Protective effect and mechanism of Qishiwei Zhenzhu pills on cerebral ischemia-reperfusion injury via blood-brain barrier and metabonomics.

Stroke is an acute cerebrovascular disease caused by the sudden rupture of cerebral blood vessels or vascular obstruction from brain tissue damage or dysfunction, thereby preventing blood flow into the brain. Cerebral ischemia-reperfusion injury (CI/RI), a common syndrome of ischemic stroke, is a complex pathological process whose physiological mechanism is still unclear. Qishiwei Zhenzhu pills (QSW), a famous Tibetan medicine preparation, has the effect of tranquilizing by heavy settling, dredging channels and activating collaterals, harmonizing Qi and blood, restoring consciousness, and inducing resuscitation. Here, we investigated the protective effect of QSW on CI/RI in rats and its potential mechanism. First, the volatile and liposoluble components in QSW were determined using gas chromatography-mass spectrometry (GCMS). After 24 h of CI/RI, the neuroprotective effect was determined by evaluating the neurological function, cerebral infarction, histopathology, and blood-brain barrier (BBB) function. Immunofluorescence, real-time quantitative PCR (RT-qPCR), and western blot (WB) were used to detect the expression of matrix metalloproteinase 9 (MMP-9), claudin-5, and occludin. Finally, GCMS metabonomics was used to identify different metabolites and analyze metabolic pathways. The results showed that 88 volatile components and 63 liposoluble components were detected in QSW. Following the experimental stroke operation, it was observed that rats administered QSW pretreatment had improved neurological function, reduced infarct volume (P < 0.01), increased Nissl bodies (P < 0.05), improved histopathology, and reduced BBB disruption. Immunofluorescence, RT-qPCR, and WB results showed that MMP-9 level in the brain tissue of the QSW pretreatment group had a decreasing trend and the expression of claudin-5 and occludin had a tendency to increase. Eleven metabolites related to lipid metabolism, fatty acid metabolism, and energy metabolism, were identified via GC-MS metabonomics. Our study shows that QSW preconditioning has a neuroprotective effect on CI/RI; however, its mechanism requires further study.

Protective effect of (Xenical+GSF) against I/R-induced blood brain barrier disruption, ionic edema, lipid deregulation and neuroinflammation.

Ischemic stroke is a leading cause of mortality worldwide that occurs following the reduction or interruption of blood brain supply, characterized by a cascade of early events as oxidative stress and ensuing neuro-inflammation, energy failure and the burst of intracellular Ca++ resulting in activation of phospholipases and large increase in FFA including arachidonic acid, ultimately leading to nervous cell death. Grape Seed Flour (GSF) is a complex polyphenolic mixture harboring antioxidant, anti-inflammatory and neuroprotective properties. Orlistat (Xenical ™,Xe) is a gastro-intestinal lipase inhibitor and an anti-obesity agent. In an earlier study we reported the higher efficiency in neuroprotection against HFD-induced brain lipotoxicity when combining the two drugs (GSF + Xe). As a result repurposing Xe as an adjunct to GSF therapy against stroke appeared relevant and worthy of investigation. I/R insult disrupted the blood brain barrier (BBB) as assessed by EB dye extravasation, increased water and Na+ within the brain. Ultrastructurally I/R altered the brain blood capillaries at the vicinity of hippocampus dentate gyrus area as assessed by transmission and scanning electron microscopy. I/R altered lipid metabolism as revealed by LDL/HDL ratio, lipase activity, and FFA profiles. Moreover, I/R induced neuro-inflammation as assessed by down-regulation of anti-inflammatory CD 56 and up-regulation of pro-inflammatory CD 68 antigen. Importantly almost all I/R-induced disturbances were retrieved partially upon Xe or GSF on their own, and optimally when combining the two drugs. Xe per se is protective against I/R injury and the best neuroprotection was obtained when associating low dosage Xe with high dosage GSF, enabling neuroprevention and cell survival within hippocampus dentate gyrus area as revealed by increased staining of Ki 67 proliferation biomarker.

Transplantation of human bone marrow stem cells into symptomatic ALS mice enhances structural and functional blood-spinal cord barrier repair.

Accumulating evidence shows alterations in the blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB) in ALS patients and in animal models of disease, mainly by endothelial cell (EC) damage. Repair of the altered barrier in the CNS by replacement of ECs via cell transplantation may be a new therapeutic approach for ALS. Recently, we demonstrated positive effects towards BSCB repair by intravenous administration of unmodified human bone marrow CD34+ (hBM34+) cells at different doses into symptomatic ALS mice. However, particular benefits of these transplanted cells on microvascular integrity in symptomatic ALS mice are still unclear. The aim of the present study was to determine the structural and functional spinal cord capillary integrity in symptomatic ALS mice after intravenous administration of hBM34+ cells. The G93A mice at 13 weeks of age intravenously received one of three different cell doses (5 × 104, 5 × 105, or 1 × 106) and were euthanized at 17 weeks of age (4 weeks post-transplant). Control groups were media-treated and non-carrier mutant SOD1 gene mice. Capillary ultrastructural (electron microscopy), immunohistochemical (laminin and HuNu), and histological (myelin and capillary density) analyses were performed in the cervical and lumbar spinal cords. Capillary permeability in the spinal cords was determined by Evans Blue (EB) injection. Results showed significant restoration of ultrastructural capillary morphology, improvement of basement membrane integrity, enhancement of axonal myelin coherence, and stabilization of capillary density in the spinal cords primarily of ALS mice receiving the high dose of 1 × 106 cells. Moreover, substantial reduction of parenchymal EB levels was determined in these mice, confirming our previous results on capillary permeability. Additionally, transplanted cells were detected in blood smears of sacrificed late symptomatic mice by HuNu marker. Altogether, these results provide novel evidence that unmodified bone marrow hematopoietic stem cell treatment at optimal dose might be beneficial for structural and functional repair of the damaged BSCB in advanced stage of ALS, potentially resulting in delayed disease progression by increased motor neuron survival.

Jia-Jian-Di-Huang-Yin-Zi decoction exerts neuroprotective effects on dopaminergic neurons and their microenvironment.

As a classical prescription of Traditional Chinese medicine, the Jia-Jian-Di-Huang-Yin-Zi (JJDHYZ) decoction has long been used to treat movement disorders. The present study evaluated the effects of JJDHYZ on dopaminergic (DA) neurons and their survival-enhancing microenvironment as well as the possible mechanisms involved using a mouse model of Parkinson's disease. In MPTP-lesioned mice, a high dosage of JJDHYZ (34 g/kg/day) attenuated the loss of DA neurons, reversed the dopamine depletion, and improved the expression of glial-derived neurotrophic factor (GDNF) compared to the untreated model group. JJDHYZ also protected the ultrastructure of the blood-brain barrier (BBB) and tight junction proteins by inhibiting the activation of microglia and astrocytes besides the increase in three types of matrix metalloproteinases in the substantia nigra. In conclusion, the JJDHYZ-high dosage (JJDHYZ-H) group exhibited the neuroprotection of DA neurons, and the underlying mechanism may be related to the survival-enhancing microenvironment of the DA neurons.

Corilagin Attenuates Radiation-Induced Brain Injury in Mice.

Cranial irradiation-induced inflammation plays a critical role in the initiation and progression of radiation-induced brain injury (RIBI). Anti-inflammation treatment may provide therapeutic benefits. Corilagin (beta-1-O-galloyl-3, 6-(R)-hexahydroxydiphenoyl-D-glucose, C27H22O18) was a novel member of the tannin family with anti-inflammatory properties and is isolated from some medicinal plants, such as Phyllanthus amarus and Caesalpinia coriaria. In this study, the effect of Corilagin on RIBI was investigated and the underlying mechanisms were explored. Spatial learning and memory ability of mice were investigated by the Morris water maze test. Evans blue leakage and electron microscopy were used to assess the integrity of blood-brain barrier (BBB). The mRNA and protein expressions of inflammatory cytokines, TNF-α and IL-1ß, were measured by using real-time PCR and Western blotting. The activation of microglial cells and expression of TNF-α were examined by immunofluorescence staining. Phosphorylated signal transducers and activators of transcription 3 (p-STAT3) and IκBα, and the translocation of p65 from cytoplasm to nucleus were detected by using Western blotting. Morris water maze test showed that Corilagin ameliorated the neurocognitive deficits in RIBI mice. Evans blue leakage and electron microscopy exhibited that Corilagin partially protected the BBB integrity from cranial irradiation-caused damage; immunofluorescence staining showed that Corilagin could inhibit microglial activation and TNF-α expression. Real-time PCR and Western blotting revealed that Corilagin downregulated the expression of TNF-α and IL-1ß and inhibited the irradiation-induced activation of NF-κB pathways by upregulating p-STAT3 expression. In conclusion, Corilagin could attenuate RIBI through inhibiting microglial activation and the expressions of inflammatory cytokines. Corilagin might inhibit the activation of NF-κB pathway in a STAT3-associated manner, thereby downregulating the inflammatory cytokine expressions.

Remote control of the permeability of the blood-brain barrier by magnetic heating of nanoparticles: A proof of concept for brain drug delivery.

Despite advances in neurology, drug delivery to the brain remains a substantial challenge. This is mainly due to the insurmountable and selective nature of the blood-brain barrier (BBB). In this study, we show that the thermal energy generated by magnetic heating (hyperthermia) of commercially available magnetic nanoparticles (MNPs) in the brain capillaries of rats can transiently increase barrier permeability. Here, the fluorescent Evans Blue (EB) dye was used to verify the BBB integrity. Results indicate a substantial but reversible opening of the BBB where hyperthermia is applied. Also, in this investigation, analysis of CD68 immunoreactivity, an indicator of inflammation, implies that this technique is not associated with any inflammation. We have previously investigated theranostic (therapeutic and diagnostic) capabilities of the MNPs, therefore, the findings presented in this investigation are particularly encouraging for a novel targeted drug delivery system to the brain.

The effects of hyperbaric air and hyperbaric oxygen on blood-brain barrier integrity in rats.

Hyperbaric oxygen (HBO) treatment yields conflicting results on blood-brain barrier (BBB) integrity under various pathological conditions and the effects of HBO on healthy brain is poorly understood. In this experimental study, the effects of HBO on BBB integrity were investigated in comparison with hyperbaric air (HBA) in intact rats. Four sessions of HBA or HBO were applied to intact rats in 24h. BBB integrity was functionally and structurally evaluated by determining extravasation of Evans blue (EB) dye and horseradish peroxidase (HRP) tracers. In immunohistochemical evaluation, relative staining intensity for occludin, a tight junction (TJ) protein, and aquaporin 4 (AQP4), a water-channel protein, was detected in the barrier type of microvessels of brain by image analysis. BBB permeability to EB dye significantly increased in animals in HBO treatment group compared to those in HBA and control groups (p<0.05). The immunoreactivity of occludin, a tight junction protein, remained essentially unaltered in capillaries of hippocampus in all groups. In animals exposed to HBO, AQP4 immunoreactivity significantly increased in parietal cortex compared to those in HBA and control groups (p<0.01). Ultrastructurally, frequent vesicles containing HRP reaction products were observed in capillary endothelial cells in cerebral cortex and hippocampus of rats subjected to both HBA and HBO. Our results indicate that the HBO administration to intact rats increased BBB permeability to both EB and HRP while HBA increased only HRP extravasation in these animals. The results of this study suggest that HBA also impairs the BBB integrity in intact rats as well as HBO.

The mechanism of the opening of the blood-brain barrier by borneol: a pharmacodynamics and pharmacokinetics combination study.

ETHNOPHARMACOLOGICAL RELEVANCE: Borneol is widely used in traditional Chinese medicine to facilitate the distribution of central nervous system (CNS) drugs in brain due to its ability to open blood-brain barrier (BBB), however, the underlying mechanism is still unclear. In this study, the effect of borneol on different brain regions were investigated to explore the mechanism. MATERIALS AND METHODS: After oral administration of borneol (0.1, 0.2 g/kg) for seven consecutive days, SD rats were injected with Rh123 (1.0 mg/kg). The concentrations of Rh123 were detected in four brain regions of cortex, hippocampus, hypothalamus and striatum by a small animal vivo imaging system and a fluorescence microplate reader respectively. The ultrastructures of BBB were examined. Moreover, the expressions of the four transporters of ATP-binding cassette (ABC) family, multidrug resistance 1a (Mdr1a), multidrug resistance 1b (Mdr1b), multidrug resistance protein 1 (Mrp1), Mrp4, Mrp5 and breast cancer resistance protein (Bcrp) in the four brain regions were analyzed. Finally, the deliveries of borneol in the plasma and the four brain regions were examined by a pharmacokinetics study. RESULTS: Administration of 0.2 g/kg borneol produced loose structure in the tight junction and void structure between the endothelial cell and mesangial cell. Borneol at 0.1 g/kg and 0.2 g/kg increased the delivery of Rh123 in hippocampus and hypothalamus obviously. Permeability index followed a similar trend. Protein expression assays showed that borneol decreased the expression of Mdr1 and Mrp1 in hippocampus and hypothalamus. Further RT-PCR study showed that borneol decreased the expressions of both Mdr1a and Mdr1b in hippocampus and hypothalamus. The pharmacokinetics study demonstrated that the delivery of borneol in cortex was the most and that in striatum the least, with the deliveries of borneol in hippocampus and hypothalamus in between. CONCLUSIONS: Borneol showed tissue specific BBB-opening effect, which was associated with its regulation of the ultrastructure of brain tissues and the expressions of Mdr1a, Mdr1b and Mrp1. The present study indicated that borneol should be used in concert with drugs targeting hippocampus or hypothalamus to exert its synergistic effect to the maximum.

[Effects of aromatic resuscitation drugs on blood brain barrier in cerebral ischemia-reperfusion injury model rats].

OBJECTIVE: To research the effects of moschus, borneol, styrax and benzoinum on the structure and function of blood brain barrier in cerebral ischemia-reperfusion injury model rats. METHOD: Focal middle cerebral artery occlusion (MCAO) was introduced as an in vivo ischemic model in rats. After 2 h MCAO, nylon suture was pulled up 1 cm to give blood reperfusion. After 22 h reperfusion, all animals were decapitated. The ultramicrostructure of blood brain barrier of ischemia hemisphere side in fronto-parietal cortex region by transmission electron microscope, and the content of VEGF and MMP-9 in ischemia side brain tissue were measured by ELISA. RESULT: In model and solvent group rats, the capillary endothelium cells, astro-glial cells and nerve cells in ischemia hemisphere side in fronto-parietal region were emerged in different degree compared with sham-operated groups, which exhibited tight junction between endothelial cells being opened, basal lamina being dissolved, and permeability increasing, and cellularedema. In borneol (0.2 g x kg(-1)) group rats, the structure of three kinds of cells were nearly normal, which tight junction structure was clear, rough endoplasmic reticulum and polyribosome could be found in cytoplasm. In moschus (66.6 mg x kg(-1)) group rats, the structure of capillary endothelium cells and astrocytes were nearly normal as well as the basal lamina, but the electrons in neurons was maldistribution. In styrax (1.332 g x kg(-1)) group rats, astrocytes were nearly normal, while capillary endothelial cells and neurons exhibited oedema in different degrees. And the basal lamina was discontinuous, augmentation of cell spaces in endothelial cells increased the permeability, some endoplasmic reticulum broadened and ribosome ablated. In benzoinum (1.0 g x kg(-1)) group rats, oedema of capillary endothelial cells and astrocytes was significant, basal lamina broke. Meanwhile endoplasmic reticulum broadened as vacuole, the number of ribosome in rough endoplasmic reticulum decreased, crista mitochondriales in some neurons disappeared as vacuole which hint oedema happened. Results also showed that borneol decrease the level of VEGF in ischemia side brain tissue significantly, while has little influence on the level of MMP-9. Moschus showed the tendency to decrease the level of VEGF and MMP-9 in ischemia side brain tissue. CONCLUSION: Aromatic resuscitation drugs showed the protection effect on blood brain barrier in cerebral ischemia-reperfusion injury rats, which the protection effect of moschus and borneol were better than that of styrax and benzoinum. The mechanism of protection effect maybe related to decrease the level of VEGF and MMP-9.

The design of barriers in the hypothalamus allows the median eminence and the arcuate nucleus to enjoy private milieus: the former opens to the portal blood and the latter to the cerebrospinal fluid.

The blood-brain barrier (BBB) is a single uninterrupted barrier that in the brain capillaries is located at the endothelial cells and in the circumventricular organs, such as the choroid plexuses (CP) and median eminence (ME), is displaced to specialized ependymal cells. How do hypothalamic hormones reach the portal circulation without making the BBB leaky? The ME milieu is open to the portal vessels, while it is closed to the cerebrospinal fluid (CSF) and to the arcuate nucleus. The cell body and most of the axons of neurons projecting to the ME are localized in areas protected by the BBB, while the axon terminals are localized in the BBB-free area of the ME. This design implies a complex organization of the intercellular space of the median basal hypothalamus. The privacy of the ME milieu implies that those neurons projecting to this area would not be under the influence of compounds leaking from the portal capillaries, unless receptors for such compounds are located at the axon terminal. Amazingly, the arcuate nucleus also has its private milieu that is closed to all adjacent neural structures and open to the infundibular recess. The absence of multiciliated cells in this recess should result in a slow CSF flow at this level. This whole arrangement should facilitate the arrival of CSF signal to the arcuate nucleus. This review will show how peripheral hormones can reach hypothalamic targets without making the BBB leaky.

Protein components of the blood-brain barrier (BBB) in the mediobasal hypothalamus.

The blood-brain barrier (BBB) plays an important role in controlling the access of substances to the brain. Of the circumventricular organs (CVO), i.e. areas that lack a BBB, the median eminence and its close relationship with the hypothalamic arcuate nucleus plays an important role in controlling the entry of blood-borne substances to neurons of the mediobasal hypothalamus. In order to clarify the nature of the BBB in the median eminence-arcuate nucleus complex, we have used immunohistochemistry and antisera to protein components of the BBB-(1) tight junctions, claudin-5 and zona occludens-1 (ZO-1); (2) endothelial cells: (a) all endothelial cells: rat endothelial cell antigen-1 (RECA-1), (b) endothelial cells at BBB: endothelial barrier antigen (EBA), glucose transporter 1 (GLUT1) and transferrin receptor (TfR), and (c) endothelial cells at CVOs: dysferlin; (3) basal lamina: laminin; (4) vascular smooth muscle cells: smooth muscle actin (SMA); (5) pericytes: chondroitin sulfate proteoglycan (NG2); (6) glial cells: (a) astrocytes: glial fibrillary acidic protein (GFAP), (b) tanycytes: dopamine- and cAMP-regulated phosphoprotein of 32kDA (DARPP-32), (c) microglia: CD11b. Neuronal cell bodies located in the ventromedial aspect of the arcuate nucleus were visualized by antiserum to agouti-related protein (AgRP). The study provides a detailed analysis on the cellular localization of BBB components in the mediobasal hypothalamus. Some vessels in the ventromedial aspect of the arcuate nucleus lacked the BBB markers EBA and TfR, suggesting an absence of an intact BBB. These vessels may represent a route of entry for circulating substances to a subpopulation of arcuate nucleus neurons.

[Effects of paeoniflorin on pathological changes in global brain ischemia model rats].

OBJECTIVE: To explore the effects of paeoniflorin on blood brain barrier and pathological changes in brain ischemia. METHOD: Mice were divided into sham operation group, model group, positive control group-Deng zhanhua tablet group and three different dose (high, middle, low-dose) groups of paeoniflorin. The neurological symptoms of rats were observed. The SOD of ischemic brain tissue, MDA BBB and EAA contents were determined. The ultrastructure on the brain cells was inspected by transmission electron microscope. RESULT: Paeoniflorin had the protetive effects on 4VO model of total cerebral ischemia. Paeoniflorin could obviously increase SOD content, reduce MDA content in rat brain-tissue and alleviate oxidative stress damage by cerebral ischemia on rat brain. Paeoniflorin could improve pathological changes of cell nuclear, perikaryon, mitochondria and myelin sheath, which was the morphologic basis of the protective effect on ischemia. Paeoniflorin could alleviate the incrense of EAA content caused by and hypoxia and inhibit the excitatory neural toxicity by EAA. CONCLUSION: Paeoniflorin has the protection effect on the brain edema after cerebral ischemia, the oxidative stress damage on brain tissue, the ultrastructure lesions of cells and the BBB. The protective mechanism may be related to inhibiting intracellular calcium overload, anti-free radical and reducing EAA content.

Effects of excess vitamin B6 intake on cerebral cortex neurons in rat: an ultrastructural study.

The aim of this study was to investigate whether excess of vitamin B6 leads to ultrastructural changes in cerebral cortex of forty-eight healthy albino rats which were included in the study. Saline solution was injected to to the control groups (CG-10, n = 12 for 10 days; CG-15, n = 12 for 15 days; CG-20, n=12 for 20 days). The three experimental groups (EG-10, n = 12; EG-15, n = 12; EG-20, n = 12) were treated with 5 mg/kg vitamin B6 daily for 10 days (EG-10), 15 days (EG-15) and 20 days (EG-20). Brain tissues were prepared by glutaraldehyde-osmium tetroxide double fixation for ultrastructural analysis. No significant changes were observed in the control groups. The ultrastructural analysis revealed that the numbers of damaged mitochondria, lipofuscin granules and vacuoles were significantly higher in all the experimental groups than in the control groups (p < 0.05). However, synaptic density was significantly decreased in the experimental groups as compared to the control groups (p < 0.05). The results suggest that the excess of vitamin B6 intake causes damage to the cerebral cortex due to cellular intoxication and decreased synaptic density. Thus, careful attention should be paid to the time and dose of vitamin B6 recommended for patients who are supplemented with this vitamin.