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Pinocembrin attenuates blood–brain barrier injury induced by global cerebral ischemia–reperfusion in rats
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Pinocembrin attenuates blood–brain barrier injury induced by global cerebral ischemia–reperfusion in rats Fanrui Menga,b , Rui Liua , Mei Gaoa , Yuehua Wanga , Xiaoyan Yua , Zhaohong Xuana,b , Jialin Suna , Fan Yanga , Chunfu Wub,⁎, Guanhua Dua,⁎⁎ a
National Center for Pharmaceutical Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, P.R. China b Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang 110016, P.R. China
A R T I C LE I N FO
AB S T R A C T
Article history:
Blood–brain barrier (BBB) disruption is a major consequence of cerebral ischemia/
Accepted 3 March 2011
reperfusion. Several studies have reported the neuroprotection of pinocembrin on
Available online 22 March 2011
cerebral ischemia in vivo and in vitro, but the effects of pinocembrin on BBB and its underlying mechanisms are not clear. In this study, we investigated the effects of
Keywords:
pinocembrin on BBB functions in the global cerebral ischemia/reperfusion (GCI/R) model
Pinocembrin
in rats. Neurological scores and brain edema were evaluated. BBB permeability was assessed
Global cerebral ischemia
by detecting the concentrations of Evan's blue (EB) and fluorescein sodium (NaF) in brain
Reperfusion
tissue. The pathological changes of BBB ultrastructure were observed by transmission
Blood–brain barrier
electron microscopy. Cerebral blood flow (CBF) was measured by laser Doppler flowmetery. The effects of pinocembrin on primary cultured rat cerebral microvascular endothelial cells (RCMECs) against oxygen–glucose deprivation/reoxygenation (OGD/R) were also investigated. The results showed pinocembrin decreased neurological score and lessened brain edema induced by GCI/R. Pinocembrin also reduced the concentrations of EB and NaF in brain tissue of the GCI/R rats. And pinocembrin alleviated the ultrastructural changes of cerebral microvessels, astrocyte end-feet and neurons, and improved CBF in the GCI/R rats. In addition, pinocembrin increased the viability and mitochondrial membrane potential of cultured RCMECs induced by OGD/R. In conclusion, these data demonstrate that pinocembrin alleviates blood–brain barrier injury induced by GCI/R in rats. © 2011 Elsevier B.V. All rights reserved.
⁎ Correspondence to: C.F. Wu, Department of Pharmacology, Shenyang Pharmaceutical University, No. 103 Wenhua Road, Shenyang 110016, P.R. China. Fax: + 86 24 23843567. ⁎⁎ Correspondence to: G.H. Du, National Center for Pharmaceutical Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 2 Nanwei Road, Beijing 100050, P.R. China. Fax: +86 10 63165184. E-mail addresses: [email protected] (C.F. Wu), [email protected] (G.H. Du). Abbreviations: GCI/R, global cerebral ischemia/reperfusion; OGD/R, oxygen–glucose deprivation/reoxygenation; RCMECs, rat cerebral microvascular endothelial cells; EB, Evan's blue; NaF, fluorescein sodium; BBB, blood–brain barrier; CBF, cerebral blood flow; MBP, mean blood pressure; △ ψm, mitochondrial membrane potential 0006-8993/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2011.03.010
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1.
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Introduction
In clinical emergencies, many accidents result in global ischemia, such as drowning, electric shock and cardiac arrest. Brain tissue is vulnerable to hypoxia, leading to irreversible injury in a short period of ischemia (Lee et al., 2000). Reperfusion has the potential consequences to aggravate the injury in cerebral ischemia, such as reactive oxygen species overproduction, calcium overload (Kuroda and Siesjö, 1997; Nakamura et al., 1999) and blood–brain barrier (BBB) injury (Abbott et al., 2010). BBB consists of cerebral microvascular endothelial cells and tight joint, the abluminal membrane, and the astrocyte endfeet which ensheathe cerebral capillaries. BBB prevents many macromolecules from entering the brain. Plasma proteins such as albumin, pro-thrombin and plasminogen are deleterious to brain tissue, leading to glial cell activation and neuron apoptosis (Abbott et al., 2010). The injury of any BBB component leads to the increase of the permeability. The permeability of BBB is often evaluated by the value of Evan's blue (EB) and fluorescein sodium (NaF) in brain tissue (Lenzsér et al., 2005). Pinocembrin (5, 7-dihydroxyflavanone) is a flavonoid abundant in propolis. Our previous studies showed that pinocembrin reduced glutamate-induced SH-SY5Y cell injury and primary cultured cortical neuron damage in oxygen–glucose deprivation/reoxygenation (OGD/R) (Gao et al., 2008a; Liu et al., 2008). And pinocembrin alleviates cerebral ischemic injury in the middle cerebral artery occlusion rats (Gao et al., 2008b, 2010; Liu et al., 2008). Pinocembrin also improved cognition by protecting cerebral mitochondria structure and function against chronic cerebral hypoperfusion in rats (Guang and Du, 2006). Furthermore, pinocembrin alleviates brain injury in the global cerebral ischemia/reperfusion (GCI/R) rats (Shi et al., 2011). In the present study, GCI/R was induced by four-vessel occlusion (4-VO) in rats and the neurological scores were evaluated referring to previous reports (Xu et al., 2005; Shi et al., 2011). The aim was to determine the effects of pinocembrin on BBB injury in the GCI/R rats. We tested the hypothesis that pinocembrin would reduce BBB permeability and alleviate the pathological changes of BBB ultrastructure in the GCI/R rats. Pinocembrin could protect the rat cerebral microvascular endothelial cells (RCMECs) against OGD/R injury. In addition, we also determined whether pinocembrin had effects on cerebral blood flow (CBF) in the GCI/R rats.
2.
Results
2.1. Effects of pinocembrin on neurological scores in the GCI/R rats The rats were scored twice immediately after reperfusion and at 24 h after reperfusion. As shown in Fig. 1, the scores of the sham rats were zero, which showed no neurological deficits, while the scores increased in other groups after reperfusion (P < 0.001, Fig. 1A). The scores of the GCI/R group at 24 h after reperfusion increased in comparison with the sham group (P < 0.001), which decreased in pinocembrin 5 mg/kg group (P < 0.05, Fig. 1B). The data indicated pinocembrin lessened the neurological symptom in the GCI/R rats.
2.2.
Pinocembrin alleviated brain edema in the GCI/R rats
As shown in Table 1, the GCI/R rats showed severe brain edema compared with the sham rats (P < 0.01). But brain edema was reduced significantly in pinocembrin 1 and 5 mg/ kg groups (P < 0.05).
2.3. GCI/R
Pinocembrin decreased BBB permeability induced by
In cortical tissue, little EB was detected in the sham rats (Fig. 2A). The amount of EB increased dramatically in the GCI/R rats (P < 0.01), and reduced in all groups administrated pinocembrin (P < 0.05 or P < 0.01). The amount of NaF in the GCI/R group increased compared with that of the sham group (P < 0.01), and decreased in the groups treated with pinocembrin (P < 0.05 or P < 0.01) (Fig. 2B). The data indicated that the permeability of BBB in the GCI/R rats increased. And the permeability was alleviated in the GCI/R rats treated with pinocembrin.
2.4. Pinocembrin protected BBB ultrastructure against GCI/ R injury The cortical microvessels in the sham rats have normal endothelial cells, basal lamina and astrocyte end-feet. A series of pathological changes of BBB were observed in the GCI/R rats after 24 h reperfusion: the astrocyte end-feet were markedly swollen and end-feet membrane was disrupted; the nucleoli
Fig. 1 – Pinocembrin decreased neurological scores in the global cerebral ischemia/reperfusion (GCI/R) rats. (A) Neurological scores immediately after reperfusion. (B) Neurological scores after 24 h reperfusion. Data were shown as means ± SEM. ### P < 0.001 vs. sham group; *P < 0.05 vs. GCI/R group, n = 8–9.
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Table 1 – Pinocembrin alleviated brain edema in the global cerebral ischemia/reperfusion (GCI/R) rats. Sham
Brain edema (%)
79.53 ± 0.14
Data were shown as means ± SEM.
##
GCI/R
Pinocembrin
##
80.28 ± 0.16
1 mg/kg
5 mg/kg
10 mg/kg
79.80 ± 0.13⁎
79.74 ± 0.19⁎
80.02 ± 0.10
P < 0.01 vs. sham group; ⁎P < 0.05 vs. GCI/R group, n = 8.
of neurons condensed and electron density of neurons increased; the glial cells were swollen. Pinocembrin 5 mg/kg decreased the edema of astrocyte end-feet and glial cells, and also alleviated the injury of neurons, Fig. 3 (1). And the edema of astrocyte end-feet significantly decreased in the ischemic group given pinocembrin 5 mg/kg (P < 0.05), Fig. 3 (2).
2.5. Pinocembrin enhanced CBF in the GCI/R rats after reperfusion CBF and MBP value were measured in the sham and GCI/R rats. CBF value was calculated by the ratio with basal CBF value in each rat. The CBF and MBP had no significant changes in the sham rats administrated pinocembrin or vehicle 5 mg/kg (Fig. 4). But the CBF was improved in dose-dependent manner in the GCI/R rats treated with pinocembrin 1, 5, and 10 mg/kg after reperfusion (Fig. 5A). MBP decreased during global ischemic period and restored normal level after reperfusion. MBP had no significant changes in GCI/R rats administrated pinocembrin 1, 5, and 10 mg/kg during reperfusion (Fig. 5B).
2.6. Pinocembrin protected the cultured RCMECs against OGD/R induced injury 2.6.1. Effects of pinocembrin on the viability of the RCMECs in OGD/R As shown in Fig. 6, the viability of the RCMECs significantly decreased (55.91 ± 2.22%) after being exposed to OGD/R. The survival rates increased to 72.47 ± 2.11% and 77.05 ± 2.78% due to the administration of pinocembrin at 106 and 105 M through the reperfusion phase, respectively.
2.6.2.
Effects of pinocembrin on △ ψm of the RCMECs
In the normal RCMECs, most JC-1 formed J-aggregates and showed red fluorescence at 525/590 nm. In damaged RCMECs, most JC-1 became monomer in mitochondrial matrix because △ ψm decreased, and showed green fluorescence at 490/ 530 nm. Under laser confocal scanning microscope, we merged both the JC-1 pictures in 525/590 nm and 490/530 nm in the same field to acquire the pictures of △ψm (Fig. 7A–E). The control RCMECs mainly showed red fluorescence (Fig. 7A). In OGD/R group green fluorescence increased and red fluorescence lessened (Fig. 7B), which was reversed in pinocembrin 0.1, 1.0 and 10.0 μM groups (Fig. 7C–E). The value of red fluorescence and green fluorescence was also detected in the 96 well plates. In the OGD/R cells, the ratio of red and green fluorescent intensity decreased (P < 0.001). The ratio increased in the groups treated with pinocembrin 0.1 μM (P < 0.05), 1.0 μM (P < 0.05) and 10.0 μM (P < 0.01), shown as Fig. 7F.
3.
Discussion
The present study showed pinocembrin decreased the neurological scores, alleviated brain edema, reduced the permeability of BBB and mitigated the ultrastructural changes of ‘neurovascular unit’ in the GCI/R rats. Pinocembrin also improved CBF in the GCI/R rats. In addition, pinocembrin protected the RCMECs against OGD/R injury. Brain injury following focal or global cerebral ischemia develops from a complex series of pathophysiological changes (Dirnagl et al., 1999; Lenzsér et al., 2005). Brain edema, including intracellular and vasogenic edema, exacerbates brain damage
Fig. 2 – Pinocembrin decreased the concentrations of Evan's blue (EB) and fluorescein sodium (NaF) in brain tissue of the global cerebral ischemia/reperfusion (GCI/R) rats. (A) Concentration of EB in brain tissue. (B) Concentration of NaF in brain tissue. Data were shown as mean ± SEM. ##P < 0.01 vs. sham group; *P < 0.05; **P < 0.01 vs. GCI/R group, n = 8.
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Fig. 3 – (1) Pinocembrin alleviated the pathological changes of ultrastructure in microvessels, neurons and glial cells in the global cerebral ischemia/reperfusion (GCI/R) rats. Representative electron photomicrographs were shown. A, B and C showed the microvessels in parietal cortex, ×2.0 k. (A) Sham group, with normal microvascular endothelial cells, basal lamina (a) and astrocyte end-feet (b). (B) GCI/R group, microvascular lumina was crushed and became narrow (c), end-feet of astrocyte were serous edema (d), and the membrane of end-foot was disrupted (e). (C) Pinocembrin 5 mg/kg group, the edema of end-feet were mitigated (f). D, E and F showed the neurons in parietal cortex, ×0.7 k. (D) Sham group. (E) GCI/R group, nucleolus condensed and electron density of the neuron increased. (F) Pinocembrin 5 mg/kg group, the injury was lessened. G, H and I showed the glial cells in parietal cortex, G ×1.0 k, H ×0.7 k, I ×1.2 k. (G) Sham group. (H) GCI/R group, the glial cell was serous edema (g). (I) Pinocembrin 5 mg/kg group, the edema of glial cell was lessened (h). (2) Pinocembrin alleviated the edema of astrocyte endfeet in the GCI/R rats. #P < 0.05 vs. sham group; *P < 0.05 vs. GCI/R group, n = 4.
after cerebral ischemia (Kondo et al., 1997, Abbott et al., 2010). And brain edema increased intracranial pressure which is life threatening. In the study, the GCI/R rats had serious brain edema, which was alleviated in the rats treated with pinocembrin. Neurological scores showed the degree of neurological
deficits (Shi et al., 2011; Zhang et al., 2009). The scores increased in the GCI/R rats, and pinocembrin 5 mg/kg decreased the scores. BBB serves as a dynamic regulator between brain tissue and peripheral circulation. Following hypoxia and reoxygenation,
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Fig. 4 – Effects of pinocembrin on cerebral blood flow (CBF) and mean blood pressure (MBP) in the sham rats. Data were shown as mean ± SEM, n = 6.
BBB is disrupted, leading to the increase of BBB permeability and the occurrence of vasogenic brain edema (Sandoval and Witt, 2008). And brain injury following ischemia and reperfusion is highly associated with inflammation response, involving in the extravasation of mononuclear phagocyte and the activation of astrocytes and microglial cells (Muir et al., 2007). Extravasation of albumin and other macromolecules proteins into brain tissue is correlated with the development of vasogenic edema (Abbott et al., 2010). Middle cerebral artery occlusion in rats also causes brain edema and BBB disruption (Gao et al., 2010; Masada et al., 2001). And neuron injury is associated with microvascular damage (Abbott et al., 2010; del Zoppo, 2009). EB and NaF are the dye that hardly passes through BBB in the normal rats, while they may arrive at brain tissue for the injury of BBB (Gao et al., 2010; Lenzsér et al., 2005). Therefore, EB and NaF are often used to study the permeability of BBB. In the study, the concentration of EB and NaF in brain tissue increased in the GCI/R rats, but decreased in the rats treated with pinocembrin. Similar results were obtained in previous study (Lenzsér et al., 2005). Therefore, pinocembrin decreased the permeability of BBB and alleviated vasogenic brain edema against GCI/R injury.
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Fig. 6 – Pinocembrin protected the rat cerebral microvascular endothelial cells (RCMECs) against oxygen–glucose deprivation/reoxygenation (OGD/R) injury. Pinocembrin increased the viability of the RCMECs in OGD/R. Data were shown as mean ± SEM. ##P < 0.001 vs. control group; **P < 0.001 vs. OGD/R group, n = 5.
Microvessels and neurons respond rapidly to ischemic injury (del Zoppo, 2010). The modulation of CBF by neurons bases on neurovascular coupling. The presence and participation of glial cells implies the association of microvessels, glial cells, and neurons in a ‘neurovascular unit’, which controls and modulates regional and local cerebral blood flow (del Zoppo, 2010; Zonta et al., 2003). In a ‘neurovascular unit’, microvessels were associated with neurons by the common astrocytes. The results of transmission electron microscope showed the neuroprotective action of pinocembrin on ‘neurovascular unit’. The neurons of the GCI/R rats showed abnormal morphology: the nucleoli condensed and electron density of neurons increased. However, the injury of neurons was markedly alleviated in the rats treated with pinocembrin 5 mg/kg. And the protective effects of pinocembrin on neurons were reported in the GCI/R rats with Nissl dye Nissl staining (Shi et al., 2011) and in vitro (Liu et al., 2008). Astrocyte end-feet were serous edema, and the membrane of astrocyte end-feet was disrupted. The microvessels were crushed by the swollen astrocyte end-feet and became narrow in the GCI/R rats. These pathological changes severely
Fig. 5 – Effects of pinocembrin on cerebral blood flow and mean blood pressure in the global cerebral ischemia/reperfusion (GCI/R) rats. (A) Brain blood flow. (B) Mean blood pressure. Data were shown as mean ± SEM, n = 6.
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Fig. 7 – Pinocembrin increased mitochondrial membrane potential of rat cerebral microvascular endothelial cells (RCMECs) in oxygen–glucose deprivation/reoxygenation (OGD/R). RCMECs were loaded with JC-1 in dark. (A–E) Representative confocal pictures of the RCMECs were shown. (A) Control group. (B) OGD/R group. (C) Pinocembrin 0.1 μM group. (D) Pinocembrin 1.0 μM group. (E) Pinocembrin 10.0 μM group. (F) Pinocembrin increased the ratio of red and green fluorescent intensity of the RCMECs in OGD/R in 96 well plates. Data were shown as mean ± SEM. ##P < 0.001 vs. control group; *P < 0.05; **P < 0.01 vs. OGD/R group, n = 6.
impaired the function of microcirculation. Pinocembrin alleviated the edema of end-feet and injury of neurons. Therefore, the ‘neurovascular unit’ deficits were alleviated in the GCI/R rats treated with pinocembrin. In the study, effects of pinocembrin on primary cultured RCMECs against OGD/R induced injury were investigated. Pinocembrin enhanced the viability of the RCMECs against OGD/R injury. △ψm of the RCMECs was detected with JC-1. In the normal cells JC-1 forms aggregates. JC-1 becomes monomers because of △ψm decrease in injury cells. △ψm of the RCMECs may be detected by the ratio of red and green fluorescent intensity. The control RCMECs mainly showed red fluorescence and the RCMECs of OGD/R group principally showed green fluorescence. Pinocembrin restored the △ψm of the RCMECs in OGD/R, shown by increasing the ratio. The RCMECs are the important component of BBB and maintain the function of BBB together with the astrocytes (Abbott et al., 2010). Therefore, pinocembrin alleviated the injury of the RCMECs and improved △ψm of the RCMECs in OGD/R. And
pinocembrin could reduce BBB damage and edema formation in the GCI/R rats. Hemodynamic disturbances after reperfusion, such as noreflow, hyperperfusion and hypoperfusion, cause further brain injury due to microcirculatory reperfusion deficits after cerebral ischemia (Wang et al., 2008; Xu et al., 2010). Positron emission tomography (PET) studies in both humans and experimental animals show that hyperperfusion is not a key dangerous factor in cerebral ischemia (Marchal et al., 1999); while the hypoperfusion or no-flow is harmful to the recovery of ischemic brain (Lee et al., 2000; Xu et al., 2010). Hypoperfusion during the period of reperfusion is caused by platelet activation, aggregation and coagulation (Zeller et al., 1999), and may be also because of the vessels contraction induced by the released vasoactive substances (Kis et al., 1999; Monge et al., 2010). The previous studies showed pinocembrin (5– 100 μM) relaxed aortic rings pre-contracted with norepinephrine or KCl by both endothelium-dependent pathway and endothelium-independent pathway (Zhu et al., 2007). In the
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study, pinocembrin 5 mg/kg had no significant effects on CBF in the sham rats, but improved CBF in the GCI/R rats. Therefore, pinocembrin may improve cerebral microcirculation and enhance CBF to alleviate ischemia/reperfusion injury in the GCI/R rats. This might be related to the mechanism that pinocembrin protected the RCMECs against OGD/R injury, alleviated the edema of astrocyte feet, and relaxed the cerebral microvessels contracted by vasoactive substances after GCI/R. In conclusion, pinocembrin protected BBB damage and edema formation induced by GCI/R. The effects of pinocembrin on RCMECs and CBF may be involved in the protective mechanisms against GCI/R injury.
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Medica, Chinese Academy of Medical Sciences) 1, 5 and 10 mg/ kg immediately after reperfusion, respectively. The sham and model group rats (n = 26) were treated intravenously with corresponding volume vehicle.
4.3.
Determination of brain edema
The rats were decapitated at 24 h after reperfusion. Brain tissue was weighed immediately (wet weight) and after dehydrated in 120 °C for 24 h (dry weight), respectively. Brain edema, percentage of water content, was calculated by the formula [(wet weight dry weight)/wet weight] × 100% (Kusaka et al., 2004; Shi et al., 2011).
4.
Experimental procedures
4.4.
4.1.
Surgery
BBB permeability was assessed by the concentration of EB and NaF in brain cortex (Lai et al., 2008; Lenzsér et al., 2005; Park et al., 2010). The animals were anesthetized, administered intravenously EB and NaF (2% solution, 4 ml/kg) for 60 min, and perfused with 0.9% NaCl (1000 ml/kg) at 24 h after reperfusion. The brains were stripped, weighed, and stored in 80 °C till to test. The samples were homogenized with 1 time volume PBS and then added 50% trichloroacetic acid in 10 times volume to precipitate protein. The homogenate was centrifuged for 20 min at 12,000 rpm in 4 °C. For the measurement of EB, 100 μl supernatant was detected at 620/680 nm (excitation/emission wavelength) by microplate spectrophotometer (SAFIRE II, Tecan, Austria). Another homogenate was added 5 M NaOH (1:0.8 v/v) to adjust pH value and detected in 485/535 nm (excitation/emission wavelength). Calculations were based on external standards in the same solvent. The content of the dye was quantified by linear standard curves of EB (0.0763–1.2207 μg/ml) and NaF (0.0191–0.1526 μg/ml), and expressed by per gram tissue.
Adult male SD rats, weight of 300 ± 30 g, were obtained from Vital River Company (Beijing, China). Before the operation, the rats were food-restriction for 4 h and free access to water. All the procedures obeyed institutional guidelines and ethics for animal experiments in Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College. The animal model was induced by four-vessel occlusion method previously described (Lenzsér et al., 2005; Wang et al., 2008; Shi et al., 2011). The rats were anesthetized with 10% chloral hydrate (4 ml/kg). Both common carotid arteries were isolated and embedded with silk threads (3–0). Then both vertebral arteries were occluded by electric coagulation. After 24 h, both common carotid arteries were occluded with bulldog clamps for 20 min. Reflow was achieved by removing the bulldog clamps. The body temperature of the rats was maintained at 37.5 ± 0.5 °C. The sham rats were treated similarly to the ischemic groups without blocking the arteries.
4.5. 4.2.
BBB permeability
Transmission electron microscopy
Neurological scores and experimental groups
Neurological scores were carried out by the scale of total 25 scores (Xu et al., 2005; Shi et al., 2011). In brief, the symptoms of hair roughed up, decreasing movement or bradypragia, enhancement of response to ear-palpating and eyelid ptosis were 1 score, respectively; and splayed-out hind limb, eye misclosure or patency, circling behavior, upwarping head or hunched posture, and hyperspasmia or clonus were 3 scores, respectively; and myasthenia of limbs was 6 scores. The total scores were acquired immediately after reperfusion. The rats which total scores were no less than 10 were severe ischemia, considered as success in the model for the next experiment. And the rats were scored again at 24 h after reperfusion. Neurological scores of the rats which were operated in the first day were calculated. The higher the score showed the higher neurological damage (Shi et al., 2011). A total of 128 rats (including 32 sham rats and 96 ischemic rats) were divided into five experimental groups. The rats in group 1 (n = 32) served as sham group without ischemia. Four other groups were exposed to GCI/R. Groups 3 (n = 22), 4 (n = 26) and 5 (n = 22) were administered intravenously with pinocembrin (synthesized and processed as sterile injection powder at the Department of New Drug Development, Institute of Materia
Transmission electron microscopy was used to observe the effects of pinocembrin on ultrastructural changes of BBB. The sham, GCI/R and pinocembrin 5 mg/kg group rats (four rats a group) were anesthetized, perfused with 0.9% NaCl (1000 ml/ kg), and then reperfused with 4% paraformaldehyde (1000 ml/ kg) at 24 h after reperfusion. The parietal cortex was cut into 1mm3, fixed with 2.5% glutaral for 2 h, and washed three times with PBS, 10 min once. Then the tissue was fixed with 1% osmic acid for 2 h, sequentially, washed with pure water, dehydrated with ethanol, replaced with propylene oxide and resin mixture, embedded in pure resin, and stained with uranyl acetate and lead citrate (Gao et al., 2010; Zheng et al., 2007). The cortical microvessels were observed under H-7650 transmission electron microscope (Hitachi, Japan) and photographed. And the area of astrocyte endfeet around cerebral microvessels (diameter 3–6 μm) was measured with Image Pro-plus 6.0 software.
4.6.
CBF and mean blood pressure (MBP) measurement
After 24 h, the operated rats were anesthetized with pentobarbital sodium (50 mg/kg). The calotte skin was incised in the middle. The right cranial bone between bregma and posterior fontanelle was grinded by the dental drill. The laser probe with
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a holder was attached to the right skull 2 mm caudal to bregma, 4 mm lateral to midline (Noppens et al., 2006; Wang et al., 2008). Then CBF was measured with laser Doppler flowmetery (Perimed, Sweden). And blood pressure was detected by BL-420 S physiological apparatus in femoral artery (Taimeng, China). After recording 10 min basal value of CBF and MBP, bilateral carotid arteries were clamped for 20 min. The rats which CBF decreased less 75% were excluded during ischemic period. Then the rats were administrated vehicle or pinocembrin at the beginning of reperfusion. The CBF and MBP value was recorded continuously for 90 min after the injection. And CBF and MBP value were acquired at every 5 min (CBF and MBP value of 1 min was averaged at each point). CBF value was expressed as percentage relative to baseline value (100%).
4.7.
RCMECs culture
The RCMECs were cultured referring to the previous paper (Liu et al., 2010). Briefly, the brains of 2–3 weeks old rats were stripped and placed in ice-cold D-Hank's solution. The cortices were cut into 1 mm3 sections, homogenated and centrifuged at 1800 rpm. Then deposition was homogenated at 1000 g in 20% BSA. The red deposition was microvascular fragments. The microvascular fragments were digested in 5 times volume 1 mg/ ml type II collagenase for 1.5 h at 37 °C, terminated with complete medium, and centrifuged at 1000 rpm. Then the microvascular fragments were cultured in the medium, including 10% fetal bovine serum, basic fibroblast growth factor (10 ng mL1) and heparin (100 μg mL1) at 37 °C with 95% air and 5% CO2. The cells were used from passage 4 to 6 for the experiment. The cells were identified with von-Willebrand factor antibody (Santa Cruz, USA) and FITC labeled secondary antibody (Santa Cruz, USA). The proportion of the RCMECs was over 95%. The RCMECs in confocal dishes and 96 well plates were randomly divided into 5 groups: control group, OGD/R group, and pinocembrin 0.1 μM, 1.0 μM and 10.0 μM groups. The RCMECs were washed with D-Hank's solution for 3 times, and then the culture medium was replaced with glucose free Earle's balanced salt solution with 100 μM sodium dithionite (Na2S2O4), a deoxygenated reagent, for another 2 h, and subsequently incubated with complete medium at 37 °C for 24 h (Liu et al., 2008). Pinocembrin was administrated at the start of reoxygenation. Control group was treated with Earle's balanced salt solution containing glucose without deoxygenated reagent (Liu et al., 2008).
4.8. The effects of pinocembrin on the RCMECs viability in OGD/R The RCMEC survival rates were assessed by MTS, 3-(4, 5dimethylthiazol-2-yl)-5- (3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2 H-tetrazolium (Promega, USA), assay in 96 well plates. The absorbance value was detected at 492 nm with microplate spectrophotometer. The experiment was operated in dark (Liu et al., 2010).
4.9.
Analysis of mitochondrial membrane potential (△ ψm)
△ψm of the RCMECs was monitored with JC-1, 5, 5′, 6, 6′Tetrachloro-1, 1′, 3, 3′-tetraethyl- imidacarbocyanine iodide (Beyotime, China). The RCMECs were loaded with JC-1 in dark
at 37 °C for 20 min. The cells were washed twice with buffer solution in the kit. Confocal images were acquired with Leica SP2 laser scanning microscope (Leica, Mannheim, Germany). The fields of the cells were randomly selected. The fluorescence of JC-1 monomer was detected at 490/530 nm (excitation/emission wavelength) and the fluorescence of JC-1 aggregates was detected at 525/590 nm (excitation/emission wavelength) (Chen et al., 2009). Then the pictures in the same field were merged. The fluorescent value of 96 well plates was detected with microplate spectrophotometer.
4.10.
Data analysis
All data were presented as mean ± SEM and compared by oneway ANOVA. A value of P < 0.05 was considered statistically significant.
Acknowledgments This work was supported by the Major Scientific and Technological Special Project for “Significant New Drugs Creation” (No. 2009ZX09302-003, 2009ZX09102-034).
REFERENCES
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Research Report
Pinocembrin attenuates blood–brain barrier injury induced by global cerebral ischemia–reperfusion in rats Fanrui Menga,b , Rui Liua , Mei Gaoa , Yuehua Wanga , Xiaoyan Yua , Zhaohong Xuana,b , Jialin Suna , Fan Yanga , Chunfu Wub,⁎, Guanhua Dua,⁎⁎ a
National Center for Pharmaceutical Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, P.R. China b Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang 110016, P.R. China
A R T I C LE I N FO
AB S T R A C T
Article history:
Blood–brain barrier (BBB) disruption is a major consequence of cerebral ischemia/
Accepted 3 March 2011
reperfusion. Several studies have reported the neuroprotection of pinocembrin on
Available online 22 March 2011
cerebral ischemia in vivo and in vitro, but the effects of pinocembrin on BBB and its underlying mechanisms are not clear. In this study, we investigated the effects of
Keywords:
pinocembrin on BBB functions in the global cerebral ischemia/reperfusion (GCI/R) model
Pinocembrin
in rats. Neurological scores and brain edema were evaluated. BBB permeability was assessed
Global cerebral ischemia
by detecting the concentrations of Evan's blue (EB) and fluorescein sodium (NaF) in brain
Reperfusion
tissue. The pathological changes of BBB ultrastructure were observed by transmission
Blood–brain barrier
electron microscopy. Cerebral blood flow (CBF) was measured by laser Doppler flowmetery. The effects of pinocembrin on primary cultured rat cerebral microvascular endothelial cells (RCMECs) against oxygen–glucose deprivation/reoxygenation (OGD/R) were also investigated. The results showed pinocembrin decreased neurological score and lessened brain edema induced by GCI/R. Pinocembrin also reduced the concentrations of EB and NaF in brain tissue of the GCI/R rats. And pinocembrin alleviated the ultrastructural changes of cerebral microvessels, astrocyte end-feet and neurons, and improved CBF in the GCI/R rats. In addition, pinocembrin increased the viability and mitochondrial membrane potential of cultured RCMECs induced by OGD/R. In conclusion, these data demonstrate that pinocembrin alleviates blood–brain barrier injury induced by GCI/R in rats. © 2011 Elsevier B.V. All rights reserved.
⁎ Correspondence to: C.F. Wu, Department of Pharmacology, Shenyang Pharmaceutical University, No. 103 Wenhua Road, Shenyang 110016, P.R. China. Fax: + 86 24 23843567. ⁎⁎ Correspondence to: G.H. Du, National Center for Pharmaceutical Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 2 Nanwei Road, Beijing 100050, P.R. China. Fax: +86 10 63165184. E-mail addresses: [email protected] (C.F. Wu), [email protected] (G.H. Du). Abbreviations: GCI/R, global cerebral ischemia/reperfusion; OGD/R, oxygen–glucose deprivation/reoxygenation; RCMECs, rat cerebral microvascular endothelial cells; EB, Evan's blue; NaF, fluorescein sodium; BBB, blood–brain barrier; CBF, cerebral blood flow; MBP, mean blood pressure; △ ψm, mitochondrial membrane potential 0006-8993/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2011.03.010
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1.
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Introduction
In clinical emergencies, many accidents result in global ischemia, such as drowning, electric shock and cardiac arrest. Brain tissue is vulnerable to hypoxia, leading to irreversible injury in a short period of ischemia (Lee et al., 2000). Reperfusion has the potential consequences to aggravate the injury in cerebral ischemia, such as reactive oxygen species overproduction, calcium overload (Kuroda and Siesjö, 1997; Nakamura et al., 1999) and blood–brain barrier (BBB) injury (Abbott et al., 2010). BBB consists of cerebral microvascular endothelial cells and tight joint, the abluminal membrane, and the astrocyte endfeet which ensheathe cerebral capillaries. BBB prevents many macromolecules from entering the brain. Plasma proteins such as albumin, pro-thrombin and plasminogen are deleterious to brain tissue, leading to glial cell activation and neuron apoptosis (Abbott et al., 2010). The injury of any BBB component leads to the increase of the permeability. The permeability of BBB is often evaluated by the value of Evan's blue (EB) and fluorescein sodium (NaF) in brain tissue (Lenzsér et al., 2005). Pinocembrin (5, 7-dihydroxyflavanone) is a flavonoid abundant in propolis. Our previous studies showed that pinocembrin reduced glutamate-induced SH-SY5Y cell injury and primary cultured cortical neuron damage in oxygen–glucose deprivation/reoxygenation (OGD/R) (Gao et al., 2008a; Liu et al., 2008). And pinocembrin alleviates cerebral ischemic injury in the middle cerebral artery occlusion rats (Gao et al., 2008b, 2010; Liu et al., 2008). Pinocembrin also improved cognition by protecting cerebral mitochondria structure and function against chronic cerebral hypoperfusion in rats (Guang and Du, 2006). Furthermore, pinocembrin alleviates brain injury in the global cerebral ischemia/reperfusion (GCI/R) rats (Shi et al., 2011). In the present study, GCI/R was induced by four-vessel occlusion (4-VO) in rats and the neurological scores were evaluated referring to previous reports (Xu et al., 2005; Shi et al., 2011). The aim was to determine the effects of pinocembrin on BBB injury in the GCI/R rats. We tested the hypothesis that pinocembrin would reduce BBB permeability and alleviate the pathological changes of BBB ultrastructure in the GCI/R rats. Pinocembrin could protect the rat cerebral microvascular endothelial cells (RCMECs) against OGD/R injury. In addition, we also determined whether pinocembrin had effects on cerebral blood flow (CBF) in the GCI/R rats.
2.
Results
2.1. Effects of pinocembrin on neurological scores in the GCI/R rats The rats were scored twice immediately after reperfusion and at 24 h after reperfusion. As shown in Fig. 1, the scores of the sham rats were zero, which showed no neurological deficits, while the scores increased in other groups after reperfusion (P < 0.001, Fig. 1A). The scores of the GCI/R group at 24 h after reperfusion increased in comparison with the sham group (P < 0.001), which decreased in pinocembrin 5 mg/kg group (P < 0.05, Fig. 1B). The data indicated pinocembrin lessened the neurological symptom in the GCI/R rats.
2.2.
Pinocembrin alleviated brain edema in the GCI/R rats
As shown in Table 1, the GCI/R rats showed severe brain edema compared with the sham rats (P < 0.01). But brain edema was reduced significantly in pinocembrin 1 and 5 mg/ kg groups (P < 0.05).
2.3. GCI/R
Pinocembrin decreased BBB permeability induced by
In cortical tissue, little EB was detected in the sham rats (Fig. 2A). The amount of EB increased dramatically in the GCI/R rats (P < 0.01), and reduced in all groups administrated pinocembrin (P < 0.05 or P < 0.01). The amount of NaF in the GCI/R group increased compared with that of the sham group (P < 0.01), and decreased in the groups treated with pinocembrin (P < 0.05 or P < 0.01) (Fig. 2B). The data indicated that the permeability of BBB in the GCI/R rats increased. And the permeability was alleviated in the GCI/R rats treated with pinocembrin.
2.4. Pinocembrin protected BBB ultrastructure against GCI/ R injury The cortical microvessels in the sham rats have normal endothelial cells, basal lamina and astrocyte end-feet. A series of pathological changes of BBB were observed in the GCI/R rats after 24 h reperfusion: the astrocyte end-feet were markedly swollen and end-feet membrane was disrupted; the nucleoli
Fig. 1 – Pinocembrin decreased neurological scores in the global cerebral ischemia/reperfusion (GCI/R) rats. (A) Neurological scores immediately after reperfusion. (B) Neurological scores after 24 h reperfusion. Data were shown as means ± SEM. ### P < 0.001 vs. sham group; *P < 0.05 vs. GCI/R group, n = 8–9.
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Table 1 – Pinocembrin alleviated brain edema in the global cerebral ischemia/reperfusion (GCI/R) rats. Sham
Brain edema (%)
79.53 ± 0.14
Data were shown as means ± SEM.
##
GCI/R
Pinocembrin
##
80.28 ± 0.16
1 mg/kg
5 mg/kg
10 mg/kg
79.80 ± 0.13⁎
79.74 ± 0.19⁎
80.02 ± 0.10
P < 0.01 vs. sham group; ⁎P < 0.05 vs. GCI/R group, n = 8.
of neurons condensed and electron density of neurons increased; the glial cells were swollen. Pinocembrin 5 mg/kg decreased the edema of astrocyte end-feet and glial cells, and also alleviated the injury of neurons, Fig. 3 (1). And the edema of astrocyte end-feet significantly decreased in the ischemic group given pinocembrin 5 mg/kg (P < 0.05), Fig. 3 (2).
2.5. Pinocembrin enhanced CBF in the GCI/R rats after reperfusion CBF and MBP value were measured in the sham and GCI/R rats. CBF value was calculated by the ratio with basal CBF value in each rat. The CBF and MBP had no significant changes in the sham rats administrated pinocembrin or vehicle 5 mg/kg (Fig. 4). But the CBF was improved in dose-dependent manner in the GCI/R rats treated with pinocembrin 1, 5, and 10 mg/kg after reperfusion (Fig. 5A). MBP decreased during global ischemic period and restored normal level after reperfusion. MBP had no significant changes in GCI/R rats administrated pinocembrin 1, 5, and 10 mg/kg during reperfusion (Fig. 5B).
2.6. Pinocembrin protected the cultured RCMECs against OGD/R induced injury 2.6.1. Effects of pinocembrin on the viability of the RCMECs in OGD/R As shown in Fig. 6, the viability of the RCMECs significantly decreased (55.91 ± 2.22%) after being exposed to OGD/R. The survival rates increased to 72.47 ± 2.11% and 77.05 ± 2.78% due to the administration of pinocembrin at 106 and 105 M through the reperfusion phase, respectively.
2.6.2.
Effects of pinocembrin on △ ψm of the RCMECs
In the normal RCMECs, most JC-1 formed J-aggregates and showed red fluorescence at 525/590 nm. In damaged RCMECs, most JC-1 became monomer in mitochondrial matrix because △ ψm decreased, and showed green fluorescence at 490/ 530 nm. Under laser confocal scanning microscope, we merged both the JC-1 pictures in 525/590 nm and 490/530 nm in the same field to acquire the pictures of △ψm (Fig. 7A–E). The control RCMECs mainly showed red fluorescence (Fig. 7A). In OGD/R group green fluorescence increased and red fluorescence lessened (Fig. 7B), which was reversed in pinocembrin 0.1, 1.0 and 10.0 μM groups (Fig. 7C–E). The value of red fluorescence and green fluorescence was also detected in the 96 well plates. In the OGD/R cells, the ratio of red and green fluorescent intensity decreased (P < 0.001). The ratio increased in the groups treated with pinocembrin 0.1 μM (P < 0.05), 1.0 μM (P < 0.05) and 10.0 μM (P < 0.01), shown as Fig. 7F.
3.
Discussion
The present study showed pinocembrin decreased the neurological scores, alleviated brain edema, reduced the permeability of BBB and mitigated the ultrastructural changes of ‘neurovascular unit’ in the GCI/R rats. Pinocembrin also improved CBF in the GCI/R rats. In addition, pinocembrin protected the RCMECs against OGD/R injury. Brain injury following focal or global cerebral ischemia develops from a complex series of pathophysiological changes (Dirnagl et al., 1999; Lenzsér et al., 2005). Brain edema, including intracellular and vasogenic edema, exacerbates brain damage
Fig. 2 – Pinocembrin decreased the concentrations of Evan's blue (EB) and fluorescein sodium (NaF) in brain tissue of the global cerebral ischemia/reperfusion (GCI/R) rats. (A) Concentration of EB in brain tissue. (B) Concentration of NaF in brain tissue. Data were shown as mean ± SEM. ##P < 0.01 vs. sham group; *P < 0.05; **P < 0.01 vs. GCI/R group, n = 8.
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Fig. 3 – (1) Pinocembrin alleviated the pathological changes of ultrastructure in microvessels, neurons and glial cells in the global cerebral ischemia/reperfusion (GCI/R) rats. Representative electron photomicrographs were shown. A, B and C showed the microvessels in parietal cortex, ×2.0 k. (A) Sham group, with normal microvascular endothelial cells, basal lamina (a) and astrocyte end-feet (b). (B) GCI/R group, microvascular lumina was crushed and became narrow (c), end-feet of astrocyte were serous edema (d), and the membrane of end-foot was disrupted (e). (C) Pinocembrin 5 mg/kg group, the edema of end-feet were mitigated (f). D, E and F showed the neurons in parietal cortex, ×0.7 k. (D) Sham group. (E) GCI/R group, nucleolus condensed and electron density of the neuron increased. (F) Pinocembrin 5 mg/kg group, the injury was lessened. G, H and I showed the glial cells in parietal cortex, G ×1.0 k, H ×0.7 k, I ×1.2 k. (G) Sham group. (H) GCI/R group, the glial cell was serous edema (g). (I) Pinocembrin 5 mg/kg group, the edema of glial cell was lessened (h). (2) Pinocembrin alleviated the edema of astrocyte endfeet in the GCI/R rats. #P < 0.05 vs. sham group; *P < 0.05 vs. GCI/R group, n = 4.
after cerebral ischemia (Kondo et al., 1997, Abbott et al., 2010). And brain edema increased intracranial pressure which is life threatening. In the study, the GCI/R rats had serious brain edema, which was alleviated in the rats treated with pinocembrin. Neurological scores showed the degree of neurological
deficits (Shi et al., 2011; Zhang et al., 2009). The scores increased in the GCI/R rats, and pinocembrin 5 mg/kg decreased the scores. BBB serves as a dynamic regulator between brain tissue and peripheral circulation. Following hypoxia and reoxygenation,
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Fig. 4 – Effects of pinocembrin on cerebral blood flow (CBF) and mean blood pressure (MBP) in the sham rats. Data were shown as mean ± SEM, n = 6.
BBB is disrupted, leading to the increase of BBB permeability and the occurrence of vasogenic brain edema (Sandoval and Witt, 2008). And brain injury following ischemia and reperfusion is highly associated with inflammation response, involving in the extravasation of mononuclear phagocyte and the activation of astrocytes and microglial cells (Muir et al., 2007). Extravasation of albumin and other macromolecules proteins into brain tissue is correlated with the development of vasogenic edema (Abbott et al., 2010). Middle cerebral artery occlusion in rats also causes brain edema and BBB disruption (Gao et al., 2010; Masada et al., 2001). And neuron injury is associated with microvascular damage (Abbott et al., 2010; del Zoppo, 2009). EB and NaF are the dye that hardly passes through BBB in the normal rats, while they may arrive at brain tissue for the injury of BBB (Gao et al., 2010; Lenzsér et al., 2005). Therefore, EB and NaF are often used to study the permeability of BBB. In the study, the concentration of EB and NaF in brain tissue increased in the GCI/R rats, but decreased in the rats treated with pinocembrin. Similar results were obtained in previous study (Lenzsér et al., 2005). Therefore, pinocembrin decreased the permeability of BBB and alleviated vasogenic brain edema against GCI/R injury.
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Fig. 6 – Pinocembrin protected the rat cerebral microvascular endothelial cells (RCMECs) against oxygen–glucose deprivation/reoxygenation (OGD/R) injury. Pinocembrin increased the viability of the RCMECs in OGD/R. Data were shown as mean ± SEM. ##P < 0.001 vs. control group; **P < 0.001 vs. OGD/R group, n = 5.
Microvessels and neurons respond rapidly to ischemic injury (del Zoppo, 2010). The modulation of CBF by neurons bases on neurovascular coupling. The presence and participation of glial cells implies the association of microvessels, glial cells, and neurons in a ‘neurovascular unit’, which controls and modulates regional and local cerebral blood flow (del Zoppo, 2010; Zonta et al., 2003). In a ‘neurovascular unit’, microvessels were associated with neurons by the common astrocytes. The results of transmission electron microscope showed the neuroprotective action of pinocembrin on ‘neurovascular unit’. The neurons of the GCI/R rats showed abnormal morphology: the nucleoli condensed and electron density of neurons increased. However, the injury of neurons was markedly alleviated in the rats treated with pinocembrin 5 mg/kg. And the protective effects of pinocembrin on neurons were reported in the GCI/R rats with Nissl dye Nissl staining (Shi et al., 2011) and in vitro (Liu et al., 2008). Astrocyte end-feet were serous edema, and the membrane of astrocyte end-feet was disrupted. The microvessels were crushed by the swollen astrocyte end-feet and became narrow in the GCI/R rats. These pathological changes severely
Fig. 5 – Effects of pinocembrin on cerebral blood flow and mean blood pressure in the global cerebral ischemia/reperfusion (GCI/R) rats. (A) Brain blood flow. (B) Mean blood pressure. Data were shown as mean ± SEM, n = 6.
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Fig. 7 – Pinocembrin increased mitochondrial membrane potential of rat cerebral microvascular endothelial cells (RCMECs) in oxygen–glucose deprivation/reoxygenation (OGD/R). RCMECs were loaded with JC-1 in dark. (A–E) Representative confocal pictures of the RCMECs were shown. (A) Control group. (B) OGD/R group. (C) Pinocembrin 0.1 μM group. (D) Pinocembrin 1.0 μM group. (E) Pinocembrin 10.0 μM group. (F) Pinocembrin increased the ratio of red and green fluorescent intensity of the RCMECs in OGD/R in 96 well plates. Data were shown as mean ± SEM. ##P < 0.001 vs. control group; *P < 0.05; **P < 0.01 vs. OGD/R group, n = 6.
impaired the function of microcirculation. Pinocembrin alleviated the edema of end-feet and injury of neurons. Therefore, the ‘neurovascular unit’ deficits were alleviated in the GCI/R rats treated with pinocembrin. In the study, effects of pinocembrin on primary cultured RCMECs against OGD/R induced injury were investigated. Pinocembrin enhanced the viability of the RCMECs against OGD/R injury. △ψm of the RCMECs was detected with JC-1. In the normal cells JC-1 forms aggregates. JC-1 becomes monomers because of △ψm decrease in injury cells. △ψm of the RCMECs may be detected by the ratio of red and green fluorescent intensity. The control RCMECs mainly showed red fluorescence and the RCMECs of OGD/R group principally showed green fluorescence. Pinocembrin restored the △ψm of the RCMECs in OGD/R, shown by increasing the ratio. The RCMECs are the important component of BBB and maintain the function of BBB together with the astrocytes (Abbott et al., 2010). Therefore, pinocembrin alleviated the injury of the RCMECs and improved △ψm of the RCMECs in OGD/R. And
pinocembrin could reduce BBB damage and edema formation in the GCI/R rats. Hemodynamic disturbances after reperfusion, such as noreflow, hyperperfusion and hypoperfusion, cause further brain injury due to microcirculatory reperfusion deficits after cerebral ischemia (Wang et al., 2008; Xu et al., 2010). Positron emission tomography (PET) studies in both humans and experimental animals show that hyperperfusion is not a key dangerous factor in cerebral ischemia (Marchal et al., 1999); while the hypoperfusion or no-flow is harmful to the recovery of ischemic brain (Lee et al., 2000; Xu et al., 2010). Hypoperfusion during the period of reperfusion is caused by platelet activation, aggregation and coagulation (Zeller et al., 1999), and may be also because of the vessels contraction induced by the released vasoactive substances (Kis et al., 1999; Monge et al., 2010). The previous studies showed pinocembrin (5– 100 μM) relaxed aortic rings pre-contracted with norepinephrine or KCl by both endothelium-dependent pathway and endothelium-independent pathway (Zhu et al., 2007). In the
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study, pinocembrin 5 mg/kg had no significant effects on CBF in the sham rats, but improved CBF in the GCI/R rats. Therefore, pinocembrin may improve cerebral microcirculation and enhance CBF to alleviate ischemia/reperfusion injury in the GCI/R rats. This might be related to the mechanism that pinocembrin protected the RCMECs against OGD/R injury, alleviated the edema of astrocyte feet, and relaxed the cerebral microvessels contracted by vasoactive substances after GCI/R. In conclusion, pinocembrin protected BBB damage and edema formation induced by GCI/R. The effects of pinocembrin on RCMECs and CBF may be involved in the protective mechanisms against GCI/R injury.
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Medica, Chinese Academy of Medical Sciences) 1, 5 and 10 mg/ kg immediately after reperfusion, respectively. The sham and model group rats (n = 26) were treated intravenously with corresponding volume vehicle.
4.3.
Determination of brain edema
The rats were decapitated at 24 h after reperfusion. Brain tissue was weighed immediately (wet weight) and after dehydrated in 120 °C for 24 h (dry weight), respectively. Brain edema, percentage of water content, was calculated by the formula [(wet weight dry weight)/wet weight] × 100% (Kusaka et al., 2004; Shi et al., 2011).
4.
Experimental procedures
4.4.
4.1.
Surgery
BBB permeability was assessed by the concentration of EB and NaF in brain cortex (Lai et al., 2008; Lenzsér et al., 2005; Park et al., 2010). The animals were anesthetized, administered intravenously EB and NaF (2% solution, 4 ml/kg) for 60 min, and perfused with 0.9% NaCl (1000 ml/kg) at 24 h after reperfusion. The brains were stripped, weighed, and stored in 80 °C till to test. The samples were homogenized with 1 time volume PBS and then added 50% trichloroacetic acid in 10 times volume to precipitate protein. The homogenate was centrifuged for 20 min at 12,000 rpm in 4 °C. For the measurement of EB, 100 μl supernatant was detected at 620/680 nm (excitation/emission wavelength) by microplate spectrophotometer (SAFIRE II, Tecan, Austria). Another homogenate was added 5 M NaOH (1:0.8 v/v) to adjust pH value and detected in 485/535 nm (excitation/emission wavelength). Calculations were based on external standards in the same solvent. The content of the dye was quantified by linear standard curves of EB (0.0763–1.2207 μg/ml) and NaF (0.0191–0.1526 μg/ml), and expressed by per gram tissue.
Adult male SD rats, weight of 300 ± 30 g, were obtained from Vital River Company (Beijing, China). Before the operation, the rats were food-restriction for 4 h and free access to water. All the procedures obeyed institutional guidelines and ethics for animal experiments in Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College. The animal model was induced by four-vessel occlusion method previously described (Lenzsér et al., 2005; Wang et al., 2008; Shi et al., 2011). The rats were anesthetized with 10% chloral hydrate (4 ml/kg). Both common carotid arteries were isolated and embedded with silk threads (3–0). Then both vertebral arteries were occluded by electric coagulation. After 24 h, both common carotid arteries were occluded with bulldog clamps for 20 min. Reflow was achieved by removing the bulldog clamps. The body temperature of the rats was maintained at 37.5 ± 0.5 °C. The sham rats were treated similarly to the ischemic groups without blocking the arteries.
4.5. 4.2.
BBB permeability
Transmission electron microscopy
Neurological scores and experimental groups
Neurological scores were carried out by the scale of total 25 scores (Xu et al., 2005; Shi et al., 2011). In brief, the symptoms of hair roughed up, decreasing movement or bradypragia, enhancement of response to ear-palpating and eyelid ptosis were 1 score, respectively; and splayed-out hind limb, eye misclosure or patency, circling behavior, upwarping head or hunched posture, and hyperspasmia or clonus were 3 scores, respectively; and myasthenia of limbs was 6 scores. The total scores were acquired immediately after reperfusion. The rats which total scores were no less than 10 were severe ischemia, considered as success in the model for the next experiment. And the rats were scored again at 24 h after reperfusion. Neurological scores of the rats which were operated in the first day were calculated. The higher the score showed the higher neurological damage (Shi et al., 2011). A total of 128 rats (including 32 sham rats and 96 ischemic rats) were divided into five experimental groups. The rats in group 1 (n = 32) served as sham group without ischemia. Four other groups were exposed to GCI/R. Groups 3 (n = 22), 4 (n = 26) and 5 (n = 22) were administered intravenously with pinocembrin (synthesized and processed as sterile injection powder at the Department of New Drug Development, Institute of Materia
Transmission electron microscopy was used to observe the effects of pinocembrin on ultrastructural changes of BBB. The sham, GCI/R and pinocembrin 5 mg/kg group rats (four rats a group) were anesthetized, perfused with 0.9% NaCl (1000 ml/ kg), and then reperfused with 4% paraformaldehyde (1000 ml/ kg) at 24 h after reperfusion. The parietal cortex was cut into 1mm3, fixed with 2.5% glutaral for 2 h, and washed three times with PBS, 10 min once. Then the tissue was fixed with 1% osmic acid for 2 h, sequentially, washed with pure water, dehydrated with ethanol, replaced with propylene oxide and resin mixture, embedded in pure resin, and stained with uranyl acetate and lead citrate (Gao et al., 2010; Zheng et al., 2007). The cortical microvessels were observed under H-7650 transmission electron microscope (Hitachi, Japan) and photographed. And the area of astrocyte endfeet around cerebral microvessels (diameter 3–6 μm) was measured with Image Pro-plus 6.0 software.
4.6.
CBF and mean blood pressure (MBP) measurement
After 24 h, the operated rats were anesthetized with pentobarbital sodium (50 mg/kg). The calotte skin was incised in the middle. The right cranial bone between bregma and posterior fontanelle was grinded by the dental drill. The laser probe with
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a holder was attached to the right skull 2 mm caudal to bregma, 4 mm lateral to midline (Noppens et al., 2006; Wang et al., 2008). Then CBF was measured with laser Doppler flowmetery (Perimed, Sweden). And blood pressure was detected by BL-420 S physiological apparatus in femoral artery (Taimeng, China). After recording 10 min basal value of CBF and MBP, bilateral carotid arteries were clamped for 20 min. The rats which CBF decreased less 75% were excluded during ischemic period. Then the rats were administrated vehicle or pinocembrin at the beginning of reperfusion. The CBF and MBP value was recorded continuously for 90 min after the injection. And CBF and MBP value were acquired at every 5 min (CBF and MBP value of 1 min was averaged at each point). CBF value was expressed as percentage relative to baseline value (100%).
4.7.
RCMECs culture
The RCMECs were cultured referring to the previous paper (Liu et al., 2010). Briefly, the brains of 2–3 weeks old rats were stripped and placed in ice-cold D-Hank's solution. The cortices were cut into 1 mm3 sections, homogenated and centrifuged at 1800 rpm. Then deposition was homogenated at 1000 g in 20% BSA. The red deposition was microvascular fragments. The microvascular fragments were digested in 5 times volume 1 mg/ ml type II collagenase for 1.5 h at 37 °C, terminated with complete medium, and centrifuged at 1000 rpm. Then the microvascular fragments were cultured in the medium, including 10% fetal bovine serum, basic fibroblast growth factor (10 ng mL1) and heparin (100 μg mL1) at 37 °C with 95% air and 5% CO2. The cells were used from passage 4 to 6 for the experiment. The cells were identified with von-Willebrand factor antibody (Santa Cruz, USA) and FITC labeled secondary antibody (Santa Cruz, USA). The proportion of the RCMECs was over 95%. The RCMECs in confocal dishes and 96 well plates were randomly divided into 5 groups: control group, OGD/R group, and pinocembrin 0.1 μM, 1.0 μM and 10.0 μM groups. The RCMECs were washed with D-Hank's solution for 3 times, and then the culture medium was replaced with glucose free Earle's balanced salt solution with 100 μM sodium dithionite (Na2S2O4), a deoxygenated reagent, for another 2 h, and subsequently incubated with complete medium at 37 °C for 24 h (Liu et al., 2008). Pinocembrin was administrated at the start of reoxygenation. Control group was treated with Earle's balanced salt solution containing glucose without deoxygenated reagent (Liu et al., 2008).
4.8. The effects of pinocembrin on the RCMECs viability in OGD/R The RCMEC survival rates were assessed by MTS, 3-(4, 5dimethylthiazol-2-yl)-5- (3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2 H-tetrazolium (Promega, USA), assay in 96 well plates. The absorbance value was detected at 492 nm with microplate spectrophotometer. The experiment was operated in dark (Liu et al., 2010).
4.9.
Analysis of mitochondrial membrane potential (△ ψm)
△ψm of the RCMECs was monitored with JC-1, 5, 5′, 6, 6′Tetrachloro-1, 1′, 3, 3′-tetraethyl- imidacarbocyanine iodide (Beyotime, China). The RCMECs were loaded with JC-1 in dark
at 37 °C for 20 min. The cells were washed twice with buffer solution in the kit. Confocal images were acquired with Leica SP2 laser scanning microscope (Leica, Mannheim, Germany). The fields of the cells were randomly selected. The fluorescence of JC-1 monomer was detected at 490/530 nm (excitation/emission wavelength) and the fluorescence of JC-1 aggregates was detected at 525/590 nm (excitation/emission wavelength) (Chen et al., 2009). Then the pictures in the same field were merged. The fluorescent value of 96 well plates was detected with microplate spectrophotometer.
4.10.
Data analysis
All data were presented as mean ± SEM and compared by oneway ANOVA. A value of P < 0.05 was considered statistically significant.
Acknowledgments This work was supported by the Major Scientific and Technological Special Project for “Significant New Drugs Creation” (No. 2009ZX09302-003, 2009ZX09102-034).
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