Effects of ghrelin on brain edema and aquaporin 4 expression after cerebral ischemia/reperfusion in rats

AIM: To explore the effects of ghrelin on the brain edema, the permeability of blood-brain barrier (BBB) and the expression of aquaporin 4 (AQP4) after cerebral ischemia/reperfusion in rats. METHODS: Adult male Sprague-Dawley rats were randomly divided into sham operation group, middle cerebral artery occlusion (MCAO) group and ghrelin treatment group. The MCAO model was made with nylon thread for 2 h of occlusion following 22 h of reperfusion. Ghrelin at a dose of 10 nmol/kg was injected via femoral vein at the beginning of reperfusion. The cerebral infarct volume was measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Brain functional deficits were evaluated by determining the neurological scores. The changes of brain swelling and water content were analyzed through volume calculation and dry/wet weight measurement. The permeability of BBB was detected by collecting extravascular Evans blue (EB) in the brain cortex. The changes of AQP4 expression were assessed by the methods of immunohistochemistry and Western blotting. RESULTS: Compared with MCAO group, the rats in ghrelin treatment group had smaller brain infarct volume, lower EB exudation content and neurological scores. The percentage of brain swelling, water content and AQP4 expression were lower in ghrelin treatment group than those in MCAO group. CONCLUSION: Ghrelin reduces the injury of cerebral ischemia/reperfusion, and lightens the brain edema and BBB damage in rats. Ghrelin also inhibits the expression of AQP4 in brain tissue.     

Effect of 3-n-butylphthalide pretreatment on structure and function chan-ges of blood brain barrier in rat model of focal cerebral ischemia-reperfusion injury

AIM: To investigate whether pretreatment with 3-n-butylphthalide (NBP) ameliorates blood brain barrier (BBB) dysfunction in a rat model of focal cerebral ischemia-reperfusion injury (CIRI). METHODS: Male SD rats (n=120, 24 rats in each group) were randomly divided into sham operation group (sham group), model group (IR group), low dose group of NBP pretreatment (NBP I group), medium dose group of NBP pretreatment (NBP II group) and high dose group of NBP pretreatment (NBP III group). The model of CIRI was established by a suture method. After ischemia for 2 h and reperfusion for 24 h, the contents of water and Evans blue (EB) were detected. The pathological changes of the BBB ultrastructure were observed under transmission electron microscope. The protein level of matrix metalloproteinases 9 (MMP-9) was measured by immunohistochemical technique. The mRNA expression of MMP-9 was determined by real-time PCR. RESULTS: After CIRI, the content of water and EB was progressively increased, the BBB was damaged seriously, and the expression of MMP-9 was significantly up-regulated compared with sham group (all P<0.01). Pretreatment with NBP significantly decreased the contents of water and EB, relieved morphological damage of the BBB, and reduced the expression of MMP-9 obviously (all P<0.01). Compared with NBP I group, the changes in NBP II and III group were remarkable (P<0.05), but the difference between NBP II group and NBP III group was not obvious (P＞0.05). CONCLUSION: Pretreatment of 3-n-butylphthalide has preventive effect against cerebral ischemia reperfusion injury in the rats, which may be related to decrease the expression of MMP-9 and reduce the permeability of blood brain barrier.     

Astrocytic connexin 43 is involved in development of cryoinjury-induced cerebral edema

ATM: To discuss the change of astrocytic connexin 43 (Cx43) expression in cerebral edema and the potential roles of Cx43 in the development of cerebral edema. METHODS: Cryoinjury-induced cerebral edema model was established in the right parietal lobe cortex in the rats. The animals were divided into sham group, cryoinjury-induced cerebral edema model group, and cryoinjury-induced cerebral edema model injected with carbenoxolone (or octanol) group. Dry and wet weight method was used to measure the content of brain water. Methanamide method was used to evaluate the permeability of blood-brain barrier (BBB) after cryoinjury. HE staining showed the physiological changes in injured brain cortex. The protein expression of Cx43 was detected by Western blot and immunohistochemical staining. RESULTS: The brain water content in the injured cortex of rats was increased and cerebral edema developed to the peak at 24 h after cryoinjury. The permeability of BBB was increased in the injured cortex of the rats, and the area where BBB was damaged was larger than injured area. HE staining showed that many cells were dead in the central area of cryoinjury, and at the same time cerebral edema was appeared obviously in the surrounding area of injured brain cortex. The protein expression of Cx43 was up-regulated in the surrounding area of injured brain cortex, while Cx43 expression decreased in the injured cortex at different periods after cryoinjury. Both the content of brain water in the injured cortex and the permeability of BBB at 24 h after cryoinjury were reduced after injecting carbenoxolone or octanol intraperitoneally to block Cx43. CONCLUSION: The cerebral edema up-regulates the expression of Cx43 and also strengthens its function. Once Cx43 is blocked, the degree of cerebral edema in rats is attenuated.     

Effect of hyperbaric oxygen on gelatinase,nitric oxide synthase and the permeability of brain blood barrier in cerebral ischemia-reperfusion mice

AIM:To investigate the effect of hyperbaric oxygen (HBO) on gelatinase,nitric oxide synthase and the permeability of brain blood barrier(BBB) in ischemia-reperfusion(I/R) mice.METHODS:Using cerebral I/R models, during the reperfusion period, 0.25 MPa (ATA) HBO were applied 5 times. matrix metalloproteinase(MMP)-2,9, nitric oxide synthase(NOS) and evans blue (EB) in brain were measured.RESULTS:①HBO had significanty effect on MMP-9, but had little effect on MMP-2. ② HBO decreased the activity of NOS.③ The maxium amount of EB in IR group was at 4 hours after reperfusion and gradually decreased at 11 h, 23 h,48 h, 72 h.CONCLUSION:HBO may decrease the activities of MMP-9,NOS and the permeability of BBB in cerebral ischemia-reperfusion mice.     

Matrix metalloproteinase-9 and cystoskeleton actin are involved in the increased permeability induced by hypoxia/ischemia status in a blood-brain barrier model

AIM: To understand the effects and approach the mechanisms of matrix metalloproteinase-9 (MMP-9) and cystoskeleton actin on the permeability increasing of blood-brain barrier (BBB) model which was induced by hypoxia/ischemia status in vitro. METHODS: The BBB model was build by the co-culture of cell ECV304 and astrocytes in vitro, then divided randomly into control group, hypoxia/ischemia group and BB-1101 pretreatment group. The permeability of BBB was determined by ［125I］- BSA. The expression and the disposition of actin were detected by direct-immunofluorescence and Western blotting. BB-1101, the MMPs inhibitor, was used to investigate if MMP-9 participate the process of the increasing of BBB models permeability in hypoxia/ischemia status. RESULTS: Post-stimulation of hypoxia/ischemia for 5 h, the permeability of ［125I］-BSA and amount expression of MMP-9 in hypoxia-ischemia group was increased compared with control group (P<0.01). The change of actin that stained by direct immunofluotescence, the floss tape blured, the cell-cell junction among cells loosed and fissure appeared. However, the amount of actin expression was unchanged. BB-1101 pretreatment extenuated the destruction of the actin-conjunction, also decreased the BBBs permeability of ［125I］-BSA induced by hypoxia/ischemia (P<0.01). CONCLUSION: The increased expression of MMP-9 which leads to the recombination of BBB-actin protein is one of the mechanisms that hypoxia/ischemia induces the increasing of BBB permeability.     

Expression and histogenesis of matrix metalloproteinase-9 and transforming growth factor-beta 1 in acute cerebral ischemia model of rats

AIM:To observe the expression and tissue localization of matrix metalloproteinase 9 (MMP-9) and transforming growth factor beta 1 (TGF-β1) in the rat acute cerebral ischemia model. METHODS:Male Wistar rats were used to establish acute cerebral ischemia model by a suturing method. The rats were divided into normal control group, sham group and ischemia 6 h, 12 h, 1 d, 2 d, 6 d and 14 d groups. The rat cerebral cortex and hippocampus of the brain were collected at different time points.The mRNA and protein levels of MMP-9 and TGF-β1 in the brain tissues were detected by real-time PCR and in situhistochemistry staining, respectively. The levels of MMP-9 and TGF-β1 in the plasma were also measured by ELISA. RESULTS:The results of real-time PCR showed that the mRNA levels of MMP-9 began to increase 6 h after acute ischemia and reached to a peak 2 d after acute ischemia. Similarly, the mRNA level of TGF-β1began to rise 12 h after acute ischemia and reached to the highest level 6 d after acute ischemia. Compared with the sham rats, the mRNA levels of MMP-9 and TGF-β1 in the rat brains that collected at ischemic time of 12 h, 1 d, 2 d, 6 d and 14 d were significantly increased. Moreover, results of in situhistochemical staining showed that the expression of MMP-9 was detected at cerebral cortex and hippocampus 1 d after acute cerebral ischemia.Further studies showed that MMP-9 dyeing of the rat cerebral cortex was most obvious 2 d after the acute cerebral ischemia. Similarly, the rat cortex and hippocampus began to express TGF-β1 2 d after acute ischemia and TGF-β1 staining at rat cerebral cortex was most obvious 6 d after the acute cerebral ischemia. In addition, ELISA showed that the increase in MMP-9 and TGF-β1 was detected in the plasma 12 h after ischemia. Compared with the sham rats, the level of these 2 factors significantly upregulated since 1 d after ischemia. CONCLUSION: The brain tissue itself contributes to the upregulation of MMP-9 and TGF-β1 post acute cerebral ischemia, which shed light on the related research in the field.     

Effect of edaravone on expression of Aβ and AQP4 and activity of MMP2 and MMP9 in acute cerebral ischemia/reperfusion rats

AIM: To investigate the effect of edaravone on acute cerebral ischemia/reperfusion rats. METHODS: SD rats were randomly divided into sham operation group (saline), model group (modeling given saline), low dose group (edaravone at 6 mg/kg) and high dose group (edaravone at 10 mg/kg). The rat model was established by Zea Longa suture method. The nerve function scores were evaluated after operation, and the infarct volume was measured by TTC assay. The mRNA expression of aquaporin 4 (AQP4) and amyloid β-protein (Aβ) in the brain tissue was detected by RT-qPCR. The protein levels of APQ4 and Aβ were determined by Western blot. The activity of matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9(MMP9) was detected by gelatin zymography. RESULTS: Compared with model group, edaravone administration markedly alleviated neurological deficits, histological damages and brain edema. The mRNA and protein levels of AQP4 and Aβ, and the activity of MMP2 and MMP9 were downregulated (P<0.05). Furthermore, the improvements in high dose group were significantly more effective than those in low dose group. CONCLUSION: Edaravone significantly reduces neurological deficits and brain edema in the rats with acute ischemic stroke, and the mechanisms may be related to the downregulation of AQP4 and Aβ, and the activation of MMP2 and MMP9.     

Protective effect of gabexate mesilate on blood-brain barrier in rats with cerebral ischemia-reperfusion

AIM To investigate the protective effects of gabexate mesilate （GM） on blood-brain barrier （BBB） permeability in rat model with cerebral ischemia-reperfusion （I/R）. METHODS Adult male SD rats （n=180） were randomly divided into sham group, I/R group, nimodipine （NMP; 2 mg·kg^-1·d^-1） group and GM （5, 10 and 20 mg·kg^-1·d^-1） groups （n=30 in each group）. The rat model of cerebral I/R was established by blocking the middle cerebral artery with thread plug for 2 h. Ten min before modeling, the drugs were given intraperitoneally. The nerve function was detected by Longa scoring method. The permeability of BBB was measured by Evans blue permeation method, and the brain water content was measured by dry-wet weight method. The activity of superoxide dismutase （SOD） and glutathione peroxidase （GSH-Px）, and the content of malondialdehyde （MDA） in brain tissue were determined by biochemical analysis. The content of tumor necrosis factor-α （TNF-α）, interleukin-1β （IL-1β） and IL-6 was measured by ELISA. The mRNA expression of matrix metalloproteinase-2 （MMP-2）, MMP-9 and nuclear factor-κB （NF-κB） was detected by RT-PCR. The protein levels of MMP-2, MMP-9 and NF-κB were determined by Western blot. RESULTS Compared with I/R group, the Longa score, permeability of Evans blue and brain water content of the rats in GM （10 and 20 mg·kg^-1·d^-1） and NMP （2 mg·kg^-1·d^-1） groups were decreased. The activity of SOD and GSH-Px was increased, while the content of MDA was decreased. The content of TNF-α, IL-1β and IL-6 was decreased, and the mRNA and protein expression levels of MMP-2, MMP-9 and NF-κB were significantly down-regulated. Compared with NMP （2 mg·kg^-1·d^-1） group, the Longa score and permeability of Evans blue were decreased in GM （20 mg·kg^-1·d^-1） group, the activity of SOD was increased, and the content of MDA and TNF-α was decreased. The mRNA and protein expression levels of MMP-2, MMP-9 and NF-κB were down-regulated. All of the differences were significant （P<0.05 or P<0.01）. CONCLUSION GM has protective effect on BBB in the rats with cerebral I/R. Its mechanism may be related to inhibition of oxidative stress and inflammation, and down-regulation of MMP-2, MMP-9 and NF-κB expression.     

Effects of cerebral ischemia and postconditioning on signaling molecules PERK and GRP78 in hippocampus tissue of tree shrew during endoplasmic reticulum stress

AIM: To investigate the effects of cerebral ischemia and postconditioning on protein kinase R-like endoplasmic reticulum kinase (PERK) and glucose-regulated protein 78 (GRP78) in the hippocampus tissue of tree shrew during endoplasmic reticulum stress and the mechanism of post-conditioning protecting the brain from damage. METHODS: The focal cerebral ischemic model was duplicated by photochemical reaction in tree shrew and the postconditioning was induced by alternatively occluding and opening the carotid artery of ischemic side for 3 cycles (5 min each cycle) at 3.5 h after ischemia. The damage and ultrastructural changes of the hippocampal neurons were observed by HE staining. The expression of PERK and GRP78 at mRNA and protein levels in the hippocampal tissue at different time points after cerebral ischemia and postconditioning was determined by RT-PCR, immunohistochemistry and Western blot. RESULTS: The injuries of hippocampal neurons were aggravated with prolonged cerebral ischemia, which was most severe at 24 h after ischemia while the postconditioning alleviated these damages correspondingly. The expression of PERK at mRNA and protein levels was upregulated at 4 h, 24 h and 72 h after ischemia (P<0.05), while postconditioning downregulated the expressions of PERK at ischemia and postconditioning 4 h (IP4 h) gruop and IP24 h group (P<0.05). The expression of GRP78 at mRNA and protein levels was not changed at 4 h, 24 h and 72 h after ischemia, while postconditioning upregulated the expressions of GRP78 at IP24 h group (P<0.05). CONCLUSION: The focal thrombotic cerebral ischemia activates the endoplasmic reticulum stress in ischemic hippocampus of tree shrews, leading to the changes in mRNA and protein expression of PERK in the PERK/eIF2α signal transduction pathway. The postconditioning treatment alleviates endoplasmic reticulum stress and neuronal damages by downregulating PERK and upregulating GRP78, thereby protecting the brain from injury.     

Glutamate transport across blood brain barrier after transient global ischemia/reperfusion in rats

AIM: to study the change of glutamate(Glu) transport across blood brain barrier(BBB) in rat following forebrain ischemia/reperfusion. METHODS: BBB unidirectional transfer constant(K_i) for [³H]-Glu in rat hippocampus, cerebral cortex and striatum were determined after rats were subjected to cerebral ischemia 10 min (two-carotid occlusion plus hypovolemic hypotension) followed by 0.17, 2, 6 and 24 h of reperfusion. The recovery of [³H]-Glu in cerebrum was also determined after intracerebral injection of [³H]-Glu in another experiment. RESULTS: Compared with control rat brain, K_i for [³H]-Glu significantly(P＜0.05) decreased at 10 min cerebral ischemia followed by 0.17, 2 and 6 h of reperfusion. At 5 min after intracerebrally injecting [³H]-Glu, recovery of [³H]-Glu in control rat brain was 23.83%. The result indicted that there is a Glu efflux mechanism on BBB. This efflux was not significantly inhibited by pretreatment of 200 mg/L probenecid. After 10 min cerebral ischemia followed by 2 h of reperfusion, the recovery(13.13%) was significantly lower than contro(P＜0.05), its recovery was only 55% of the control. The result indicated that cerebral ischemia/reperfusion may enhanced the efflux of [³H]-Glu from brain. CONCLUSION: Cerebral ischemia/reperfusion significantly reduced Glu BBB transport from plasma to brain and enhanced efflux of Glu from brain.     

Effect of idazoxan on permeability of blood-brain barrier and expression of MMP-9/TIMP-1 in mouse experimental autoimmune encephalomyelitis

AIM：To study the effect of idazoxan (IDA) on the permeability of blood-brain barrier (BBB) and the expression of matrix metalloproteinase 9 (MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1) in mouse experimental autoimmune encephalomyelitis (EAE).METHODS：Female C57BL/6 mice (n=36) were randomly divided into control group, EAE group and IDA group, with 12 mice in each group. EAE was induced by myelin oligodendrocyte glycoprotein 35-55 (MOG35-55). IDA (2 mg/kg, ip, bid) was administered for 15 d after immunization. The neurological defects of the mice were observed daily and scored. The pathological changes were observed under microscope with HE staining and LFB myelin staining. The BBB permeability was detected by Evans blue extravasation. The expression of MMP-9 and TIMP-1 in the brain of EAE mice was determined by Western blotting.RESULTS：Compared with EAE group, the score of neurological defects in IDA group was decreased, the inflammation was relieved, the BBB permeability was reduced, and the expression MMP-9 and the ratio of MMP-9/TIMP-1 were decreased (P<0.05).CONCLUSION：The neuroprotective effect of IDA on mouse EAE might be related to the down-regulation of MMP-9 and the ratio of MMP-9/TIMP-1, thus reducing the degradation of BBB and the permeability of BBB, and ameliorating the pathologic process of EAE.     

Effect of idazoxan on permeability of inflammatory blood-brain barrier model in vitro

AIM: To study the effect of idazoxan on the permeability of inflammatory blood-brain barrier (BBB) model in vitro and the expression of tight junction protein ZO-1.METHODS: In vitro BBB model was established by murine brain endothelial cell line bEnd.3 incubated for 7 d. The cells were treated with TNF-α (10 nmol/L) for additional 24 h to establish the inflammatory BBB model, which was pretreated with IDA at doses of 50, 100 and 200 μmol/L, respectively. The permeability was measured using fluorescein isothiocyanate-conjugated dextran (FD-40, MW 40,000), the expression of ZO-1 was detected by Western blot analysis, the distribution of ZO-1 was observed by immunofluorescence, and the mRNA expression of MMP-9/TIMP-1 was measured by RT-PCR.RESULTS: After incubated for 7 d, b.End3 cells converged to be confluent monolayer with low permeability. The inflammatory BBB model induced by TNF-α treatment displayed much higher permeability with decreased expression of tight junction protein ZO-1, destroyed distribution of ZO-1 and increased mRNA expression of MMP-9. When pretreated with IDA, the permeability was greatly decreased, the expression of ZO-1 was greatly increased, the abnormal distribution of ZO-1 was greatly ameliorated and the mRNA expression of MMP-9 was obviously reduced. The effect was most significant in IDA (200 μmol/L)-pretreated group (P<0.01).CONCLUSION: IDA directly acts on brain endothelial cells to reduce the expression of MMP-9, increase the expression of tight junction protein ZO-1 and ameliorate the destroyed distribution of ZO-1 in the inflammatory BBB, thus reversing the abnormally elevated permeability in a inflammatory BBB model in vitro induced by TNF-α.     

All-trans retinoic acid attenuates blood-brain barrier injury induced by cerebral ischemia-reperfusion in rats through JNK/P38 MAPK signaling pathway

AIM: To investigate the effect of all-trans retinoic acid (ATRA) on blood-brain barrier after cerebral ischemia-reperfusion (CIR) injury in rats and its possible role mechanism.METHODS: Male SD rats were randomly divided into sham group, model (CIR) group and CIR+ATRA (10, 30 and 90 mg/kg) groups. The rat model of CIR injury was established by MCAO thread occlusion method. After ischemia for 1.5 h and reperfusion for 24 h, the neurological functional behavioral score, cerebral infarction volume, brain water content and Evans blue content were determined. The activity of matrix metalloprotein-9 (MMP-9) was measured by gelatin zymography. The protein levels of claudin-5, occludin, ZO-1, JNK, p-JNK, P38, p-P38 and MMP-9 in the brain tissues were determined by Western blot.RESULTS: Compared with CIR model group, ATRA at 30 mg/kg significantly improved neurological function, and decreased cerebral infarction volume, brain water content, Evans blue content and the degradation of tight junction proteins in ischemic area (P<0.01). The activity and protein expression of MMP-9 in ischemic brain tissue were decreased (P<0.01). The phosphorylation of JNK and P38 was inhibited and the protein levels of p-JNK and p-P38 were decreased (P<0.01).CONCLUSION: ATRA reduces the damage of brain tissue and the destruction of blood-brain barrier induced by CIR in rats. The protective effect may be related to inhibiting the activation of JNK/P38 MAPK signaling pathway and MMP-9.     

Effect of aquaporin 4 on lymphatic brain edema in rats

AIM：To investigate the role of aquaporin 4 (AQP4) in the formation and elimination of lymphatic brain edema (LBE). METHODS：Sprague-Dawley rats were randomly divided into sham group and LBE group. LBE was induced by ligating bilateral cervical lymphatic tubes and removing the corresponding lymphatic nodes. The water contents in the cerebral cortex were measured by wet-dry weight method. The expression of AQP4 in the cerebral cortex was detected 1 d, 3 d, 7 d, 11 d and 15 d after a cervical lymphatic blockade or a sham operation by immunohistofluorescence staining and Western blotting. Furthermore, correlation analysis was made between the protein expression of AQP4 and the brain water content. RESULTS：Compared with sham group, cerebral water content, and the protein expression of AQP4 in the LBE rats increased significantly 3 d after operation, peaked at 7 d, followed by gradually stepping down. Furthermore, the expression of AQP4 was positively correlated with brain water content (r=0.8024, P<0.05). CONCLUSION：AQP4 may play an important role in the formation and elimination of LBE.     

Effect of carbon monoxide on permeability of brain blood barrier in cerebral local ischemia rats

AIM:To evaluate the effect of carbon monoxide(CO) on the permeability of brain blood barrier(BBB) in cerebral ischemic rats. METHODS:SD rats were divided into three groups. Saline, hemin or ZnPP were injected intraperitoneally 12 h before middle cerebral artery occlusion (MCAO), respectively. The concentration of blood CO and the permeability of BBB at 24 h after MCAO were measured. RESULTS:The CO concentration in blood in hemin group was higher than that in saline group(P＜0.01), but those in ZnPP group was lower than that in saline group(P＜0.01). In infracted regions, the permeability of BBB in hemin group was lower than that in saline group, and those in ZnPP group was higher than that in saline group (P＜0.05). There was no significant changes in BBB permeability among hemin, ZnPP and saline groups in noninfarcted side(P＞0.05). CONCLUSION:CO reduced the permeability of BBB as a messenger gas molecular when its intrinsic concentration was elevated.     

Ischemia postconditioning induces tight junction protein expression and inhibits brain edema after thrombotic cerebral ischemia in tree shrews

AIM: To assess whether the expression of tight junction(TJ) proteins, occludin/zonula occludins(ZO)-1, and regional cerebral blood flow(rCBF) link to brain edema in tree shrews during thrombotic cerebral ischemia and ischemic postconditioning(PC), and to explore how TJ affects brain edema and cerebral infarction. METHODS: Tree shrews were randomly grouped into control, ischemia and cerebral ischemia+PC(n=23), and the remaining 3 animals were used for magnetic resonance imaging(MRI). The local cerebral thrombosis were induced by photochemical reaction in the tree shrews, and ischemic PC was established at 4 h after induction of cerebral ischemia followed by clipped ipsilateral common carotid artery(5 min×3). The changes of the neural ultrastructure were observed under electron microscope. The neuronal apoptosis was analyzed by the method of TUNEL. Laser Doppler brain flowmetry was used to monitor the rCBF. The protein levels of occludin/ZO-1 were determined by immunochemistry and Western blot. The cerebral infarction volume was detected by MRI. The brain water content was measured by dry-wet weight method. RESULTS: Induction of cerebral ischemia led to a significant reduction of the normal neuron numbers in the hippocampal CA1 area, and conversely, the number of neurons with abnormal ultrastructure was increased. The TUNEL positive cells were increased significantly(P<0.01) in ischemia group. Moreover, the rCBF decreased significantly(P<0.01), and occludin/ZO-1 protein expression decreased(P<0.01). The brain water content and cerebral infarction volume were significantly increased(P<0.01). Ischemic PC increased the rCBF and the occludin/ZO-1 expression, but reduced the brain water content, the TUNEL positive cells, and the infarction volume(P<0.01). CONCLUSION: Ischemic PC increases the rCBF but not the local water content, suggesting that reduced cerebral infarction volume after ischemia PC is associated with the attenuation of cerebral edema by the enhancement of occludin/ZO-1 protein expression.     

Effects of Xingnaojing injection on expression of ZO-1 in blood-brain barrier after global ischemia-reperfusion

AIM: To investigate the effect of Xingnaojing (XNJ) injection on the permeability of blood-brain barrier (BBB) and zonula occludens-1 (ZO-1) protein expression after global ischemia-reperfusion in rats. METHODS: Improved Pulsinelli four-vessel occlusion method was adopted to establish the global ischemia-reperfusion model in the rats. Male Wistar rats were randomly divided into sham group, model group, solvent group and XNJ group. The observations were conducted at the time points of 24 h, 48 h and 72 h after ischemia reperfusion. The water content of the brain tissues was determined by dry-wet weight method, while the Evans blue (EB) content of brain tissue was detected by spectrophotometry. The protein levels of ZO-1 in the cerebral cortex were analyzed by Western blot. RESULTS: The water contents in the brain tissues in model group, solvent group and XNJ group were significantly higher than those in sham group (P<0.05) 24 h after ischemia reperfusion. However, the brain water contents in model group and solvent group were significantly higher than those in XNJ group and sham group (P<0.05) 48 h and 72 h after ischemia reperfusion. The EB contents in the brain tissues in model group, solvent group and XNJ group were entirely higher than that in sham group 24 h after ischemia reperfusion (P<0.05). The EB contents in sham group and XNJ group were significantly lower than those in model group and solvent group 48 h and 72 h after ischemia reperfusion (P<0.05). The protein expression of ZO-1 in the rat cerebral cortex in model group, solvent group and XNJ group was significantly lower than that in sham group 24 h after ischemia-reperfusion (P<0.05). Similarly, 48 h and 72 h after ischemia reperfusion, ZO-1 protein level in the cortex in sham group and XNJ group was significantly higher than that in model group and solvent group (P<0.05). CONCLUSION: At 48 h and 72 h after global ischemia-reperfusion, Xingnaojing injection play a protective role in blood-brain barrier and this role may be associated with the increase in ZO-1 protein expression by Xingnaojing injection.     

Protective effect of atorvastatin on blood brain barrier in rats with focal cerebral ischemia-reperfusion injury

AIM: To explore the protective effects of atorvastatin on blood brain barrier(BBB) in cerebral ischemia-reperfusion(IR) injury and the potential mechanisms involved. METHODS: SD rats were divided into sham group, IR group and atorvastain group. Intraluminal suture method was used to establish cerebral IR model, and the ischemic brain was reperfused for 72 h after the occlusion. The rats in atorvastatin group were administered with atorvastatin(20 mg·kg^-1·d^-1) by gavage once a day for 3 consecutive days after operation. At 72 h after reperfusion, neurological function scores, the water content of the brain tissue, Evans blue(EB) content of ischemic hemisphere, the expression of tight junction(TJ)-associated protein occludin and inflammation factor phosphatidylinositiol 3-kinase-p110 gamma(PI3K-p110γ) were tested and analyzed. RESULTS: In IR group, the rats showed elevated neurological function scores(P<0.01), brain tissue water content(P<0.01) and EB content(P<0.01), accompanied with the down-regulation of occludin expression(P<0.01) and up-regulation of PI3K-p110γ(P<0.01) at 72 h after reperfusion. Compared with IR group, decreased brain edema(P<0.01) and EB leakage(P<0.01) were observed in atorvastatin group, accompanied with increased occludin expression(P<0.01) and decreased PI3K-p110γ expression(P<0.01). However, no statistical difference of the neurological function scores between the 2 groups was observed. CONCLUSION: Atorvastain attenuates cerebral IR injury, which may be associated with the inhibition of inflammatory reactions and the up-regulation of TJ-asso-ciated proteins to maintain the stability of BBB.     

Effects of hyperglycemia and cerebral ischemia on VEGF expression in different subfield of cerebral cortex in tree shrews

AIM: To observe the changes of VEGF expression in different subfield of brain in tree shrews during hyperglycemia and focal cerebral ischemia, in order to explore the relationship between cerebral ischemia, hyperglycemia and VEGF. METHODS: High blood glucose in tree shrews was induced by intraperitoneal injection of streptozotoctin. Focal cortical thrombotic cerebral ischemia was induced by photochemical method in tree shrews. At 4 h, 24 h and 72 h after cerebral ischemia, the histopathological changes and hippocampal neuronal density were examined. VEGF expressions in the ischemic core, penumbra and contralateral cerebral cortex were detected by immunohistochemistry technique at different times after cerebral ischemia. RESULTS: The results of histopathological study showed that there was infarction zone in the exposured cerebral cortex at 4 h after photochemical reaction, and the damage was most severe at 24 h, subsequently accompanied with the glia multiplication and rehab reaction at 72 h. The animals in hyperglycemic ischemic group suffered from greater neurological lesion than the normoglycemic stroke animals, especially at 24 h (P<0.01) and 72 h (P<0.05) after cerebral ischemia. Immunohistochemical analyses of VEGF expression revealed that it started to increase at 4 h after brain ischemia in the penumbra, reached a peak at 24 h, and weakened at 72 h. The stimulated VEGF production was also observed in hyperglycemic only group. When hyperglycemia and brain ischemia were combined, the VEGF expression was higher than that in hyperglycemic only group (P<0.05). Compared to normoglycemic ischemic group, no additivity of the effects of hyperglycemia combined with brain ischemia was observed. CONCLUSION: (1) The model of experimental hyperglycemia and cerebral ischemia is replicated successfully by applying the method combined in vivo injection of streptozotocin in the lower primate tree shrew with thrombotic focal cerebral ischemia. (2) This study shows that hyperglycemia aggravates the focal cerebral ischemia damage. (3) Cerebral ischemia and hyperglycemia both can independently up-regulate VEGF expression, but there is no additional increase in VEGF expression when hyperglycemia combined with brain ischemia is applied.