Evans Blue Extravasation Method Research Articles

Effect of mesenchymal stem cells on small intestinal injury in a rat model of acute necrotizing pancreatitis

BackgroundAcute necrotizing pancreatitis (ANP) is often complicated by multiple organ failure. The small intestine is frequently damaged during ANP. Capillary leakage in multiple organs during ANP is one of the most important causes of multiple organ dysfunction. Damage to the capillary endothelial barrier and impaired water transportation could lead to capillary leakage in ANP.MethodsSprague–Dawley (SD) rats were randomized into a control group, the ANP group, the culture media-treated group, or the bone marrow-derived mesenchymal stem cell (BMSC)-treated group (30 rats in each group). Ten rats in each group were sacrificed at 6, 12, and 24 h after induction of experimental models. Serum, ascites, pancreatic, and small intestinal samples were collected. The levels of serum and ascites albumin and amylases were measured, pancreatic histology was assessed, and the connection changes between vessel endothelial cells were evaluated using scanning electron microscopy (SEM). Capillary leakage in small intestinal tissue was observed visually by tracking fluorescein isothiocyanate (FITC)-albumin, and was measured by the Evans blue extravasation method. The location and expression of aquaporin 1 (AQP1) in the small intestine was analyzed using immunohistochemistry, real-time polymerase chain reaction (PCR), and Western blot.ResultsThe outcomes showed that the level of serum and ascites amylase is elevated. Conversely, the level of serum albumin is decreased while ascites albumin is elevated. There is damage to pancreatic tissue, and the small intestinal capillary endothelial barrier was aggravated. Furthermore, the expression of AQP1 was reduced significantly after induced ANP. Following treatment with MSCs, the elevation of amylase and the decrease of serum albumin were inhibited, the damage to pancreatic tissue and the level of small intestinal capillary leakage was alleviated, and the downregulation of AQP1 was reversed.ConclusionsIn conclusion, MSC therapy could alleviate small intestinal injury in rats with ANP, the mechanism of which might be related to reduction of damage to the small intestinal capillary endothelial barrier, and increased expression of AQP1 in the small intestine.

A highly reproducible mice model of chronic kidney disease: Evidences of behavioural abnormalities and blood-brain barrier disruption

AimsIn the present study, a novel mice model of chronic kidney disease (CKD) was developed, and psycho-motor behavioural abnormalities, blood-brain barrier (BBB) integrity and brain histology were studied. Main methodsSwiss albino female mice were given high adenine diet (0.3% w/w mixed with feed) for 4weeks. Serum urea and creatinine levels and renal histological studies were performed to validate the model. Psycho-motor behavioural abnormalities and neurological severity were studied. BBB integrity was assessed using Evans blue extravasation method. Nissl staining was performed to see possible morphological aberrations in brain. Key findingsThere was a significant increase in serum urea and creatinine levels in mice given high adenine diet, and the mice had abnormal kidney morphology. Deposition of adenine and 2,8-dihydroxyadenine crystals, and increased collagen deposits in the renal tissues were found, which validate induction of CKD in the mice. Motor behavioural abnormalities, depression-like and anxiolytic behaviour and increase in neurological severity were prevalent in mice with CKD. Evans Blue dye extravasation was found to occur in the brain, which signifies disruption of BBB. However, Nissl staining did not reveal any morphological aberration in brain tissue. SignificanceThe present study puts forward a highly reproducible mice model of CKD validated with serum parameters and renal histopathological changes. The mice showed psycho-motor behavioural abnormalities and BBB disruption. It is a convenient model to study the disease pathology, and understanding the associated disorders, and their therapeutic interventions.

Placental Ischemia Impairs Cerebral Blood Flow Autoregulation and Increases Blood‐Brain Barrier Permeability in Pregnant Rats

Cerebrovascular events contribute to ~40% of preeclampsia/eclampsia related deaths, and neurological symptoms are common among preeclamptic patients. We previously reported that placental ischemia, induced by reducing utero‐placental perfusion pressure, leads to impaired cerebrovascular myogenic reactivity and cerebral edema in the pregnant rat. Whether the impaired myogenic reactivity is associated with altered cerebral blood flow (CBF) autoregulation and the edema is due to altered blood‐brain barrier (BBB) permeability is unclear. Therefore, we tested the hypothesis that placental ischemia leads to impaired CBF autoregulation and a disruption of the BBB. CBF autoregulation, measured in vivo by laser Doppler flowmetry, was significantly impaired in placental ischemic rats compared to normal pregnant rats. Brain water content was increased in the anterior cerebrum of placental ischemic rats and BBB permeability, assayed using the Evans blue extravasation method, was also increased in the anterior cerebrum. The expression of the tight junction proteins: claudin‐1 was increased in the posterior cerebrum while zonula occludens‐1, and occludin, were not significantly altered in either the anterior or posterior cerebrum. These results are consistent with the hypothesis that placental ischemia mediates anterior cerebral edema through impaired CBF autoregulation and the associated increased transmission of pressure to microvessels, resulting in increased BBB permeability and cerebral edema.

Placental ischemia in pregnant rats impairs cerebral blood flow autoregulation and increases blood-brain barrier permeability.

Cerebrovascular events contribute to ~40% of preeclampsia/eclampsia‐related deaths, and neurological symptoms are common among preeclamptic patients. We previously reported that placental ischemia, induced by reducing utero‐placental perfusion pressure, leads to impaired myogenic reactivity and cerebral edema in the pregnant rat. Whether the impaired myogenic reactivity is associated with altered cerebral blood flow (CBF) autoregulation and the edema is due to altered blood–brain barrier (BBB) permeability remains unclear. Therefore, we tested the hypothesis that placental ischemia leads to impaired CBF autoregulation and a disruption of the BBB. CBF autoregulation, measured in vivo by laser Doppler flowmetry, was significantly impaired in placental ischemic rats. Brain water content was increased in the anterior cerebrum of placental ischemic rats and BBB permeability, assayed using the Evans blue extravasation method, was increased in the anterior cerebrum. The expression of the tight junction proteins: claudin‐1 was increased in the posterior cerebrum, while zonula occludens‐1, and occludin, were not significantly altered in either the anterior or posterior cerebrum. These results are consistent with the hypothesis that placental ischemia mediates anterior cerebral edema through impaired CBF autoregulation and associated increased transmission of pressure to small vessels that increases BBB permeability leading to cerebral edema.

The Effect of Butylphthalide on the Brain Edema, Blood–Brain Barrier of Rats After Focal Cerebral Infarction and the Expression of Rho A

The aim of this study was to explore the effect of butylphthalide on the brain edema, blood-brain barrier of rats of rats after focal cerebral infarction and the expression of Rho A. A total of 195 sprague-dawley male rats were randomly divided into control group, model group, and butylphthalide group (40 mg/kg, once a day, by gavage). The model was made by photochemical method. After surgery 3, 12, 24, 72, and 144 h, brain water content was done to see the effect of butylphthalide for the cerebral edema. Evans blue extravasation method was done to see the changes in blood-brain barrier immunohistochemistry, and Western blot was done to see the expression of Rho A around the infarction. Compared with the control group, the brain water content of model group and butylphthalide group rats was increased, the permeability of blood-brain barrier of model group and butylphthalide group rats was increased, and the Rho A protein of model group and butylphthalide group rats was increased. Compared with the model group, the brain water content of butylphthalide group rats was induced (73.67 ± 0.67 vs 74.14 ± 0.46; 74.89 ± 0.57 vs 75.61 ± 0.52; 77.49 ± 0.34 vs 79.33 ± 0.49; 76.31 ± 0.56 vs 78.01 ± 0.48; 72.36 ± 0.44 vs 73.12 ± 0.73; P < 0.05), the permeability of blood-brain barrier of butylphthalide group rats was induced (319.20 ± 8.11 vs 394.60 ± 6.19; 210.40 ± 9.56 vs 266.40 ± 7.99; 188.00 ± 9.22 vs 232.40 ± 7.89; 288.40 ± 7.86 vs 336.00 ± 6.71; 166.60 ± 6.23 vs 213.60 ± 13.79; P < 0.05), and the Rho A protein of butylphthalide group rats was decreased (western blot result: 1.2230 ± 0.0254 vs 1.3970 ± 0.0276; 1.5985 ± 0.0206 vs 2.0368 ± 0.0179; 1.4229 ± 0.0167 vs 1.7930 ± 0.0158;1.3126 ± 0.0236 vs 1.5471 ± 0.0158; P < 0.05). The butylphthalide could reduce the brain edema, protect the blood-brain barrier, and decrease the expression of Rho A around the infarction.

Tumor Necrosis Factor induces cerebral edema and increased cerebrovascular permeability in normal pregnant rats

Cerebrovascular events contribute to ~40% of preeclampsia/eclampsia deaths and preeclamptic patients commonly present with neurological symptoms. Our laboratory recently showed that placental ischemia, induced by reducing uterine perfusion pressure, closely mimics preeclamptic patients resulting in cerebral edema and increased inflammatory cytokines such as tumor necrosis factor (TNFα). In this study, we examined whether TNFα, infused via intraperitoneal implanted mini‐osmotic pumps (100ng/day from gestational day 14 to 19), contributes to cerebral edema and blood‐brain barrier (BBB) disruption in normal pregnant rats. Mean arterial pressure, measured via carotid catheters, was significantly increased in TNFα infused rats (112 vs. 107 mmHg; p=0.01). Brain water content, calculated as (wet weight ‐ dry weight)/ wet weight × 100, was also significantly increased in TNFα infused rats (78.2 vs. 77.9%; p=0.01). BBB permeability, assayed using the Evans blue extravasation method, was increased in the hippocampus of TNFα‐infused rats (p&lt;0.05) but was unchanged in the striatum, cerebellum, and anterior brain. Our results indicate that TNFα released from ischemic placentas, could mediate cerebral edema through increased BBB permeability. TNFα may be an important clinical target for preventing cerebrovascular complications associated with preeclampsia.Funding: NIH Grant HL108618

Down-Regulation of Aquaporin 1 in Rats With Experimental Acute Necrotizing Pancreatitis

The objectives of this study were to investigate the expression of aquaporin 1 in capillary endothelial cells of rats with experimental acute necrotizing pancreatitis (ANP) and to explore its pathogenic role in capillary leak. Sixty-four male Sprague-Dawley rats were randomly divided into control (n = 32) and ANP groups (n = 32). Eight rats in each group were killed at 3, 6, 12, and 18 hours after induction of experimental models. Quantity of ascites and levels of serum amylases were measured. Capillary permeability in pancreas, lung, and intestinal tissue was detected by Evans blue extravasation method. Aquaporin 1 expression in pancreas, lung, and intestinal tissue was detected by real-time polymerase chain reaction, immunohistochemical staining, and Western blot. Serum amylase level was significantly higher in ANP group than in controls (P < 0.05). Evans blue concentration in tissues in the ANP group was significantly higher than that in controls (P < 0.05). Aquaporin 1 mRNA and protein expressions in tissues were significantly less in the ANP group than in the controls (P < 0.05). The expression of aquaporin 1 was down-regulated in the pancreas, lung, and intestinal tissue of ANP rats, which could play an important role in the pathogenesis of capillary leak syndrome.

Inhibition of Secretory Phospholipase A2 Activity Attenuates Acute Cardiogenic Pulmonary Edema Induced by Isoproterenol Infusion in Mice After Myocardial Infarction

Several types of secretory phospholipase A2 (sPLA2) are expressed in lung tissue, yielding various eicosanoids that might cause pulmonary edema. This study examined whether inhibition of sPLA2 activity attenuates acute cardiogenic pulmonary edema in mice. Acute cardiogenic pulmonary edema was induced in C57BL/6J male mice by an increase in heart rate with continuous intravenous infusion of isoproterenol (ISP) (10 mg/kg/h) at 2 weeks after the creation of myocardial infarction by left coronary artery ligation. Just before ISP infusion, a single intraperitoneal injection of 100 mg/kg LY374388, a prodrug of LY329722 that inhibits sPLA2 activity, or vehicle was administered. The ISP infusion after myocardial infarction induced interstitial and alveolar edema on lung histology. Furthermore, it increased the lung-to-body weight ratio, pulmonary vascular permeability evaluated by the Evans blue extravasation method, lung activity of sPLA2, and lung content of thromboxane A2 and leukotriene B4. These changes were significantly attenuated by LY374388 treatment. In Kaplan-Meier analysis, the survival rate during the ISP infusion after myocardial infarction was significantly higher in LY374388- than in vehicle-treated mice. Similar results were obtained with another inhibitor of sPLA2 activity, para-bromophenacyl bromide. In conclusion, inhibition of sPLA2 activity suppressed acute cardiogenic pulmonary edema.

Effects of hydroxyethyl starch (130 kD) on brain inflammatory response and outcome during normotensive sepsis

Background During sepsis, the dysfunction of blood–brain barrier (BBB) was mediated by inflammation and subsequently caused sepsis-associated encephalopathy. Hydroxyethyl starch (HES, 130/0.4) is most widely used for volume replacement to maintain or improve tissue perfusion in patients with sepsis, trauma, and shock. This study was undertaken to investigate the effects of HES on BBB permeability, brain edema, inflammatory response and clinical outcome in septic rats. Methods Using the cecal ligation and puncture (CLP) model, Sprague–Dawley rats were treated with 15 ml/kg HES or normal saline 4 h after the operation. Two hours later, expressions of brain toll-like receptor (TLR)-2, TLR4 and intercellular adhesion molecule (ICAM)-1 mRNA was determined by real-time reverse transcription-polymerase chain reaction; inflammatory cytokines like tumor necrosis factor (TNF)-α and interleukin (IL)-6 by enzyme-linked immunosorbent assay; activity of nuclear factor-kappa B (NF-κB) by electrophoretic mobility shift assay; BBB permeability by Evans blue extravasation method; brain edema by wet/dry weight ratio. Weight loss, and clinical symptoms were also observed. Results Without obvious influence on systemic macrohemodynamics, HES could markedly attenuate BBB dysfunction and brain edema. Meanwhile, HES could significantly reduce TNF-α, IL-6, and ICAM-1 mRNA, inhibit NF-κB activation, and down-regulate TLR2 and TLR4 expression in the brain. In addition, CLP-induced increase in weight loss, and clinical symptoms was not reduced after treatment with HES. Conclusions HES could ameliorate BBB dysfunction and inflammation mediators by modulating brain TLR2 and TLR4 expression during sepsis. However, HES could not improve clinical outcome.

Abstract 2617: Inhibition of Secretory Phospholipase A 2 Activity Attenuates Acute Cardiogenic Pulmonary Congestion Induced by Isoproterenol Infusion in Mice with Post-Myocardial Infarction

Several types of secretory phospholipase A 2 (sPLA 2 ) are expressed in airway epithelial cells and alveolar inflammatory cells, yielding various eicosanoids that might cause pulmonary edema. This study examined whether inhibition of sPLA 2 activity attenuates acute cardiogenic pulmonary congestion in mice. Acute cardiogenic pulmonary congestion in 10-week-old male C57BL/6J mice was induced by continuous intravenous infusion of isoproterenol (ISP) (10 mg/kg/hr) at 2 weeks after the creation of myocardial infarction by left coronary artery ligation. Just before ISP infusion, a single intraperitoneal injection of 100 mg/kg of LY374388 (LY), a prodrug of LY329722 that is a specific inhibitor of activity of all sPLA 2 , or vehicle was administered. Arterial systolic pressure, heart rate, and LV end-diastolic pressure were comparably increased after ISP in both LY-mice and vehicle-mice. Also, lung expression levels of mRNA and protein of group V and X sPLA 2 were comparably increased from baseline by 3 ~ 5-fold in both groups of mice. sPLA 2 activity in lung tissue after ISP was increased from baseline by 5-fold in vehicle-mice, while the activity was not detected in LY-mice. In isolated neutrophils, incubation with LY329722 inhibited the respiratory burst and migration in response to fMLP or C5a by 40~50%. LY significantly reduced the lung-to-body weight ratio after ISP (71% of vehicle-mice) and attenuated alveolar neutrophil accumulation based on the lung histology (56% of vehicle-mice). Using the Evans Blue extravasation method, pulmonary vascular permeability was suppressed in LY-mice (Evans Blue content in lung; 66% of vehicle-mice). The lung content of thromboxane A 2 and leukotriene B 4 was increased in both groups, but was lower in LY-mice (71% and 64% of vehicle-mice, respectively). In Kaplan-Meyer analysis, the survival rate during the 12hr ISP infusion was higher in LY-mice than vehicle-mice (p &lt; 0.01, n = 15 mice per group). Similar results were obtained with another inhibitor of sPLA 2 activity, para -bromophenacyl bromide. Inhibition of sPLA 2 activity suppressed acute cardiogenic pulmonary edema, and sPLA 2 inhibition may have therapeutic value in acute cardiogenic pulmonary congestion.

Early appearance of activated matrix metalloproteinase-9 and blood–brain barrier disruption in mice after focal cerebral ischemia and reperfusion

Blood–brain barrier (BBB) disruption is thought to play a critical role in the pathophysiology of ischemia/reperfusion. Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that can degrade all the components of the extracellular matrix when they are activated. Gelatinase A (MMP-2) and gelatinase B (MMP-9) are able to digest the endothelial basal lamina, which plays a major role in maintaining BBB impermeability. The present study examined the expression and activation of gelatinases before and after transient focal cerebral ischemia (FCI) in mice. Adult male CD1 mice were subjected to 60 min FCI and reperfusion. Zymography was performed from 1 to 23 h after reperfusion using the protein extraction method with detergent extraction and affinity-support purification. MMP-9 expression was also examined by both immunohistochemistry and Western blot analysis, and tissue inhibitors to metalloproteinase-1 was measured by reverse zymography. The BBB opening was evaluated by the Evans blue extravasation method. The 88-kDa activated MMP-9 was absent from the control specimens, while it appeared 3 h after transient ischemia by zymography. At this time point, the BBB permeability alteration was detected in the ischemic brain. Both pro-MMP-9 (96 kDa) and pro-MMP-2 (72 kDa) were seen in the control specimens, and were markedly increased after FCI. A significant induction of MMP-9 was confirmed by both immunohistochemistry and Western blot analysis. The early appearance of activated MMP-9, associated with evidence of BBB permeability alteration, suggests that activation of MMP-9 contributes to the early formation of vasogenic edema after transient FCI.

Malnutrition and experimental lung allergy

Although it is commonly accepted that the immune response is affected by malnutrition there are very few data about its effect in allergic diseases. The aim of the present study was to investigate the effect of malnutrition in allergic lung inflammation. An anaphylactic reaction was induced in rat lungs and the increased vascular permeability was measured in the trachea, internal and external bronchi and parenchyma by the Evans blue extravasation method. These studies were conducted in two dietary groups: one fed a normoproteic diet (18%) and the other a hypoproteic diet (4.5%). When the animals were 60 days old the group fed the hypoproteic diet presented a reduction of 77.86% in bodyweight, 63.3% in food intake and 36% in plasma protein concentration characterizing a severe protein-calorie malnutrition. The anaphylactic reaction in the lungs induced a significant increase in vascular permeability in the trachea and bronchi of both dietary groups. However, the intensity of this effect was significantly lower in the malnourished group. Analysis of immunoglobulin isotypes in the serum by ELISA showed that whereas IgG1 and IgG2a levels were similar in both groups, the levels of IgE were significantly lower in the malnourished animals. Moreover, the levels of antigen-specific IgG1, IgG2a and IgE were all significantly inhibited by the protein-calorie malnutrition. When antibodies were passively transfered to the malnourished rats, they developed a reaction as intense as the normoproteic group. These results suggest that the capacity to release inflammatory mediators and the vascular response to these mediators is not affected by this type of malnutrition and, therefore, the diminished response of the airways reported here is probably due to the lower levels of anaphylactic antibodies produced by the malnourished rats.

Inhibition of cigarette smoke-induced oedema in the nasal mucosa by capsaicin pretreatment and a substance P antagonist.

The effect of cigarette smoke on vascular permeability in the rat nasal mucosa was studied using the Evans blue extravasation method. Exposure to smoke from cigarettes induced a significant extravasation of Evans blue in the nasal mucosa of normal rats, suggesting an increased vascular permeability to plasma proteins. The oedema response was correlated to tar, nicotine and vapour phase components in the smoke. The smoke-induced permeability effect was abolished in rats pretreated neonatally with capsaicin. Also, systemic or local pretreatment with [D-Arg, D-Pro, D-Trp, Leu]Substance P, a substance P antagonist, inhibited the permeability response to cigarette smoke. Insertion of a glass-fibre filter, which removes the particulate phase of the smoke (including nicotine), did not significantly reduce the permeability response. The present findings suggest that the smoke-induced oedema in the rat nasal mucosa is not caused by nicotine but by vapour-phase irritants, which activate capsaicin-sensitive C-fibre afferents. These neurons then release agents such as substance P or a related tachykinin which increase permeability to plasma proteins.