Blue Extravasation Research Articles

Abstract 142: Transgenic Overexpression of Krüppel-Like Factor 11 in Endothelium Ameliorates Ischemic Brain Injury

Introduction: Krüppel-like factors (KLFs) belong to zinc finger family of transcription factors and their roles in stroke are poorly explored. KLF11 is highly enriched in vascular endothelium, and we have previously documented that peroxisome proliferator-activated receptor gamma-mediated cerebral protection during ischemic insults needs KLF11 as a critical coactivator. However, the role of endothelial KLF11 itself in cerebrovascular function and stroke outcome is unclear. Hypothesis: We hypothesize that endothelium-targeted overexpression of KLF11 stabilizes the BBB, reduces the progression of brain damage, and improves neurological outcomes after ischemic stroke. Methods: Transient middle cerebral artery occlusion was performed in endothelial cell-selective KLF11 transgenic mice (EC-KLF11 Tg), and WT controls. BBB integrity was assessed by using Evans Blue and TMR-Dextran extravasation assays. Brain water content was measured by using a dry-wet method. Brain infarct was analyzed by 2%TTC staining. A battery of neurobehavioral tests, including rotarod, adhesive tape removal, and foot fault, were performed to assess sensorimotor functions. Total RNA was extracted from brain tissues and the expression of BBB tight junctions (TJs) was detected by qPCR and western blotting. Results: Compared to WT controls, EC-selective transgenic overexpression of KLF11 led to reduced BBB leakage in ischemic brains, evidenced by significantly reduced extravasation of BBB tracers and less brain water content. EC-KLF11 Tg mice also exhibited a smaller brain infarct and improved neurological functions in response to ischemic insults. Mechanistically, we found KLF11 binding sites in the promoter region of major endothelial TJs including Claudin 5 and ZO-1. EC-selective transgenic overexpression of KLF11 significantly increased cerebral TJ levels in mice after ischemic stroke. Conclusions: The present study has demonstrated that KLF11 functions at the EC level to preserve BBB structural and functional integrity and confers brain protection in ischemic stroke. KLF11 may be a novel therapeutic target for the treatment of ischemic stroke.

HMGB1 a-Box Reverses Brain Edema and Deterioration of Neurological Function in a Traumatic Brain Injury Mouse Model

Background/Aims: Traumatic brain injury (TBI) is a complex neurological injury in young adults lacking effective treatment. Emerging evidences suggest that inflammation contributes to the secondary brain injury following TBI, including breakdown of the blood brain barrier (BBB), subsequent edema and neurological deterioration. High mobility group box-1 (HMGB1) has been identified as a key cytokine in the inflammation reaction following TBI. Here, we investigated the therapeutic efficacy of HMGB1 A-box fragment, an antagonist competing with full-length HMGB1 for receptor binding, against TBI. Methods: TBI was induced by controlled cortical impact (CCI) in adult male mice. HMGB1 A-box fragment was given intravenously at 2 mg/kg/day for 3 days after CCI. HMGB1 A-box-treated CCI mice were compared with saline-treated CCI mice and sham mice in terms of BBB disruption evaluated by Evan’s blue extravasation, brain edema by brain water content, cell death by propidium iodide staining, inflammation by Western blot and ELISA assay for cytokine productions, as well as neurological functions by the modified Neurological Severity Score, wire grip and beam walking tests. Results: HMGB1 A-box reversed brain damages in the mice following TBI. It significantly reduced brain edema by protecting integrity of the BBB, ameliorated cell degeneration, and decreased expression of pro-inflammatory cytokines released in injured brain after TBI. These cellular and molecular effects were accompanied by improved behavioral performance in TBI mice. Notably, HMGB1 A-box blocked IL-1β-induced HMGB1 release, and preferentially attenuated TLR4, Myd88 and P65 in astrocyte cultures. Conclusion: Our data suggest that HMGB1 is involved in CCI-induced TBI, which can be inhibited by HMGB1 A-box fragment. Therefore, HMGB1 A-box fragment may have therapeutic potential for the secondary brain damages in TBI.

Cerebral Edema and Blood Brain Barrier Dysfunction in the Dahl S Rat Model of Superimposed Preeclampsia

Preeclampsia, characterized by hypertension and renal dysfunction in pregnancy, is associated with placental ischemia, which has been linked to increased blood‐brain barrier permeability and cerebral edema. We recently demonstrated that pregnant Dahl salt sensitive rats (Dahl S) closely mimic the maternal syndrome of preeclampsia. In this study, we tested the hypothesis that pregnant Dahl S rats develop cerebral vascular dysfunction associated with encephalopathies commonly observed in preeclamptic women. To test this, pregnant Dahl S and Sprague Dawley (SD, healthy pregnancy control) rats were studied during late pregnancy (gestational day 20, n=4–6). Mean arterial pressure (MAP) was significantly greater on day 20 of pregnancy in the Dahl S rat compared to SD (153±11 vs. 107±3 mmHg, p<0.05), and there was no drop in MAP during pregnancy in the Dahl S compared to virgin littermates (141±12, p=0.48). To assess blood‐brain barrier function, Evan's Blue extravasation into the brain was measured. Evan's Blue concentration was increased in both posterior (0.04±0.012 vs. 0.018±0.001 ng/g tissue/plasma concentration, p=0.07) and anterior (0.021±0.001 vs. 0.034±0.006, p=0.06) brain in pregnant Dahl S rats compared to virgin littermates, respectively. No difference was observed in either brain region in SD rats. In a separate group of Dahl S pregnant and virgin rats (n=5), brains were harvested and weighed on day 20, heated at 50°C for 72 hours, and weighed again to determine the dry:wet weight ratio as a measure of brain water content (edema). Greater water content was found in the posterior brain of the pregnant Dahl S compared to virgin littermates (78.0±0.1 vs. 77.4±0.2, p<0.05) with no significant difference in the anterior brain (79.2±0.2 vs. 78.8±0.3, p=0.33). These results suggest that the Dahl S rat model of superimposed preeclampsia exhibits blood brain barrier dysfunction and cerebral edema. Therefore, pregnant Dahl S rats may be a useful model to investigate mechanisms leading to cerebral vascular abnormalities that occur during preeclampsia.Support or Funding InformationNational Institutes of Health, American Society of Nephrology, UMMCIRSP

Abstract TP98: Genetic Deletion of Kruppel-Like Factor 11 Aggravates Ischemic Brain Injury

Kruppel-like factors (KLFs) are members of the zinc finger family of transcription factors and the function of the KLFs in the central nervous system is largely unexplored. KLF11 is a member of the KLF family and we have previously demonstrated that peroxisome proliferator-activated receptor gamma-mediated cerebral protection during ischemic insults needs recruitment of KLF11 as its critical coactivator. In this study we sought to determine the role of KLF11 itself in cerebrovascular function and the pathogenesis of ischemic stroke. Transient middle cerebral artery occlusion (MCAO) was performed in KLF11 knockout and wild-type control mice, and brain infarction size was analyzed by TTC staining. BBB integrity was assessed by using Evans Blue and TMR-Dextran extravasation assays. KLF11 KO mice exhibited significantly larger brain infarct volume and worse neurological outcomes in response to ischemic insults. Genetic deficiency of KLF11 in mice also significantly aggravated ischemia-induced BBB disruption by increasing cerebrovascular permeability and edema in comparison with wild-type mice. Mechanistically, KLF11 was found to directly regulate several key inflammatory cytokines in the brains of ischemic mice. These findings suggest that KLF11 acts as a novel protective factor in ischemic stroke. Elucidating the functional importance of KLF11 in ischemic process may lead us to discover novel pharmaceutical targets for the development of effective therapies against ischemic stroke.

Dynamic contrast-enhanced MRI in the evaluation of blood-brain barrier permeability following acute cerebral ischemia in rats

Objective To investigate the dynamic changes of blood-brain barrier(BBB) permeability after acute cerebral ischemia in rats with middle cerebral artery occlusion (MCAO) by dynamic contrast-enhanced(DCE)-MRI. Methods Sixty MCAO rat models were established by suture-occlusion method. All rats were divided randomly into twelve groups with different ischemia duration (3 hours, 6 hours, permanent) and reperfusion times (2, 6, 12 and 24 hours after reperfusion). Each group was examined by MRI at the time points. The BBB permeability parameters(Ktrans, Ve, Kep, rKtrans, rVe, rKep) were calculated by Siemens workstation and compared with Evans blue(EB) extravasation results. Multivariate analysis of variance (M-ANOVA), one-way analysis of variance (one-way ANOVA), Pearson analysis were respectively used to verify the influences of ischemia duration and reperfusion time on BBB permeability parameters, EB extravasation and relationships between parameters. Results In 3 hours and 6 hours ischemia duration groups, change of BBB permeability after reperfusion appeared biphasic. At 2 hours and 6 hours after reperfusion, BBB permeability increased, while rKtrans values and rVe values rose and rKep values dropped. BBB permeability decreased at 12 hours and increased again at 24 hours after reperfusion. The highest BBB permeability was observed at 6 hours after reperfusion. However, BBB permeability in permanent ischemia groups had uniphasic change, as its increase was rather mild as ischemia time went on. rKtrans values(1.99±0.79)were positively correlated with rVe values(2.88±1.78)(r=0.93, P<0.01) and negatively correlated with rKep values(0.66±0.21)(r=-0.84, P<0.01). The negative correlation between rVe values and rKep valueswas also significant(r=-0.80, P<0.01). EB extravasation results were consistent with MRI findings. Conclusions BBB permeability change was biphasic in reperfusion groups, while it was uniphasic in permanent ischemia groups. DCE-MRI may accurately reflect the changes of BBB permeability after acute cerebral ischemia. Both ischemic duration and reperfusion time had influences on BBB permeability. With prolongation of ischemic time, the duration of BBB permeability increase became shorter, BBB damage appeared earlier, with increased degree of ischemic damage. Key words: Blood brain barrier; Reperfusion injury; Infarction, middle cerebral artery; Magnetic resonance imging

Zinc contributes to acute cerebral ischemia-induced blood–brain barrier disruption

Zinc ions are stored in synaptic vesicles and cerebral ischemia triggers their release from the terminals of neurons. Zinc accumulation in neurons has been shown to play an important role in neuronal death following ischemia. However, almost nothing is known about whether zinc is involved in ischemia-induced blood–brain barrier (BBB) disruption. Herein, we investigated the contribution of zinc to ischemia-induced acute BBB disruption and the possible molecular mechanisms using both cellular and animal models of cerebral ischemia. Zinc greatly increased BBB permeability and exacerbated the loss of tight junction proteins (Occludin and Claudin-5) in the endothelial monolayer under oxygen glucose deprivation conditions. In cerebral ischemic rats, a dramatically elevated level of zinc accumulation in microvessels themselves was observed in isolated microvessels and in situ, showing the direct interaction of zinc on ischemic microvessels. Treatment with a specific zinc chelator N,N,N′,N′-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN), even at 60-min post-ischemia onset, could greatly attenuate BBB permeability in the ischemic rats as measured by Evan's Blue extravasation, edema volume and magnetic resonance imaging. Furthermore, zinc accumulation in microvessels activated the superoxide/matrix metalloproteinase-9/-2 pathway, which leads to the loss of tight junction proteins (Occludin and Claudin-5) and death of endothelial cells in microvessels themselves. Our findings reveal a novel mechanism of cerebral ischemia-induced BBB damage, and implicate zinc as an effective and viable new target for reducing acute BBB damage following ischemic stroke.

Normobaric Hyperoxia Extends Neuro- and Vaso-Protection of N-Acetylcysteine in Transient Focal Ischemia.

N-acetylcysteine (NAC), a precursor of glutathione that reduces reperfusion-induced injury, has been shown protection when it was administered pre-ischemia. However, less is known about the effect when it was given post-ischemia and there is no positive result associated with anti-oxidant in clinical trials. This study investigated the neuro- and vaso-protection of post-ischemia NAC administration as well as combining NAC with normobaric hyperoxia (NBO). Male Sprague-Dawley rats were exposed to NBO or normoxia during 2-h occlusion of the middle cerebral artery, followed by 48-h reperfusion. NAC or vehicle was intraperitoneally administered to rats immediately before reperfusion onset. NAC and NBO treatments produced 1.2 and 30% reduction of infarction volume, respectively, and combination treatment showed greater reduction (59.8%) as well as more decrease of hemispheric swelling volume. Of note, combination therapy showed improved neurological assessment and motor function which were sustained for 7days after reperfusion. We also determined that the combination therapy showed greater inhibitory effects on tight junction protein degradation accompanied by Evan's blue extravasation, hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) induction, and poly ADP-ribose polymerase (PARP)-1 activation in ischemic brain tissue. Our results showed that although post-ischemia NAC administration had limited protection, combination treatment of NAC plus NBO effectively prevented blood-brain barrier (BBB) damage and significantly improved the outcome of brain injury, providing new evidence to support the concept that "cocktail" treatment targeting different stages provides better neuro- and vaso-protection than current individual treatment that has all failed in their clinical trials.

Effect of obesity on early blood–brain barrier disruption following transient focal cerebral ischemia

Summary Objective We determined the effect of obesity on early post-ischemic blood–brain barrier (BBB) disruption. Methods Male C57BL/6J mice were fed a high-fat diet (HFD) or standard chow for 16 weeks. Transient focal cerebral ischemia was induced by directly ligating the middle cerebral artery for 2 h. Early BBB disruption was assessed by measuring Evans Blue and sodium fluorescein extravasation at 3 h of reperfusion. Results Obesity produced an increase in cerebral vasodilation/hyperaemia during reperfusion. N(omega)-propyl-L-arginine and 7-nitroindazole (neuronal nitric oxide synthase inhibitors) failed to alter the cerebral vasodilation/hyperaemia in lean mice, but significantly inhibited the cerebral vasodilation/hyperaemia in obese mice. The magnitude of early post-ischemic BBB disruption was significantly greater in obese mice compared with lean mice. Topical treatment with N(omega)-propyl-L-arginine, 7-nitroindazole or N(omega)-propyl-L-arginine, 7-nitroindazole (a non-specific nitric oxide synthase [NOS] inhibitor) completely abolished the BBB disruption in lean mice, but only partially suppressed the BBB disruption in obese mice. Furthermore, a reduced matrix metallopeptidase (MMP)-9 activity and increased endothelial NOS accompanied with unchanged protein expression of tight/adherens junctions were found in cerebral cortex of obese mice. Conclusions Our findings suggest that obesity exacerbates early post-ischemic BBB disruption via a mechanism independent of MMP or NOS.

Abstract TP110: Polymerase δ-Interacting Protein 2 Regulates Astrocyte Activation in Ischemic Stroke

Introduction: Polymerase δ-Interacting Protein 2 (Poldip2) is a binding partner of Nox4 NADPH oxidase and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). Nox4 and CEACAM1 have been implicated in stroke, but with different outcomes: both contribute to neurodegeneration but only CEACAM1 contributes to blood brain barrier (BBB) dysfunction. However, the underlying mechanisms are still obscure. Hypothesis: Poldip2+/- mice may be protected from the consequences of transient middle cerebral artery occlusion (tMCAO). Methods: tMCAO was induced in wild type (WT) and Poldip2+/- mice. The volume of the ischemic lesion was measured in TTC-stained sections. BBB breakdown was evaluated by Evans blue dye extravasation. Poldip2 protein expression was evaluated by immunofluorescence and western blotting. RT-PCR was used to measure mRNA levels of cytokines, MMPs and TIMPs. Astrocytes were transfected with Poldip2 siRNA in culture and mRNA levels of cytokines were evaluated. Results: Poldip2+/- and WT mice displayed comparable infarct sizes following tMCAO (n=6). A decrease in Evans blue dye extravasation was observed in Poldip2+/- mice (25±3 vs 6±2uM/g) (n=7). Upregulation of cytokine mRNA following tMCAO was also attenuated in Poldip2+/- mice (n=5): MCP-1(253±34 vs 83±23AU), IL-6 (134±38 vs 38±10AU), TNFα (39±12 vs 12±3AU), MMP-2 (19±3 vs 10±1AU), MMP-9 (253±12 vs 13±3AU) and TIMP-1 (286±110 vs 61±15AU). Poldip2 protein expression increased in the ischemic brain of WT mice after tMCAO (69±4 vs 20±5AU) and was predominantly located in astrocytes (n=4). Poldip2 protein expression was also increased in astrocytes following oxygen and glucose deprivation (79±15 vs 27±5AU) (n=6), and Poldip2 siRNA prevented cytokine induction under these conditions. Conclusions: In conclusion, Poldip2 contributes to stroke-induced BBB breakdown via its ability to inhibit a pro- inflammatory response in perivascular astrocytes.

Abstract WP105: Therapeutic Time Window for Conivaptan Treatment of Cerebral Edema and Blood-Brain Barrier Breakdown in Mice

Introduction: Stroke is complicated by brain edema, blood-brain barrier (BBB) disruption, and is often accompanied by increased release of arginine-vasopressin (AVP). AVP, acting through V1a and V2 receptors can trigger hyponatremia, vasospasm, and platelet aggregation which can exacerbate brain edema. We recently reported that conivaptan, V1a and V2 receptor blocker, reduces stroke-evoked brain edema and blood-brain barrier (BBB) disruption in mice when administered immediately after reperfusion. However, these pre-clinical results can be translated into clinical applications if the therapeutic time window for conivaptan is explored. Hypothesis: We hypothesized that conivaptan will alleviate brain edema and BBB disruption even if the treatment is delayed. Methods: Mice underwent the filament model of middle cerebral artery occlusion (MCAO) with reperfusion. After 60 minutes occlusion, mice were recovered and randomly allocated into the following groups: conivaptan or vehicle-treated at 3 or 5 hours after MCAO. Treatments were administered via a jugular catheter and continued for 48 hours. Physiological variables, plasma and urine sodium and osmolality, brain water content (BWC) and Evans Blue (EB) extravasation were evaluated at the end point. Results: Conivaptan produced aquaresis as indicated by changes in plasma and urine sodium and osmolality. Conivaptan treatment, when initiated at 3 hours after MCAO reduced BWC in the ipsilateral hemisphere from 80.2 ± 0.2% (vehicle) to 78.1 ± 0.1% (p &lt; 0.05 vs vehicle, n=10), and attenuated the EB extravasation index from 1.3 ± 0.2 (vehicle) to 1.01 ± 0.1 (p &lt; 0.05, n=5-7). However, a 5-hour delay of conivaptan treatment after MCAO did not affect ipsilateral BWC (n=10). Conclusion: A 3-hour delay of conivaptan treatment after experimental stroke is effective at reducing brain edema and protecting BBB integrity. Further delay of treatment up to 5 hours after stroke abolishes the beneficial effect of conivaptan on postischemic brain edema. In conclusion, these results present an opportunity for potential use of conivaptan to treat post-ischemic brain edema in ICU patients.

The NACHT, LRR and PYD Domains-Containing Protein 3 (NLRP3) Inflammasome Mediates Inflammation and Voiding Dysfunction in a Lipopolysaccharide-Induced Rat Model of Cystitis.

NOD-like receptors (NLRs) sense sterile and non-sterile signals and form inflammasomes which trigger an inflammatory response through the activation of caspase-1 and release of IL-1β. Recently we have shown the presence of several NLRs in the bladder urothelia and demonstrated the importance of NLRP3 in bladder outlet obstruction and cyclophosphamide-induced cystitis, both models of sterile inflammation. In this study we explore a role for NLRP3 in mediating the response to LPS, a key antigen of uropathogenic bacteria. In order to bypass the protective glycosaminoglycan layer lining the urothelium, LPS was directly injected into the bladder wall of Sprague-Dawley rats. Glyburide (a NLRP3 inhibitor) or vehicle was administered orally prior to and after injection. Rats were analyzed 24 h later. Inflammasome activity (caspase-1 activity, IL-1β release) and inflammation (Evan's Blue extravasation, bladder weight) were assessed, as was physiological bladder function (urodynamics). Injection of LPS stimulated inflammasome activation (caspase-1 activity) and the release of IL-1β into the urine which was prevented by glyburide. Likewise, LPS increased inflammation, (bladder weight and the extravasation of Evan's blue dye), and this was reversed by glyburide. Functionally, animals injected with saline alone demonstrated decreased voiding volume as measured by urodynamics. In the presence of LPS, additional urinary dysfunction was evident with decreased voiding pressures and threshold pressures. The decrease in voiding pressure was blocked by glyburide but the decrease in threshold pressure was not, suggesting that LPS has significant effects mediated by inflammasome-dependent and -independent mechanisms. Overall, the results demonstrate the potential importance of inflammasomes in bacterial cystitis as well as the ability of the bladder wall injection technique to isolate the in vivo effects of specific inflammasome ligands to the physiological changes associated with cystitis.

Neuroprotective effects of novel antiepileptic drug lacosamide via decreasing glial activation in the hippocampus of a gerbil model of ischemic stroke

neuroprotective effects of HQ against ischemic damage in a focal cerebral ischemia rat model. Results: It was proven that preand post-treatment with 100mg/kg of HQ protects from ischemia-induced cerebral damage, which was confirmed by evaluation of neurological deficit, PET (Positron-emission tomography) and TTC (2,3,5triphenyltetrazoliumchloride) staining. In addition, preand post-treatment with 100mg/kg of HQ significantly attenuated ischemia-induced Evans blue dye extravasation, and significantly increased the immunoreactivities and protein levels of SMI-71 and glucose transporter-1 (GLUT-1), which were well-known as useful makers of endothelial cell, in ischemic cortex compared to vehicletreated-group. Conclusion: Briefly, these results indicate that preand posttreatment with HQ can protect from ischemic damage induced by transient focal cerebral ischemia, and the neuroprotective effects of HQmaybe closely associatedwith thepreventionof BBBdisruption via increasing of SMI-71 and GLUT-1 expressions.

Relationship of ultrasound cavitation pressure amplitude and rat blood brain barrier permeability

Objective To explore the effectiveness of different peak negative pressure ultrasound irradiated microbubble destruction on the control of rat blood brain barrier permeability. Methods Thirty healthy SD rats were divided into 5 groups according to different ultrasound pressure level (62 kPa, 86 kPa, 181 kPa, 324 kPa, 708 kPa). After intravenous injection of microbubble, the rats were underwent ultrasonic irradiation for 9 minutes, and the contralateral hemispheres were served as controls. The permeability of blood brain barrier was determined by the quantitative analysis of Evans blue and lanthanum nitrate extravasation. Results Quantitative analysis results revealed a leakage in Evans blue about(4.52±0.11) μg/g and (10.56±0.13)μg/g in group 181 kPa and 708 kPa, respectively. A wide endothelial cell gap with lanthanum nitrate trace leaked out was shown at the group of ultrasound pressure of 181 kPa, and erythrocytes extravasations were found when ultrasound pressure at 708 kPa. Conclusions The pressure amplitude in microbubble enhanced cavitation can effectively regulate and control blood-brain barrier opening in rats. Key words: Blood-brain barrier; Microbubblest; Cavitation; Hemicerebrum; Rat

TGFβ1 exacerbates blood–brain barrier permeability in a mouse model of hepatic encephalopathy via upregulation of MMP9 and downregulation of claudin-5

Recent studies have found that vasogenic brain edema is present during hepatic encephalopathy following acute liver failure and is dependent on increased matrix metalloproteinase 9 (MMP9) activity and downregulation of tight junction proteins. Furthermore, circulating transforming growth factor β1 (TGFβ1) is increased following liver damage and may promote endothelial cell permeability. This study aimed to assess whether increased circulating TGFβ1 drives changes in tight junction protein expression and MMP9 activity following acute liver failure. Blood–brain barrier permeability was assessed in azoxymethane (AOM)-treated mice at 6, 12, and 18 h post-injection via Evan's blue extravasation. Monolayers of immortalized mouse brain endothelial cells (bEnd.3) were treated with recombinant TGFβ1 (rTGFβ1) and permeability to fluorescein isothiocyanate-dextran (FITC-dextran), MMP9 and claudin-5 expression was assessed. Antagonism of TGFβ1 signaling was performed in vivo to determine its role in blood–brain barrier permeability. Blood–brain barrier permeability was increased in mice at 18 h following AOM injection. Treatment of bEnd.3 cells with rTGFβ1 led to a dose-dependent increase of MMP9 expression as well as a suppression of claudin-5 expression. These effects of rTGFβ1 on MMP9 and claudin-5 expression could be reversed following treatment with a SMAD3 inhibitor. AOM-treated mice injected with neutralizing antibodies against TGFβ demonstrated significantly reduced blood–brain barrier permeability. Blood–brain barrier permeability is induced in AOM mice via a mechanism involving the TGFβ1-driven SMAD3-dependent upregulation of MMP9 expression and decrease of claudin-5 expression. Therefore, treatment modalities aimed at reducing TGFβ1 levels or SMAD3 activity may be beneficial in promoting blood–brain barrier integrity following liver failure.

Inhibition of multidrug resistance-associated protein 4 protects against pulmonary vascular endothelial barrier dysfunction in septic rats

Objective To explore the effects of multidrug resistance-associated protein 4 (MRP4) inhibition on pulmonary vascular endothelial barrier dysfunction in septic rats. Methods Sixty Sprague Dawley rats were randomly (random number) divided into three groups: sham-operated group, sepsis group, and sepsis plus MRP4 inhibitor treatment group, with 20 rats in each group. Sepsis was induced by cecal ligation and puncture. MRP4 inhibitor MK571 (20 mg/kg) was administrated by intraperitoneal injection 30 minutes before induction of sepsis. Twenty-four later, serum interlukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) levels were measured by enzyme-linked immunosorbent assay. Lung injury was assessed by histopathological examination. Lung vascular permeability was evaluated by quantitation of Evans blue dye extravasation from vascular space to lung parenchyma. Results Compared with sham group, serum IL-6 and TNF-α levels were significantly higher in sepsis group. In addition, lung injury and lung vascular permeability were elevated in sepsis group compared to sham group. Importantly, MRP4 inhibitor treatment significantly decreased serum IL-6 and TNF-α levels, improved lung injury and reduced lung vascular permeability in septic rats. Conclusions Inhibition of MRP4 protects against pulmonary vascular endothelial barrier dysfunction in septic rats. Key words: Sepsis; Vascular endothelial barrier; Multidrug resistance-associated protein 4

Lack of dystrophin results in abnormal cerebral diffusion and perfusion in vivo

Dystrophin, the main component of the dystrophin–glycoprotein complex, plays an important role in maintaining the structural integrity of cells. It is also involved in the formation of the blood–brain barrier (BBB). To elucidate the impact of dystrophin disruption in vivo, we characterized changes in cerebral perfusion and diffusion in dystrophin-deficient mice (mdx) by magnetic resonance imaging (MRI). Arterial spin labeling (ASL) and diffusion-weighted MRI (DWI) studies were performed on 2-month-old and 10-month-old mdx mice and their age-matched wild-type controls (WT). The imaging results were correlated with Evan's blue extravasation and vascular density studies. The results show that dystrophin disruption significantly decreased the mean cerebral diffusivity in both 2-month-old (7.38±0.30×10-4mm2/s) and 10-month-old (6.93±0.53×10-4mm2/s) mdx mice as compared to WT (8.49±0.24×10-4, 8.24±0.25×10-4mm2/s, respectively). There was also an 18% decrease in cerebral perfusion in 10-month-old mdx mice as compared to WT, which was associated with enhanced arteriogenesis. The reduction in water diffusivity in mdx mice is likely due to an increase in cerebral edema or the existence of large molecules in the extracellular space from a leaky BBB. The observation of decreased perfusion in the setting of enhanced arteriogenesis may be caused by an increase of intracranial pressure from cerebral edema. This study demonstrates the defects in water handling at the BBB and consequently, abnormal perfusion associated with the absence of dystrophin.

Effects of hypoxia-inducible factor-1α and matrix metalloproteinase-9 on alveolar-capillary barrier disruption and lung edema in rat models of severe acute pancreatitis-associated lung injury

The aim of this study was to investigate the effects of hypoxia-inducible factor-1α (HIF-1α) and matrix metalloproteinase-9 (MMP-9) on alveolar-capillary barrier disruption and lung edema in rat models of severe acute pancreatitis-associated lung injury (PALI). A total of 40 male Sprague-Dawley rats were randomly divided into a sham surgery group (n=10) and three PALI groups, in which acute pancreatitis was induced by the retrograde infusion of 5% sodium taurocholate (1 ml/kg). The PALI groups were as follows: i) Untreated PALI group (n=10); ii) 2-methoxyestradiol (2ME2) group (5 mg/kg body mass; n=10); and iii) 2ME2 group (15 mg/kg body mass; n=10). In the two 2ME2 groups, the HIF-1α inhibitor 2ME2 was administered intraperitoneally 1 h after the induction of AP. The severity of the pancreatitis was evaluated by the serum amylase levels and pathology. The severity of the lung injury was evaluated by the wet/dry ratio, blood gas analysis and pathology. The alveolar-capillary barrier disruption was assessed by Evans blue dye extravasation. The protein and mRNA expression levels of HIF-1α and MMP-9 were studied using enzyme-linked immunosorbent assays (ELISAs), western blot analysis and reverse transcription-polymerase chain reaction. The active tumor necrosis factor-α levels were measured using an ELISA. The HIF-1α inhibitor 2ME2 attenuated the severity of the pancreatitis and PALI, while the lung edema and alveolar-capillary barrier disruption were significantly ameliorated compared with those in the untreated PALI group. Administration of the higher dose of 2ME2 significantly suppressed the protein expression of MMP-9 in the lung tissues. The results indicate that HIF-1α has a major function in alveolar-capillary barrier disruption and lung edema in PALI via a molecular pathway cascade involving MMP-9. Inhibition of HIF-1α by 2ME2 attenuates alveolar-capillary barrier disruption and lung edema. Pharmacological blockade of this pathway in patients with PALI may provide a novel therapeutic strategy.

Abstract W P90: Insulin-like Growth Factor-1-mediated Reduction of Stroke-induced Brain Infarction is Preceded by Improvement of Blood Brain Barrier Integrity and Inflammatory Cytokines

Background and Purpose: Although insulin-like growth factor (IGF)-1 treatment has been shown to reduce stroke-induced infarct volume, the mechanisms underlying its neuroprotective actions are not fully understood. The present study tested the hypothesis that IGF-1 may regulate the neuroinflammatory cascade initiated by ischemic stroke, specifically, by promoting the integrity of the blood brain barrier. Methods: Middle-aged (10-12 month old) female rats were implanted with a cannula directed toward the lateral ventricle. One week later, animals were anesthetized using isoflurane and the middle-cerebral artery was occluded using an intraluminal suture maintained in place for 90 mins. IGF-1 or vehicle was delivered icv, via a mini-pump, following reperfusion. All animals were terminated at 4h or 24h post MCAo. Blood brain barrier permeability was determined using Evan’s blue extravasation and infarct volumes were determined from TTC-stained brain sections. Inflammatory cytokines were analyzed from brain lysates by ELISA. Results: IGF-1 treatment to middle-aged females resulted in a 39% reduction in infarct volume when measured 24h post MCAo, confirming the neuroprotective action of this peptide hormone. In order to assess the effects of IGF-1 on early stroke induced effects, we next determined blood brain permeability 4h post stroke as well as the expression of cytokines in the ischemic hemisphere. IGF-1 treatment resulted in a significant reduction of blood brain barrier permeability at 4h post-stroke compared to vehicle treated animals. Moreover, multiplex cytokine analysis showed that IGF-1 significantly reduced inflammatory markers such as IL-6, IFN-gamma, TNF-alpha, eotaxin, GM-CSF. Brain infarct volume at 4h was similar in both IGF-1 and vehicle groups, indicating that the effects of IGF-1 on inflammatory markers precede its effects on infarct volume. IGF-1 also suppressed IL-6, MIP-2, GRO-KC, MCP-1, EGF and MIP-1-alpha expression at 24h post stroke, indicating a persistent anti-inflammatory effect for this growth factor. Conclusions: The above findings support the hypothesis that IGF-1 has unique anti-inflammatory action in the aging female brain, and suggest that brain endothelial cells may be a primary target of IGF-1.

Neuroprotective effects of low dose minocycline against focal cerebral ischemia reperfusion injury in rats

Objective To investigate the neuroprotective effects of low dose intravenous minocycline against ischemia reperfusion injury in rats after focal cerebral ischemia reperfusion and to explore the possible mechanism.Methods Seventy-two rats were randomly divided into sham-operated group(S group),cerebral ischemia/reperfusion group(I/R group),and minocycline intervention group(I/R+ MC group).Focal cerebral ischemia was induced by filament medial cerebral artery occlusion method.Minocycline(3mg/kg)in saline was administered intravenously via the caudal vein twice a day for 14 days in the I/R+ MC group.At 2days after ischemia reperfusion,the infarct volume was evaluated using TTC staining,the permeability of blood-brain barrier was assessed by Evan's blue(EB)dye extravasation,and the expressions of high mobility group box-1protein(HMGB1)and Iba1,a marker of activated microglia,were analyzed by using Western blotting analysis.The neurological function recovery was evaluated using the modified neurological severity score(mNSS)at 2d,7d,and 14 d after ischemia/reperfusion.Results Two days after cerebral ischemia reperfusion,the brain infarction volume and the extravasations of EB were significantly increased and the expressions of HMGB1 and Iba1were significantly up-regulated in I/R group compared with sham group(P0.05).Compared with I/R group,minocycline at 3 mg/kg via the caudal veinsignificantly reduced the infarct volume in ischemic brain(P0.05),extravasation of EB and expressions of HMGB1 and Iba1(P0.05).Additionally,the rats in I/R+MC group exhibited a significantly decreased neurological severity score compared with I/R group(P0.05).Conclusion Minocycline exerts a neuroprotective effect in rats with cerebral ischemia reperfusion injury,which may be related to decrease of infarct size,inhibition of EB extravasation and microglia activation.

Vardenafil ameliorates immunologic- and non-immunologic-induced allergic reactions

Cyclic nucleotides, such as cAMP and cGMP, play a protective role in the modulation of the activity of some inflammatory cells in allergic disorders. Their intracellular concentrations are tightly regulated by the phosphodiesterases (PDEs). The protective efficacy of the selective PDE5 inhibitor vardenafil against mast-cell-mediated allergic reactions in murine models has been investigated. Compound 48/80 was used as a direct mast cell degranulator to induce anaphylaxis. Vardenafil (administered orally at 5, 10, 20, 40, and 80 mg/kg body mass) significantly (P < 0.05, n = 12) increased protection against compound-48/80-induced anaphylaxis in mice to 33.33%, 66.67%, 66.67%, 83.33%, and 66.67% respectively compared with the control (vehicle). In passive cutaneous anaphylaxis (PCA) in rats, vardenafil (10 mg/kg body mass) significantly (P < 0.05, n = 6) decreased Evans' blue dye extravasation (4.6-fold). Pre-incubation of isolated rat peritoneal mast cells (RPMCs) with vardenafil (10 and 100 μmol/L) significantly (P < 0.05, n = 6) reduced compound-48/80-induced histamine release by 2.8- and 3-fold, respectively. Moreover, histamine release by immunogenic stimulation of sensitized RPMCs by egg albumin significantly declined following pre-incubation with vardenafil (10 and 100 μmol/L) by 1.94- and 1.99-fold, respectively. In conclusion, inhibition of PDE5 by vardenafil ameliorated immunologic and non-immunologic mast-cell-mediated allergic reactions and reduced histamine release, providing evidence for the potential anti-allergic properties of vardenafil.