. Middle cerebral artery occlusion (MCAO) model was established to induce permanent cerebral infarction. The neurological function was observed and scored by the modified longa score method after model establishment. Besides, the water content of brain tissue was measured by the standard dry weight method. Evans blue exudation rate was used to evaluate the effect of 8-MOP on the permeability of the blood-brain barrier. Western-blot and quantitative polymerase chain reaction (qPCR) were used to detect the expression of MMP-9, claudin-5, vascular endothelial growth factor (VEGF), as well as the NFE2-related factor 2 (Nrf-2)/hemeoxygenase 1 (HO-1) pathway. 8-MOP could reduce the neurological deficit scores in a dose-dependent manner, thereby reducing cerebral edema. After 8-MOP treatment, the expression of MMP-9 decreased in ischemic brain tissue, whereas the expression of claudin-5, VEGF, and GFAP increased, suggesting that the blood-brain barrier ultrastructure was improved. In addition, the expression of Nrf-2 and HO-1 decreased after the model establishment of cerebral infarction. However, the expression of Nrf-2 and HO-1 increased in ischemic brain tissue after 8-MOP treatment. 8-MOP may protect the blood-brain barrier via the Nrf-2/HO-1 pathway.

Effects of N-butylphthalide on the expressions of ZO-1 and claudin-5 in blood-brain barrier of rats with acute carbon monoxide poisoning

To explore the effects of N-butylphthalide on the expressions of ZO-1 and claudin-5 in blood-brain barrier (BBB) in rats with acute carbon monoxide (CO) poisoning. A total of 144 adult healthy male Sprague-Dawley (SD) rats were randomly divided into normal control group, CO poisoning group, and NBP treatment group, with 48 rats in each group. The acute CO poisoning model was reproduced in hyperbaric oxygen chamber, and all model rats were given hyperbaric oxygen therapy once daily. The rats in the normal control group were free to breathe fresh air. The rats in NBP treatment group were administered orally NBP 60 mg/kg twice a day at 2 hours after poisoning until death. The rats in normal control group and CO poisoning group were treated with equal amount of pure olive oil. Four rats were sacrificed from each group at 1, 3, 7, 14 days after model reproducing, respectively. The changes in ultrastructure of BBB were observed under transmission electron microscope. The expressions of ZO-1 and claudin-5 proteins were determined by immunofluorescence staining and Western Blot. The localization of the two target proteins was observed by immunofluorescence double staining. The correlation between the two proteins was analyzed by linear regression. The ultrastructure of BBB was normal in normal control group, some ZO-1 and a large number of claudin-5 positive cells were observed. The ultrastructure of BBB was seriously injured, ZO-1 and claudin-5 positive cells in brain tissue were significantly decreased, and the expressions of ZO-1 and claudin-5 proteins in brain tissue at 1 day after poisoning in CO poisoning group were significantly lower than those of normal control group (ZO-1 protein: 3.38±0.30 vs. 24.50±5.62, claudin-5 protein: 11.38±0.93 vs. 46.35±6.88, both P < 0.05), and although gradually restored, they were maintained at relatively lower levels until 14 days as compared with those in normal control group (ZO-1 protein: 10.35±0.80 vs. 24.63±3.57, claudin-5 protein: 32.35±3.11 vs. 46.43±7.20, both P < 0.05). NBP treatment could significantly alleviate the ultrastructure injury of BBB induced by acute CO poisoning, the amount of ZO-1 and claudin-5 positive cells in brain tissue were significantly increased, as well as the expressions of ZO-1 and claudin-5 proteins were significantly increased, which were significantly higher than those of CO poisoning group from 1 day and 3 days on, respectively (1-day ZO-1 protein: 7.57±0.69 vs. 3.38±0.30, 3-day claudin-5 protein: 20.46±1.42 vs. 11.43±0.86, both P < 0.05), and which showed an increase tendency with time prolongation. The results of immunofluorescence double staining showed that ZO-1 and claudin-5 proteins could not only coexist in the same cell, but also could be expressed separately in different cells. Linear regression analysis showed the positive correlation between the expressions of ZO-1 and claudin-5 proteins in brain tissue of rats with acute CO poisoning (R2 = 0.917, P = 0.022). NBP could markedly improve the ultrastructure and functional integrity of BBB through up-regulating the expressions of ZO-1 and claudin-5 proteins, and then reduce brain damage caused by CO poisoning.