Comparative Efficacy and Distribution of Evans Blue Liposome Modified with DSPE-PEG,Tween80,and Brij35

Aim To improve the stability and optimize the tissue distribution of Evans blue liposome in rats, some surfactants such as DSPE-PEG, Tween80, and Brij35 were used to modify the Evans blue liposome. Methods The Evans blue liposome was prepared by the reverse-phase-evaporation method. The effect of cholesterol on the encapsulation percentage of Evans blue was studied. The effects of DSPE-PEG, Tween80, and Brij35 on the encapsulation percentage and tissue distribution of Evans blue liposome in the rat were determined. Results The top encapsulation percentage of Evans blue liposome is 25.30%. After modification by DSPE-PEG, Tween80, and Brij35, the encapsulation percentages decreased slightly, but not significantly. After modification, the Evans blue concentrations deceased in the liver, spleen, lung and kidney, but increased in the brain, especially in the EB-L-Tween80 group. Conclusion DSPE-PEG, Tween80, and Brij35 have slight effect on the encapsulation percentage of Evans blue liposome. The effect of Brij35 on the distribution of Evans blue liposome is similar to that of DSPE-PEG because they both is prevent the reticuloendothelial system (RES) from clearing liposome. The Evans blue concentration in the brain is greatly improved when Tween80 is used to modify the EB liposome, which is good information for preparing liposome targeting the brain.

Evans blue staining to detect deep blood vessels in peripheral retina for observing retinal pathology in early-stage diabetic rats

AIM: To observe and compare the statistical significance of superficial and deep vascular leakage in the pathological changes of the diabetic rats retina after the Evans blue(EB) perfusion, and utilize the modified whole-retina spreading method to make the slides while protecting the periphery of the retina. METHODS: The Sprague-Dawley(SD) rats were randomly divided into 6 groups. Each group named as the normal groups for 4, 8, and 12 wk and the diabetic groups for 4, 8, and 12 wk. The EB was injected into the cardiovascular system of the rats at the different time points. The retina of each group was obtained for observation.RESULTS: The superficial vascular leakage was found in all 6 groups. The size of leakage area of superficial retinal blood vessels was(0.54±0.23)%,(0.65±0.11)%,and(0.58±0.10)% in normal group. No notable leakage was found in the deep blood vessels [(0.03±0.04)%,(0.03±0.05)%, and(0.03±0.05)%]. The deep retinal vascular leakage was found in the peripheral retina of diabetic rats. The size of leakage area of superficial retinal blood vessels in diabetic group were(0.53±0.22)%,(0.69±0.16)%, and(0.52±0.11)%. The leakage areas of deep blood vessels were(0.54±0.50)%,(1.42±0.16)%, and(1.80±0.07)% at 4, 8, and 12 wk, respectively. There was a statistically difference of the leakage area between the 8 th week and the 4 th week of diabetes group(P=0.003). The statistically significant difference between the diabetes and the control groups was noted at 4 wk and 8 wk(P<0.001).CONCLUSION: The main retinal pathological changes of early-stage diabetic rats are the vascular leakage of the periphery of deep retina. Diabetic rats modeled after 8 wk have semi-quantitative statistical difference compared with the normal rats, thus early intervention treatment research can start at this time point.

Determination of Proteins by Measuring Total Internal-reflected Resonance Light Scattering Signals on Water/Tetrachloromethane Interface with Evans Blue and Cetyltrimethylammonium Bromide

A sensitive and selective assay of proteins is proposed based on measuring the total internal-reflected resonance light scattering(TIR-RLS) signals produced on the water/tetrachloromethane(H_2O/CCl_4) interface. In an aqueous medium with pH value in the range of 3.29—3.78, electrostatic attraction occurs between the negatively charged Evans Blue(EB) and positively charged proteins, forming hydrophobic ion associates and resulting in EB-protein adsorption on H_2O/CCl_4 interface. The presence of cetyltrimethylammonium bromide prompts this adsorption, resulting in strongly enhanced TIR-RLS signals. The intensity of the enhanced TIR-RLS at 360—370 nm was found to be proportional to the concentration of proteins. For bovine serum albumin and human serum albumin, the linear range of detection is 0.07—1.2 μg/mL and the limits of detection are 6.68 and 6.30 ng/mL(3σ), respectively, while for lysozyme, the linear range of detection is 0.06—1.0 μg/mL and the limit of detection is 6.0 ng/mL(3σ). The content of the total albumin in a human urine sample could be directly determined by using the standard addition method with a percent recovery of 97.6%—104.1%, and the RSD ranging from 1.9% to 4.2%.

Resonance Rayleigh Scattering Method for the Determination of Raloxifene with Evans Blue

The intensity of resonance Rayleigh scattering (RRS) of Evans blue (EB) or raloxifene hydrochloride (Ralo) is very weak, but it can be enhanced significantly and a new RRS spectrum appears when both of them interact to form an ion-association complex in sodium acetate-hydrochloric acid buffer solution at pH 1.8. The intensity of RRS is directly proportional to the concentration of Ralo in the range of 0–8.3 μg · mL?1, and the detection limit for Ralo (σ = 3) is 18.9 ng·mL?1. The method has high sensitivity and fairly good selectivity. Based on the above evidences, a new facile method for the determination of trace amount of Ralo has been established with satisfactory results.

Effect of Electromagnetic Pulse Exposure on Permeability of Blood-testicle Barrier in Mice

Objective To study the effect of electromagnetic pulse （EMP） exposure on the permeability of blood-testicle barrier （BTB） in mice. Methods Adult male BALB/c mice were exposed to EMP at 200 kV/m for 200 pulses with 2 seconds interval. The mice were injected with 2% Evans Blue solution through caudal vein at different time points after exposure, and the permeability of BTB was monitored using a fluorescence microscope. The testis sample for the transmission electron microscopy was prepared at 2 h after EMP exposure. The permeability of BTB in mice was observed by using Evans Blue tracer and lanthanum nitrate tracer. Results After exposure, cloudy Evans Blue was found in the testicle convoluted seminiferous tubule of mice. Lanthanum nitrate was observed not only between testicle spermatogonia near seminiferous tubule wall and sertoli cells, but also between sertoli cells and primary spermatocyte or secondary spermatocyte. In contrast, lanthanum nitrate in control group was only found in the testicle sertoli cells between seminiferous tubule and near seminifdrous tubule wall. Conclusion EMP exposure could increase the permeability of BTB in the mice.

Changes in the blood-nerve barrier after sciatic nerve cold injury:indications supporting early treatment

Severe edema in the endoneurium can occur after non-freezing cold injury to the peripheral nerve, which suggests damage to the blood-nerve barrier. To determine the effects of cold injury on the blood-nerve barrier, the sciatic nerve on one side of Wistar rats was treated with low tem- peratures （3-5℃） for 2 hours. The contralateral sciatic nerve was used as a control. We assessed changes in the nerves using Evans blue as a fluid tracer and morphological methods. Excess fluid was found in the endoneurium 1 day after cold injury, though the tight junctions between cells remained closed. From 3 to 5 days after the cold injury, the fluid was still present, but the tight junctions were open. Less tracer leakage was found from 3 to 5 days after the cold injury compared with 1 day after injury. The cold injury resulted in a breakdown of the blood-nerve barrier func- tion, which caused endoneurial edema. However, during the early period, the breakdown of the blood-nerve barrier did not include the opening of tight junctions, but was due to other factors. Excessive fluid volume produced a large increase in the endoneurial fluid pressure, prevented liquid penetration into the endoneurium from the microvasculature. These results suggest that drug treatment to patients with cold injuries should be administered during the early period after injury because it may be more difficult for the drug to reach the injury site through the microcirculation after the tissue fluid pressure becomes elevated.

Relationship of gelatinases-tight junction proteins and blood-brain barrier permeability in the early stage of cerebral ischemia and reperfusion

Gelatinases matrix metalloproteinase-2 and matrix metalloproteinase-9 have been shown to mediate claudin-5 and occludin degradation, and play an important regulatory role in blood-brain barrier permeability. This study established a rat model of 1.5-hour middle cerebral artery occlusion with reperfusion. Protein expression levels of claudin-5 and occludin gradually decreased in the early stage of reperfusion, which corresponded to the increase of the gelatinolytic activity of matrix metalloproteinase-2 and matrix metalloproteinase-9. In addition, rats that received treatment with matrix metalloproteinase inhibitor N-[（2R）-2-（hydroxamidocarbonylmethyl）-4-methylpenthanoyl]-L- tryptophan methylamide （GM6001） showed a significant reduction in Evans blue leakage and an inhibition of claudin-5 and occludin protein degradation in striatal tissue. These data indicate that matrix metalloproteinase-2 and matrix metalloproteinase-9-mediated claudin-5 and occludin degradation is an important reason for blood-brain barrier leakage in the early stage of reperfusion. The leakage of the blood-brain barrier was present due to gelatinases-mediated degradation of claudin-5 and occludin proteins. We hypothesized that the timely closure of the structural component of the blood-brain barrier （tight junction proteins） is of importance.

Progesterone is neuroprotective by inhibiting cerebral edema after ischemia

Ischemic edema can alter the structure and permeability of the blood-brain barrier. Recent studies have reported that progesterone reduces cerebral edema after cerebral ischemia. However, the underlying mechanism of this effect has not yet been elucidated. In the present study, progesterone effectively reduced Evans blue extravasation in the ischemic penumbra, but not in the ischemic core, 48 hours after cerebral ischemia in rats. Progesterone also inhibited the down-regulation of gene and protein levels of occludin and zonula occludens-1 in the penumbra. These results indicate that progesterone may effectively inhibit the down-regulation of tight junctions, thereby maintaining the integrity of the blood-brain barrier and reducing cerebral edema.

Correlation of aquaporin-4 expression to blood-brain barrier permeability in rats with focal cerebral ischemia

BACKGROUND： Ischemic cerebrovascular disease causes injury to the blood-brain barrier. The occurrence of brain edema is associated with aquaporin expression following cerebral ischemia/reperfusion. OBJECTIVE： To analyze the correlation of aquaporin-4 expression to brain edema and blood-brain barrier permeability in brain tissues of rat models of ischemia/reperfusion. DESIGN, TIME AND SETTING： The randomized control experiment was performed at the Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical College, China from December 2006 to October 2007. MATERIALS： A total of 112 adult, male, Sprague-Dawley rats, weighing 220-250 g, were used to establish rat models of middle cerebral artery occlusion and reperfusion by the suture method. Rabbit anti-aquaporin-4 （Santa Cruz, USA） and Evans blue （Sigma, USA） were used to analyze the tissue. METHODS： The rats were randomized into sham-operated （n = 16） and ischemia/reperfusion （n = 96） groups. There were 6 time points in the ischemia/reperfusion group, comprising 4, 6, 12, 24, 48, and 72 hours after reperfusion, with 16 rats for each time point. Rat models in the sham-operated group at 4 hours after surgery and rat models in the ischemia/reperfusion group at different time points were equally and randomly assigned into 4 different subgroups. MAIN OUTCOME MEASURES： Brain water content on the ischemic side and the control side was measured using the dry-wet weight method. Blood-brain barrier function was determined by Evans Blue. Aquaporin-4 expression surrounding the ischemic focus, as well as the correlation of aquaporin-4 expression with brain water content and Evans blue staining, were measured using immunohistochemistry and Western blot analysis. RESULTS： Brain water content on the ischemic side significantly increased at 12 hours after reperfusion, reached a peak at 48 hours, and was still high at 72 hours. Brain water content was greater on the ischemic hemispheres, compared with the control hemispheres at 6, 12, 24, 48, and 72 hours after reperfusion, as well as both hemispheres in the sham-operated group （P 〈 0.05）. Evans blue content significantly increased on the ischemic side at 4 hours after ischemia/reperfusion, and reached a peak at 48 hours. Evans blue content was greater on the ischemic hemispheres, compared with the control hemispheres at various time points, as well as both hemispheres in the sham-operated group （P 〈 0.05）. Aquaporin-4-positive cells were detected in the cortex and hippocampus, surrounding the ischemic penumbra focus, at 4-6 hours after ischemia/reperfusion. The number of positive cells significantly increased at 12 hours and reached a peak at 48-72 hours. Aquaporin-4 was, however, weakly expressed in the control hemispheres and the sham-operated group. The absorbance ratio of aquaporin-4 to β-actin was greater at 12, 24, 48, and 72 hours following cerebral ischemia/reperfusion, compared with the sham-operated group （P 〈 0.05）. Aquaporin-4 expression positively correlated to brain water content and Evans blue staining following cerebral ischemia/reperfusion （r1 = 0.68, r2 = 0.81, P 〈 0.05）. CONCLUSION： Aquaporin-4 is highly expressed in brain tissues, participates in the occurrence of ischemic brain edema, and is positively correlated to blood-brain barrier permeability following cerebral ischemia/reperfusion.

Vascular endothelial growth factor-165b protects the blood-retinal barrier from damage after acute high intraocular pressure in rats

AIM:To elucidate the role of vascular endothelial growth factor-165b(VEGF-165b)in blood-retinal barrier(BRB)injury in the rat acute glaucoma model.METHODS:In this study,the rat acute high intraocular pressure(HIOP)model was established before and after intravitreous injection of anti-VEGF-165b antibody.The expression of VEGF-165b and zonula occludens-1(ZO-1)in rat retina was detected by double immunofluorescence staining and Western blotting,and the breakdown of BRB was detected by Evans blue(EB)dye.RESULTS:The intact retina of rats expressed VEGF-165b and ZO-1 protein,which were mainly located in the retinal ganglion cell layer and the inner nuclear layer and were both co-expressed with vascular endothelial cell markers CD31.After acute HIOP,the expression of VEGF-165b was up-regulated;the expression of ZO-1 was down-regulated at 12h and then recovered at 3d;EB leakage increased,peaking at 12h.After intravitreous injection of anti-VEGF-165b antibody,the expression of VEGF-165b protein was no significantly changed;and the down-regulation of the expression of ZO-1 was more obvious;EB leakage became more serious,peaking at 3d.EB analysis also showed that EB leakage in the peripheral retina was greater than that in the central retina.CONCLUSION:The endogenous VEGF-165b protein may protect the BRB from acute HIOP by regulating the expression of ZO-1.The differential destruction of BRB after acute HIOP may be related to the selective loss of retinal ganglion cells.

Perihematomal endothelin-1 level is associated with an increase in blood-brain barrier permeability in a rabbit model of intracerebral hematoma

Background Endothelin-1 （ET-1） has deleterious effects on water homeostasis, cerebral edema, and blood-brain barrier （BBB） integrity. Highly expressed ET-1 was observed after intracerebral hemorrhage （ICH）; however, ET-1 changes and their relationship with BBB disruption within 24 hours of ICH have not been thoroughly investigated. The aim of the present study was to observe the changes in perihematomal ET-1 levels in various phases of ICH and their correlation with the BBB integrity in a rabbit model of ICH. Methods Twenty-five rabbits （3.2-4.3 kg body weight） were randomly divided into a normal control group （five rabbits） and a model group （20 rabbits）. Animals in the model group were equally divided into four subgroups （five rabbits each to be sacrificed at 6, 12, 18, and 24 hours following ICH establishment）. An ICH model was prepared in the model group by infusing autologous arterial blood into the rabbit brain. ET-1 expression in perihematomal brain tissues was determined using immunohistochemistry and color image analysis, and the permeability of the BBB was assayed using the Evan＇s Blue （EB） method. A repeated measures analysis of variance was used to make comparisons of the ET-1 and EB content across the entire time series. Results The number of perihematomal endothelial cells with ET-1 positive expressions following 6, 12, 18, and 24 hours ICH model establishment was 9.32, 13.05, 15.90, and 20.44, respectively, but as low as 6.67 in the control group. The average transmittance of ET-1-positive cell bodies at 6, 12, 18, and 24 hours after ICH was 99.10, 97.40, 85.70, and 80.80, respectively, but 100.12 in the control group. These data reveal that the expression of ET-1 was significantly increased at 6, 12, 18, and 24 hours after ICH compared with the control group, and a marked decrease in the average transmittance of ET-l-positive cell bodies was noted （P 〈0.05）. Similarly, the perihematomal EB content at 6, 12, 18, and 24 hours after ICH was 29.39±1.16, 32.20±0.73, 33.63±1.08, and 35.26±1.12, respectively, in the model group and 28.06±0.80 in the control group. The results indicate that a significant increase in the EB content in the model group was observed compared with that of the control group （P 〈0.05）. Moreover, a positive correlation between the number of ET-1-positive endothelial cells and BBB permeability was observed （t=0.883, P 〈0.05）. Conclusions High levels of ET-1 are closely associated with BBB disruption. ET-1 may play an important role in the pathogenesis of secondary brain injury after ICH.