Evans Blue Content Research Articles

Effect of hydrogen on blood brain barrier of mice with sepsis-associated encephalopathy

Objective To evaluate the effect of hydrogen on blood brain barrier of mice with sepsis-associated encephalopathy (SAE). Methods A total of 100 adult male ICR mice, aged 6-8 weeks, weighing 20-25 g, were divided into 4 groups(n=25 each) using a random number table method: sham operation group (group Sham), sham operation plus hydrogen group (group Sham+ H), group SAE and SAE plus hydrogen group (group SAE+ H). Sepsis was induced by cecal ligation and puncture (CLP). Sham+ H and SAE+ H groups inhaled 2% hydrogen for 1 h starting from 1 and 6 h after CLP, respectively.At 24 h after CLP, Evans blue (EB) was injected via the caudal vein, and then the mice were sacrificed and brain tissues were removed for measuring the EB and water contents, for examining the pathological changes of hippocampi (with a light microscope) and for detecting the expression of occludin and VE-cadherin (by Western blot). Morris water maze test was performed at days 10-16 after CLP. Results Compared with group Sham, the contents of EB and water in brain tissues were significantly increased, the expression of occludin and VE-cadherin was down-regulated, the escape latency was prolonged, and the number of crossing the original platform was reduced in SAE and SAE+ H groups (P<0.05). Compared with group SAE, the contents of EB and water in brain tissues were significantly decreased, the expression of occludin and VE-cadherin was up-regulated, the escape latency was shortened, the number of crossing the original platform was increased (P<0.05), and the pathological changes of hippocampi were significantly attenuated in group SAE+ H. Conclusion The mechanism by which hydrogen mitigates SAE may be related to reducing the damage to blood brain barrier of mice. Key words: Hydrogen; Sepsis; Brain; Blood-brain barrier

Does inhibition of angiotensin function cause neuroprotection in diffuse traumatic brain injury?

Objective(s):Neuroprotection is created following the inhibition of angiotensin II type 1 receptor (AT1R). Therefore, the purpose of this research was examining AT1R blockage by candesartan in diffuse traumatic brain injury (TBI).Materials and Methods:Male rats were assigned into sham, TBI, vehicle, and candesartan groups. Candesartan (0.3 mg/kg) or vehicle was administered IP, 30 min post-TBI. Brain water and Evans blue contents were determined, 24 and 5 hr after TBI, respectively. Intracranial pressure (ICP) and neurologic outcome were evaluated at -1, 1, 4 and 24 hr after TBI. Oxidant index [malondialdehyde (MDA)] was determined 24 hr after TBI.Results:Brain water and Evans blue contents, and MDA and ICP levels increased in TBI and vehicle groups in comparison with the sham group. Candesartan attenuated the TBI-induced brain water and Evans blue contents, and ICP and MDA enhancement. The neurologic score enhanced following candesartan administration, 24 hr after TBI.Conclusion:The blockage of AT1R may be neuroprotective by decreasing ICP associated with the reduction of lipid peroxidation, brain edema, and blood-brain barrier (BBB) permeability, which led to the improvement of neurologic outcome.

Effect of curcumin pretreatment on brain injury induced by intestinal ischemia-reperfusion in mice

Objective To evaluate the effect of curcumin pretreatment on brain injury induced by intestinal ischemia-reperfusion (I/R) in mice. Methods Forty-eight male C57BL/6 mice, aged 8 weeks, weighing 20-24 g, were divided into 3 groups (n=16 each) using a random number table: sham operation group (Sham group), intestinal I/R group (I/R group) and curcumin pretreatment group (CUR group). Mice were subjected to 75 min superior mesenteric artery occlusion followed by 24 h reperfusion to establish the model of brain injury induced by intestinal I/R in mice.Curcumin 200 mg/kg (in 30 ml of 10% dimethyl sulfoxide) was intraperitoneally injected at 30 min before ischemia in CUR group, while 10% dimethyl sulfoxide 30 ml was given instead of curcumin in Sham group and I/R group.Two percent Evans blue (EB) in 4 ml/kg of normal saline was injected via the caudal vein at 23 h of reperfusion, 1 h later mice were sacrificed, and hippocampi were removed for determination of EB content.Mice were sacrificed at 24 h of reperfusion, hippocampal tissues were isolated for determination of wet to dry weight ratio (W/D ratio) and cell apoptosis (by TUNEL) and for examination of the pathological changes (with a light microscope), and brain tissues were isolated for determination of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) contents (by enzyme-linked immunosorbent assay), malondialdehyde (MDA) content (by thiobarbituric acid method), superoxide dismutase (SOD) activity (by xanthine oxidase method) and expression of caspase-3 (by Western blot). Apoptosis rate was calculated. Results Compared with Sham group, the W/D ratio of hippocampal tissues, EB content and apoptosis rate were significant increased, the contents of MDA, TNF-α and IL-6 in brain tissues were increased, SOD activity was decreased, and the expression of caspase-3 was up-regulated in I/R and CUR groups (P<0.05). Compared with I/R group, the W/D ratio of hippocampal tissues, EB content and apoptosis rate were significant decreased, the contents of MDA, TNF-α and IL-6 in brain tissues were decreased, SOD activity was increased, and the expression of caspase-3 was down-regulated (P<0.05), and the pathological changes of hippocampal tissues were significantly reduced in CUR group. Conclusion Curcumin pretreatment can reduce brain injury induced by intestinal I/R in mice, and the mechanism may be related to inhibiting inflammatory responses, lipid peroxidation and cell apoptosis. Key words: CURCUMIN; Reperfusion injury; Intestinal; Brain injuries

Effects of pregabalin on brain edema, neurologic and histologic outcomes in experimental traumatic brain injury

Brain edema and increased intracranial pressure (ICP) are among the main causes of neurological disturbance and mortality following traumatic brain injury (TBI). Since pregabalin neuroprotective effects have been shown, this study was performed to evaluate the possible neuroprotective effects of pregabalin in experimental TBI of male rats. Adult male Wistar rats were divided into 4 groups: sham, vehicle, pregabalin 30 mg/kg and pregabalin 60 mg/kg. TBI was induced in vehicle and pregabalin groups by Marmarou method. Pregabalin was administered 30 min after TBI. Sham and vehicle groups received saline. Brain water and Evans blue content and histopathological changes were evaluated 24, 5 and 24 h after TBI, respectively. The ICP and neurological outcomes (veterinary coma scale, VCS) were recorded before, 1 h and 24 h post TBI. The results showed a significant reduction in brain water content and ICP, and a significant increase in VCS of pregabalin group (60 mg/kg) as compared to vehicle group (P < 0.05). Also, pregabalin reduced brain edema and apoptosis score as compared to vehicle group. Post TBI pregabalin administration revealed a delayed but significant improvement in ICP and neurological outcomes in experimental TBI. The underlying mechanism(s) was not determined and needs further investigation.

Stereo-Specific Transcript Regulation of the Polyamine Biosynthesis Genes by Enantiomers of Ornithine in Tobacco Cell Culture.

BackgroundOrnithine (Orn) plays an essential role in the metabolism of plant cells through incorporation in polyamines biosynthesis, the urea cycle and nitrogen metabolism. Physiological response of the plant cells to its two enantiomers have not been widely investigated yet.ObjectivesThis study aimed to evaluate effect of ornithine enantiomers on expression of certain polyamine (PAs) biosynthetic genes in tobacco cells.Materials and MethodsSuspension-cultured tobacco cells were treated with different concentrations of L- and D- Orn for 24 h. Cell viability was assayed by Evans Blue and hydrogen peroxide content. The changes of gene expression were analyzed by semi-quantitative RT-PCR.ResultsExogenous D-Orn resulted in enhancement of expression of genes involved in Orn, arginine and S-adenosyl methionine metabolism. Additionally, exogenous D-Orn treatment resulted in sustained viability of cultured tobacco cells and normal levels of hydrogen peroxide were maintained. Supplied L-Orn increased the hydrogen peroxide level and lowered viability of cells. Treatment with L-Orn had a negative effect on the transcript levels for most analyzed PA-related genes. It was also illustrated that transcription of putrescine methyl transferase, key enzyme for nicotine production, was highly upregulated by L-Orn.ConclusionsBased on the results, D-Orn was shown to have a stereo-selective function in regulation of the PAs-related genes.

Dynamics of Evans blue clearance from cerebrospinal fluid into meningeal lymphatic vessels and deep cervical lymph nodes

ObjectivesRecently, it has been confirmed, that excess fluid and waste products from the brain are drained into the cerebrospinal fluid (CSF) and afterwards cleared via the olfactory route and/or lymphatic vessels in the brain dura and corresponding extracranial lymphatic structures. Therefore, the aim of present study was to monitor time-dependent uptake of Evans blue (EB) tracer from subarachnoid space into the meningeal lymphatic vessels and extracranial lymph nodes in rats during 3 hours–12 days.MethodsEB was injected into the cisterna magna of anesthetized rats and after required survival, plasma, brain dura matter and corresponding lymph nodes (cervical, thoracic and lumbar) were dissected and processed for lymphatic vessels analyses using immunofluorescence and immunohistochemistry. Furthermore, we have used sensitive ultra-high-performance liquid chromatography (UHPLC) method for the determination of EB concentrations in selected samples.ResultsUsing a combination of imaging methods, we have detected two different types of the vascular structures in the brain dura and in deep cervical lymph nodes. The blood vessels, which were RECA-1 + positive and the lymphatic-like vessels, expressing bright intense red fluorescence of EB tracer. Subsequently, using UHPLC with UV detection, we have quantified the EB concentration in positive structures by 3 hours up to 12 days after tracer delivery. A significant increase of EB concentration was detected in deep cervical lymph nodes already at 3 hours with a peak at 1 day that decreased to about one-tenth of its peak value by 12 days. Similar pattern was detected in brain dura. On the contrary, the brain tissue and plasma were almost negative for EB tracer during all tested time periods.ConclusionOur results demonstrate the dynamic changes of EB in meningeal lymphatic vessels and in deep cervical lymph nodes, thus recapitulating the downstream outflow of intracisternally injected tracer during 3 hours–12 days via dura mater lymphatic vessels towards corresponding extracranial draining system, particularly the deep cervical lymph nodes.

Neuroprotective nature of adipokine resistin in the early stages of focal cerebral ischemia in a stroke mouse model

New evidence suggests that resistin may have a therapeutic potential effect in management of neurodegenerative disease; but its role in the pathophysiology of stroke-induced injuries is not understood. However, further investigations are required to elucidate the effect of resistin and explore its possible molecular mechanisms on the ischemic reperfusion injury. Transient focal cerebral ischemia was induced by the middle cerebral artery occlusion (MCAO) in mice. Animal treated with resistin at doses of 25, 50, 100, 200, and 400 ng/mouse, on the MCAO commencement. Neurological function, infarct size, brain edema and Blood-brain barrier (BBB) disruption were measured. Additionally, content of malondialdehyde (MDA), TUNEL-positive cells and apoptosis-related proteins were assessed by immunohistochemistry and western blot techniques. Resistin mRNA was detected at 3 h, 6 h, 12 h and 24 h after MCAO using real-time QRT-PCR method. Central administration of resistin only at doses of 200 and 400 ng/mouse considerably reduced the infarct size and promoted neurological function (p < 0.001). In addition, resistin (400 ng/mouse) significantly decreased brain edema (p < 0.001), evans blue (EB) leakage (p < 0.05), MDA content (p < 0.005), apoptotic cells and apoptosis-related proteins (p < 0.001). Resistin mRNA expression markedly increased at 12-h time point and then returned to basal level at 24 h after MCAO. Our findings revealed that treatment with resistin could attenuate ischemic damage in a dose-dependent approach via suppressing apoptosis and oxidative stress. Application of resistin in clinical settings to treat stroke and brain ischemia warrants further research.

RLj-RGD3, a novel recombinant toxin protein from Lampetra japonica, prevents coronary thrombosis-induced acute myocardial infarction by inhibiting platelet functions in rats

Recombinant Lampetra japonica RGD-peptide (rLj-RGD3), a soluble protein containing three RGD sequences, was acquired from the oral salivary glands of Lampetra japonica using recombinant DNA technology. The aim of this study was to investigate the protective effects of rLj-RGD3 against acute myocardial infarction (AMI) induced by coronary artery thrombosis, as well as the underlying mechanisms. A rat model of AMI caused by ferric chloride-induced thrombosis on the surface of the left anterior descending (LAD) coronary artery was successfully established. Rats were given various doses of rLj-RGD3 (12 μg/kg, 24 μg/kg and 48 μg/kg) via sublingual intravenous delivery 10 min before AMI. ST segment elevation was recorded by electrocardiogram (ECG) until the end of the model. Left ventricular Evans blue content and histopathological changes were examined. Blood samples were collected to determine 5-hydroxytryptamine (5-HT), β-thromboglobulin (β-TG), platelet factor 4 (PF4) and cAMP levels. The effects of rLj-RGD3 on platelet aggregation, adhesion and intracellular calcium concentrations were also measured. rLj-RGD3 significantly reduced ST segment elevation, prevented thrombus formation in the coronary artery and decreased Evans blue content in the left ventricular myocardium. Meanwhile, rLj-RGD3 exerted an inhibitory effect on adenosine diphosphate (ADP)-induced platelet aggregation and blocked platelet adhesion to collagen. Treatment with rLj-RGD3 prevented 5-HT, β-TG and PF4 release and significantly elevated intracellular cAMP levels in a dose-dependent manner but decreased the level of cytosolic-free Ca2+, an aggregation-inducing molecule. These results show that rLj-RGD3 can effectively reduce coronary thrombosis in AMI rats by strongly inhibiting platelet function, indicating that the recombinant RGD toxin protein rLj-RGD3 may serve as a potent clinical therapeutic agent for AMI.

Effects of limb ischemic preconditioning on intestinal injury and brain injury induced by hepatic ischemia-reperfusion in rats

Objective To evaluate the effects of limb ischemic preconditioning on intestinal injury and brain injury induced by hepatic ischemia-reperfusion(I/R)in rats. Methods Healthy male Wistar rats, aged 6-8 weeks, weighing 200-250 g, were divided into 3 groups(n=16 each)using a random number table: sham operation group(Sham group), hepatic I/R group(I/R group)and limb ischemic preconditioning group(LIP group). In LIP group, the lower limb blood flow was blocked for 10 min with an elastic rubber tourniquet at the right groin followed by 30-min reperfusion through releasing the tourniquet.Hepatic I/R injury was induced by occlusion of the portal vein, hepatic artery and common bile duct for 30 min followed by reperfusion in I/R and LIP groups.Eight rats in each group were selected at 6 h of reperfusion and samples from the cardiac apex were taken for determination of plasma concentrations of tumor necrosis factor-alpha(TNF-α)and interleukin-10(IL-10)by radioimmunoassay.Then the rats were sacrificed and the small intestine tissues and brain tissues were removed.Intestinal damage was assessed and scored according to Chiu.The activity of myeloperoxidase(MPO)in intestinal mucosa was detected by colorimetric method.The pathological changes of brain tissues were examined under a light microscope.The ultrastructure of brain tissues was observed under an electron microscope.Eight rats in each group were randomly selected at 6 h of reperfusion, and Evans blue(EB)2 mg/kg was injected through the caudal vein over 1 min.Then the rats were sacrificed, brain tissues were removed for measurement of EB content, and the brain water content was calculated. Results Compared with Sham group, Chiu′s score and MPO activity in intestinal tissues were significantly increased, the brain water content and EB content were increased, and the concentrations of TNF-α and IL-10 in plasma were increased in I/R group and LIP group(P 0.05), and the pathological changes of brain tissues were significantly attenuated in LIP group. Conclusion Limb ischemic preconditioning can attenuate intestinal injury and brain injury induced by hepatic I/R, and the mechanism may be related to inhibiting systemic inflammatory responses of rats. Key words: Ischemic preconditioning; Limbs; Reperfusion injury; Liver; Intestine; Brain injuries

Blood-brain Barrier Disruption Leads to Postoperative Cognitive Dysfunction.

Postoperative Cognitive Dysfunction (POCD) has received considerable attention as one of the main postoperative complications. The underlying mechanism of POCD in elderly subjects has not been fully elucidated to date. The Central Nervous System (CNS) is isolated from the bloodstream by the Blood Brain Barrier (BBB) that consists of endothelial cells, capillary blood vessels and tight junctions. The tight junctions carry out significant biological functions that are associated with the CNS and blood circulation. In this review, I present a hypothesis that blood-brain barrier disruption leads to postoperative cognitive dysfunction. A total of 81 healthy male Wistar rats were used for the present study. All the experimental animals were randomly divided into 3 groups: normal control group, isoflurane group and splenectomy group. The control group was not subjected to any form of treatment. The rats in isoflurane group were given 1.5-2% isoflurane under intubation and mechanical ventilation. The rats in splenectomy group underwent splenectomy under the same anesthesia as the isoflurane group. The Morris water maze was used to examine the learning and memory ability of the animals. The expression of the Tight Junctions Proteins (TJPs) in the hippocampus was analyzed using Western blotting. The concentration of Evans Blue (EB) in the supernatant was analyzed using UV spectroscopy. Ultrastructure changes in the basal laminas, the Tight Junctions (TJs), mitochondria and the endoplasmic reticulum surrounding the capillaries were assessed by Transmission Electron Microscopy (TEM). Following splenectomy, the rats displayed concomitant significant cognitive deficits in the Morris water maze test. Taken together, the results indicate that the expression levels of occludin (65KD) following splenectomy were reduced on days one and three in aged rats. No significant difference was noted in the expression levels of claudin-5, except for a reduction after surgery on day one. The leakage of EB was higher following splenectomy than control group and isoflurane group. The ultrastructure of the neurovascular unit was monitored on the day prior to surgery and on the 1st, 3rd and 7th day following surgery using a transmission electronmicroscope. The alterations in the levels of tight junction proteins following splenectomy may contribute to the BBB permeability increase, which in turn will induce postoperative cognitive dysfunction.

The effect of angiotensin receptor type 2 inhibition and estrogen on experimental traumatic brain injury

Background: Estrogen interferes with renin-angiotensin system (RAS). Increasing evidence suggests that estrogen interferes with the RAS such as decreasing angiotensin receptor in the brain. Objectives: This study aimed at investigating the mutual interaction between estrogen and candesartan (an angiotensin receptor blocker) to inhibit or amplify each other's neuroprotective effects after traumatic brain injury (TBI). Materials and Methods: Female rats were divided into 11 groups and the ovaries were removed in nine groups. Study groups included sham, TBI, oil, vehicle (Veh), a low dose (LC) and a high dose (HC) of candesartan, estrogen (E2), Veh + Veh, and a combination of estrogen with a low dose (E2 + LC) and a high dose (E2 + HC) of candesartan. TBI was induced by the Marmarou's method. Brain edema and integrity of blood–brain barrier (BBB) were assayed by calculating brain water content (BWC) and Evans blue content, respectively. The neurological outcome was evaluated using the veterinary coma scale (VCS). Results: The results showed that the BWC in the E2 group was less than that of the oil group (P

Rhubarb attenuates cerebral edema via inhibition of the extracellular signal-regulated kinase pathway following traumatic brain injury in rats

Background:Rhubarb is a traditional Chinese medicine for treating traumatic brain injury (TBI).Purpose:The purpose of this study is to elucidate the potential mechanism of rhubarb by suppressing extracellular signal-regulated kinase (ERK) to ameliorate brain edema.Materials and Methods:Sprague-Dawley rats were separated into four groups at random. One group received 3 g/kg rhubarb, and another group received 12 g/kg rhubarb, and the vehicle group and sham group were administered the same dose of saline solution. The blood–brain barrier disruption and edema were detected by Evans blue extravasation and water content, respectively. ERK, Matrix metalloproteinase 9 (MMP-9), and zonula occluden-1 (ZO-1) in the damaged tissue were measured by western blot analysis and quantitative real-time polymerase chain reaction.Results:Rhubarb attenuated the brain edema after TBI, especially at the dose of 12 g/kg. Rhubarb significantly suppressed ERK, down-regulated MMP-9, and up-regulated ZO-1. Rhubarb might be a prospective therapeutic regimen to decrease edema in TBI.Conclusions:Rhubarb alleviates the edema by restraining the ERK signaling pathway. Our results contribute to the validation of the traditional use of rhubarb in the treatment of TBI and its mechanism.SUMMARY The aim of this study was to explore the potential mechanism of rhubarb by suppressing extracellular signal-regulated kinase to ameliorate brain edema. Results: Rhubarb ameliorates edema caused by traumatic brain injury by inhibiting the ERK/Matrix metalloproteinase 9/zonula occluden-1 signaling pathway. Abbreviations used: TBI: Traumatic brain injury, ERK: Extracellular signal-regulated kinase pathway, MMP-9: Matrix metalloproteinase 9, ZO-1: Zonula occluden-1, BBB: Blood-brain barrier, PCR: Polymerase chain reaction, TCM: Traditional Chinese medicine, MAPKs: Mitogen-activated protein kinases, CCI: Controlled cortical impact, DL: Rhubarb 3 g/kg in distilled water, DH: Rhubarb 12 g/kg in distilled water, EB: Evans blue, IOD: Integral optical density, MEK: Mitogen extracellular kinase, MMPs: Matrix metalloproteinases, NADPH: Nicotinamide adenine dinucleotide phosphate: ROS, reactive oxygen species.

Regulation of paxillin tyrosine phosphorylation via inhibition of c-Abl kinase to protect ventilator induce lung injury in vivo in rats

To determine whether the inhibition of paxillin tyrosine residues 31 and tyrosine residues 118 (Pxn Y31 and Pxn Y118) phosphorylation via inhibition of c-Abl kinase will effectively block its downstream effector molecules vessel endothelium-cadherin (VE-cad), and whether Rho/Rho kinase activation which will induce the vascular barrier dysfunction. Ninety healthy male Sprague-Dawley (SD) rats were randomly divided into nine groups (each n =10). Only tracheotomy was undergone in the sham group. Groups of protective ventilation were set at a volume tidal (VT) of 6 mL/kg, a positive end-expiratory pressure (PEEP) of 5 cmH2O (1 cmH2O = 0.098 kPa) for 1 hour or 2 hours (namely group PVT 1 h and group PVT 2 h), respectively. Groups of high VT were put on mechanical ventilation (MV) at high VT 30 mL/kg, PEEP 0 for 1 hour or 2 hours (namely group HVT 1 h and group HVT 2 h), respectively. Groups UO126 and AG957 pretreatment were set on MV at HVT for 1 hour or 2 hour respectively, but they were given p42/44 mitogen-activated protein kinase (p42/44MAPK) inhibitor UO126 1 mg/kg by intraperitoneal injection or c-Abl kinase inhibitor AG957 10 mL/kg by intragastric injection 1 hour before HVT ventilation. All the animals were sacrificed after experiments and specimens of lung tissues and bronchoalveolar lavage fluid (BALF) were harvested. Pulmonary vascular permeability was measured by Evans blue (EB). The levels of tumor necrosis factor-α (TNF-α) in BALF were measured by enzyme linked immunosorbent assay (ELISA). Then the change of lung tissue pathology was observed with light microscope, diffuse alveolar damage system (DAD) score and lung wet/dry ratio (W/D) were estimated. The myeloperoxidase (MPO) activity was measured by colorimetric analysis, phosphorylations of c-Abl Y245, Pxn Y31, Pxn Y118, VE-cad Y658, p42/44MAPK Y202/Y204, myosin light chain (MLC) and myosin-associated phosphatasetype Y696 (MYPT Y696) were determined by Western Blot. (1)There were no obvious pathological changes in the lung tissue in the sham group and PVT 1 h or 2 h group, and also there were no significant differences in all the parameters between above groups. However, the injury in lung tissue was severe in the HVT groups. In addition, DAD score, lung W/D ratio, EB content, the activity of MPO, and TNF-α in BALF in HVT groups were significantly higher than those in sham group and PVT groups. After pretreatment with AG957 or UO126, all the parameters were significantly decreased as compared with those of groups HVT. (2) The levels of phosphorylation of the proteins in lung tissue in HVT groups were increased as compared with those of group sham and groups PVT, especially at 2 hours of MV. However, compared with groups HVT, the level of p-VE-cad Y658 in lung tissue decreased significantly in group AG957 and group UO126 at 2 hours after HVT. However, the levels of all phosphorylated proteins at 2 hours were significantly lowered in the AG957 group compared with those of the HVT group [p-c-Abl Y245 (gray value): 0.29±0.04 vs. 0.42±0.04, p-Pxn Y31 (gray value): 0.51±0.03 vs. 0.70±0.05, p-Pxn Y118 (gray value): 0.65±0.04 vs. 0.91±0.04, p-VE-cad Y658 (gray value): 0.77±0.07 vs. 1.32±0.07, p-p42/44MAPK Y202/Y204 (gray value): 0.38±0.06 vs. 0.61±0.03, p-MLC (gray value): 0.37±0.04 vs. 0.77±0.05, p-MYPT Y696 (gray value): 0.54±0.05 vs. 0.87±0.06, all P < 0.05]. After pretreatment with UO126, the phosphorylation level of VE-cad in lung tissue at 2 hours was significantly lower than that of HVT group (gray value: 0.74±0.04 vs. 1.32±0.07), and the phosphorylation levels of p42/44MAPK and its downstream effector molecules MLC and MYPT Y696 were also significantly decreased [p-p42/44MAPK Y202/Y204 (gray value): 0.38±0.07 vs. 0.61±0.03, p-MLC (gray value): 0.37±0.04 vs. 0.77±0.05, p-MYPT Y696 (gray value): 0.55±0.05 vs. 0.87±0.06, all P < 0.05]. Pxn Y31 and Pxn Y118 phosphorylation could be blocked by inhibition of c-Abl kinase, which could strengthen VE-cad at attachment junction and might block formation of Pxn-guanine nucleotide-exchange factor H1 (GEF-H1)-p44/42MAPK signalosome which induce activation local Rho signaling, lead to activation of MLC phosphorylation, actomyosin contraction, and increase endothelial permeability.

IMM-H004, A New Coumarin Derivative, Improved Focal Cerebral Ischemia via Blood–Brain Barrier Protection in Rats

IMM-H004 (7-hydroxy-5-methoxy-4-methyl-3-[4-methylpiperazin-1-yl]-2H-chromen-2-one) is a novel coumarin derivative that showed better effect in improving global cerebral ischemia in rats. However, the effects and mechanisms in focal cerebral ischemia were not clear. Blood-brain barrier (BBB) protection is a vital strategy for the treatment of cerebral ischemia. This study is to investigate whether IMM-H004 improves brain ischemia injury via BBB protection. Focal brain ischemia model was induced by middle cerebral artery occlusion for 1 hour and reperfusion (MCAO/R) for 24 hours in rats. IMM-H004 (1.5, 3, 6 mg/kg) and edaravone (positive drug, 6 mg/kg) were administered after 5 minutes of occlusion. Neurological score and TTC staining were used to evaluate the effect of IMM-H004. Evans Blue (EB) staining and electron microscopy were used to assess BBB permeability. Western blot, reverse transcription-polymerase chain reaction, and immunohistochemistry were used to detect the expression of BBB structure-related proteins. Compared with the model group, IMM-H004 in the focal brain ischemia model improved neurological function and reduced cerebral infarction size and edema content. IMM-H004 sharply reduced the EB content and alleviated BBB structure. In addition, IMM-H004 increased the level of zonula occludens (ZO-1) and occluding, decreased the level of aquaporin 4 and matrix metalloproteinase 9, either in cortex or in hippocampus. And all of these changed were related to BBB protection. IMM-H004 improved cerebral ischemia injury via BBB protection. For a potential therapy drug of cerebral ischemia, IMM-H004 merits further study.

Anti-nociceptive and anti-inflammatory effects of the methanolic extract of Opuntia humifusa stem.

Opuntia humifusa (O. humifusa) Raf. has been used for the prevention and treatment of rheumatoid arthritis, inflammation, and cancer. Our study was designed to unveil the anti-nociceptive and anti-inflammatory effects of the methanolic extract of O. humifusa Raf stem (OHS). The anti-nociceptive effect was measured by hot plate, acetic acid-induced writhing, and tail flick assays in mice and rats. Moreover, the anti-inflammatory effect was measured by vascular permeability and carrageenan and serotonin-induced paw edema tests in rats. Furthermore, anti-inflammatory effect was also measured using macrophage-like LPS-induced RAW 264.7 cells. OHS extract inhibited acetic acid-induced writhing (p<0.0001), and delayed the reaction time of mice to the hot plate-induced thermal stimulation (p<0.0001) and tail flick tests (p<0.05). OHS extract attenuated the carrageenan and serotonin-induced paw edema in rats (p<0.001). Similarly, OHS extract significantly decreased Evans blue concentration in acetic acid induced vascular permeability test (p<0.0001), revealing its strong anti-inflammatory effect. Finally, among four different fractions of OHS extract, n-butanol fraction strongly decreased NO production (p<0.0001) and iNOS expression in LPS-induced RAW 264.7 cells. Our results suggest that the methanolic extract of O. humifusa stem can be used to develop a therapeutic or supportive drug and/or functional food against pain and inflammation related diseases.

Effect of Bacillus subtilis on Aeromonas hydrophila-induced intestinal mucosal barrier function damage and inflammation in grass carp (Ctenopharyngodon idella)

Our study explored the effect of oral intubation of Bacillus subtilis on Aeromonas hydrophila-induced intestinal mucosal barrier function damage and inflammation in grass carp. The mid-intestine mucosal tissue was collected for ATPase activity measurement. Intestinal mucosa was also ultrastructurally examined with transmission electron microscope (TEM), and its permeability was determined using Evans blue (EB) and D-lactic acid. The mid-intestine pro-inflammation cytokine, MyD88 and tight junction (TJ) protein mRNA expression levels were measured using real-time quantitative PCR. The results revealed that B. subtilis was found to prevent the decrease in the activity of Na+, K+-ATPase and Ca2+, Mg2+-ATPase, as well as the increase in EB and D-lactic acid concentration and inflammation induced by A. hydrophila in grass carp. Compared with A. hydrophila groups, B. subtilis safeguarded the integrity of intestinal villi and tight junction structure and restrained A. hydrophila-induced down-regulation of TJ proteins zonula occludens-1 (ZO-1) and occludin. B. subtilis also restrained up-regulation of TJ protein claudin b, pro-inflammation cytokine tumour necrosis factor α (TNF-α), cytokine interleukin 8 (IL-8), IL-1β, and adaptor protein myeloid differentiation factor 88 (MyD88) mRNA levels. Thus, oral intubation of B. subtilis could reduce A. hydrophila-induced intestinal mucosal barrier function damage and inflammation.

A strategy for bypassing the blood-brain barrier: Facial intradermal brain-targeted delivery via the trigeminal nerve

Although intranasal delivery bypasses the blood-brain barrier (BBB), the anatomical location of the olfactory mucosa and respiratory airflow interference lead to less brain-targeted drug delivery. In addition to intranasal delivery, evidence indicates that facial intradermal injection might be a novel strategy for bypassing the BBB via the trigeminal nerve (TN). The hypothesis was verified by pharmacokinetic evaluation, nasal injury, lymphatic vessels inhibition and immunohistochemistry. Intradermal injection into the rat mystacial pad (i.d.) elevated the brain sub-areas and trigeminal Evans Blue (EB) concentrations, Cmax and AUC(0−t). I.d. also increased them in brain sub-areas beyond those of intranasal (i.n.) and intravenous injection (i.v.), especially the pons varolii and the medulla oblongata (sub-areas associated with TN). I.d. injection increased the brain drug targeting efficiency, brain direct transport percentage and brain bioavailability of EB while i.n. injection altered them slightly. Trigeminal transection and nasal injury reduced trigeminal EB with i.d. administration. Trigeminal perineurium, epineurium, perivascular spaces, neurons and Schwann cells were involved in the EB brain-targeted delivery. The lymphatic system mediated EB diffusion from the mystacial pad to the nasal mucosa and the brain. Thus, facial intradermal injection might be a promising strategy for brain-targeting delivery, bypassing the BBB via the trigeminal substructures.

The effects of 7-nitroindazole on serum neuron-specific enolase and astroglia-derived protein (S100β) levels after traumatic brain injury.

We investigated the possible role of 7-nitroindazole (7-NI) in regulating serum neuron-specific enolase (NSE) and S100β levels in a rat model of traumatic brain injury (TBI). We also explored the possible mechanism by which 7-NI may affect the level of NSE and S100β. A total of 160 healthy adult male Sprague-Dawley rats were randomly divided into 2 groups: i) The saline-treated group and ii) the 7-NI-treated group. Using the random number table, the groups were further divided into four subgroups: i) The sham-injured group; ii) the TBI 6 h group; iii) the TBI 12 h group; and iv) the TBI 24 h group (n=20). Controlled cortical impact in rats was established. Serum NSE and S100β levels, nitric oxide (NO) level, water content, Evans blue (EB) content, malondialdehyde (MDA) level and total superoxide dismutase (T-SOD) level in the brain tissue were measured. NO synthase (NOS) activity was measured at 6, 12 and 24 h after TBI. Pathological changes in brain tissue were studied by hematoxylin and eosin (H&E) staining at each time-point. NSE and S100β levels, NO content, water content, EB content and MDA level in the brain tissue increased significantly after TBI. NOS activity was also increased significantly after TBI while T-SOD content in brain tissue was significantly reduced after TBI. H&E staining showed that brain damage was aggravated gradually after TBI. We concluded that the early application of 7-NI significantly reduced serum NSE and S100β levels after TBI. The neuroprotective effects of 7-NI may be associated with reduced NOS activity, reduced NO content, alleviated brain edema, lower blood-brain barrier permeability and oxidative stress. Serum NSE and S100β levels can reflect the therapeutic effect of 7-NI, which suggest a good diagnostic value.

Roflumilast Reduces Cerebral Inflammation in a Rat Model of Experimental Subarachnoid Hemorrhage.

Roflumilast, a selective inhibitor for PDE4, is approved by FDA as an anti-inflammation drug for treatment of chronic obstructive pulmonary disease (COPD). This study investigates the effects of roflumilast on cerebral inflammation in the rat SAH model. Here, we show that subcutaneous administration of roflumilast (3mg/kg) significantly improved the neurological deficits. Measurement of evans blue extravasation and brain water content revealed a significant reduction of blood-brain barrier permeability and brain edema. Importantly, roflumilast treatment remarkably decreased levels of IL-1β, IL-6, and TNF-α and the number of apoptotic neurons in the brain after SAH. These results indicate that roflumilast is effective in treating cerebral inflammation following SAH.

Protective Effect of 4-Methoxy Benzyl Alcohol on the Blood–Brain Barrier after Cerebral Ischemia Reperfusion Injury

Damage of the blood-brain barrier (BBB) during the process of cerebral ischemic injury is a key factor that influences the therapeutic efficacy to the cerebral ischemic injury. This work was designed to investigate the mechanisms underlying the protective effects of 4-methoxy benzyl alcohol (4-MA) on the BBB by developing a cerebral ischemia/reperfusion model of rats (MCAO/R). The MCAO/R was developed through a thread embolism method. The neurologic scales, the brain infarct rate, and the Evans blue (EB) contents of the brains were detected. Meanwhile, the release of nitric oxide (NO) and activities of NO synthase (NOS) in brain tissues were measured. Western blotting analyses were also used to assess the protein expressions of aquaporin-4 (AQP-4), occludin, and claudin-5 in brain tissue. After rats were pretreated with different concentrations of 4-MA, the neurologic scores, the infarct rate, and the EB contents in the brain tissues were significantly decreased. The release of NO and the activities of neuronal NOS and inducible NOS were notably inhibited. Furthermore, the protein expression of AQP-4 was markedly decreased, whereas the protein expressions of claudin-5 and occludin were significantly increased. In conclusion, the 4-MA decreases the permeability of BBB when focal cerebral ischemia occurs. The inhibition of the NOS pathways, the attenuation of the protein expression of AQP-4, and the enhancement of the expressions of the tight junction proteins might contribute to the protective effects of 4-MA on the BBB.