• blood–brain barrier;
  • cerebral ischemia;
  • endothelial cells;
  • microsphere;
  • nitric oxide synthase;
  • tyrosine nitration


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References

Microsphere embolism (ME)-induced up-regulation of endothelial nitric oxide synthase (eNOS) in endothelial cells of brain microvessels was observed 2–48 h after ischemia. eNOS induction preceded disruption of the blood–brain barrier (BBB) observed 6–72 h after ischemia. In vascular endothelial cells, ME-induced eNOS expression was closely associated with protein tyrosine nitration, which is a marker of generation of peroxynitrite. Leakage of rabbit IgG from microvessels was also evident around protein tyrosine nitration-immunoreactive microvessels. To determine whether eNOS expression and protein tyrosine nitration in vascular endothelial cells mediates BBB disruption in the ME brain, we tested the effect of a novel calmodulin-dependent NOS inhibitor, 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate (DY-9760e), which inhibits eNOS activity and, in turn, protein tyrosine nitration. Concomitant with inhibition of protein tyrosine nitration in vascular endothelial cells, DY-9760e significantly inhibited BBB disruption as assessed by Evans blue (EB) excretion. DY-9760e also inhibited cleavage of poly (ADP-ribose) polymerase as a marker of the apoptotic pathway in vascular endothelial cells. Taken together with previous evidence in which DY-9760e inhibited brain edema, ME-induced eNOS expression in vascular endothelial cells likely mediates BBB disruption and, in turn, brain edema.

Abbreviations used

blood–brain barrier




3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate


Evans blue


endothelial NOS


inducible NOS


microsphere embolism


nitric oxide synthase


neuronal NOS


poly(ADP-ribose) polymerase


phosphate-buffered saline

Production of nitric oxide (NO) by nitric oxide synthase (NOS) has important roles in physiological and pathological events in the central nervous system. Accumulating evidence suggests that both neuronal NOS (nNOS) and inducible NOS (iNOS) have detrimental effects on neurons in the ischemic brain, whereas endothelial NOS (eNOS) activity has protective effects (Iadecola 1997). Endothelium-derived NO regulates blood pressure, augments regional blood flow, improves cerebral circulation and inhibits platelet aggregation (Radomski et al. 1990; Morikawa et al. 1994; Huang et al. 1995; Iadecola 1997). As several studies suggest that treatment with 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) up-regulates eNOS expression, thereby improving endothelial function (Endres et al. 1998; Laufs et al. 1998, 2000, 2002), statins are believed to reduce the risk of myocardial infarction and stroke (Ganz et al. 2000). Beasley et al. (1998) showed eNOS up-regulation via an indomethacin-sensitive mechanism in a global ischemia model induced by increasing intracranial pressure, whereas it is still controversial whether cerebral ischemia itself causes eNOS induction in vessels (Zhang et al. 1993; Limbourg et al. 2002; Veltkamp et al. 2002). We recently showed that eNOS up-regulation in vascular endothelial cells is elicited by sublethal ischemia, inducing preconditioning and lethal ischemia in the gerbil forebrain, and that the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway mediates eNOS up-regulation (Hashiguchi et al. 2004). eNOS expression following sublethal ischemia likely mediates preconditioning-induced neuroprotection.

Notably, in lethal ischemia, we found a marked increase in NO production 24–48 h after transient forebrain ischemia in the gerbil (Hashiguchi et al. 2003) and microsphere embolism (ME) in the rat (Shirakura et al. 2005). In both cases, increased NO production coincided with increased eNOS expression without changes in nNOS and iNOS. Thus, the pathological relevance of eNOS up-regulation and prolonged NO production in the late phase by lethal ischemia remains unclear. Interestingly, aberrant expression of nNOS, iNOS and eNOS has also been reported in the Alzheimer's (AD) brain (Luth et al. 2002). In that case, nNOS is mainly expressed in cortical pyramidal neurons, whereas iNOS and eNOS are highly expressed in astrocytes. It is also notable that aberrant NOS expression in the AD brain co-localized with immunoreactivity against nitrotyrosine, a marker of formation of peroxynitrite (ONOO). Peroxynitrite is produced by generation of both peroxide (inline image) and NO, thereby leading to oxidization of proteins, membrane lipid and DNA, or nitration of proteins (Ischiropoulos and al-Mehdi 1995). Peroxynitrite also modifies free tyrosine and tyrosine residues in proteins, resulting in their loss of function. For example, inactivation of manganese superoxide dismutase by tyrosine nitration occurs in rat renal ischemia/reperfusion injury (Cruthirds et al. 2003).

The blood–brain barrier (BBB) in brain microvessels maintains homeostasis of the brain microenvironment mostly through maintenance of tight junctions between brain vascular endothelial cells, thereby preventing passage of hydrophilic molecules or toxic substances from blood to brain. NO and peroxynitrite are known to elicit cerebral microvascular injury, resulting in BBB disruption, following cerebral ischemia (Janigro et al. 1994; Mayhan and Didion 1996; Greenacre et al. 1997; Tan et al. 2004). Of note, inhibition of NOS attenuates BBB disruption during middle cerebral artery occlusion (MCAO) (Nagafuji et al. 1995) and experimental meningitis (Boje 1996). However, the precise molecular mechanisms underlying NO/peroxynitrite-induced BBB disruption are not fully understood.

Here, we report that up-regulation of eNOS in ME-induced cerebral ischemia predominantly occurs in vascular endothelial cells, thereby eliciting protein tyrosine nitration in the same cells. Increased protein tyrosine nitration in microvascular endothelial cells leads to cell damage, thereby inducing BBB disruption. To confirm this hypothesis, we tested the effect of a novel calmodulin-dependent NOS inhibitor, 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate (DY-9760e) (Fukunaga et al. 2000), on ME-induced BBB disruption. ME induced multi embolic infarcts mainly in the striatum, cortex, thalamus and hippocampus, and DY-9760e treatment (50 mg/kg, i.p.) elicited a significant reduction (about 60% of reduction compared with vehicle-treated animals) in infarct area after ME (Shirakura et al. 2005). Using DY-9760e, we confirm that inhibition of protein tyrosine nitration protects endothelial cells, thereby attenuating BBB disruption. The present study also supports the idea that DY-9760e is a novel therapeutic agent to treat brain edema following multiple cerebral infarction.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References

Microsphere-induced permanent focal cerebral ischemic model

All experimental procedures using animals were approved by the Committee on Animal Experiments at Tohoku University. The ME model was prepared as described previously (Shirakura et al. 2005). Briefly, male Wistar rats weighing 220–270 g were subjected to general anesthesia by 1.5% halothane in oxygen via a face mask. After placing animals in a supine position, both external carotid and pterygopalatine arteries were temporarily occluded using aneurysm clips, and then 1000 non-radioactive microspheres (48.4 ± 0.7 µm in diameter) suspended in a 20% dextran solution were injected into the left common carotid artery. Rectal temperature was monitored and maintained at 37 ± 0.5°C with a heating blanket throughout the surgery. Sham-operated groups received the same experimental procedures except for microsphere injection. Behavioral changes, such as lack of movement, truncal curvature and forced circling during locomotion, following microsphere injection were observed as previously described (Miyake et al. 1993). After recovery from anesthesia, animals were returned to their cages with free access to food and water for the duration of observation. Rats were decapitated at 15 min, 2, 6, 12, 24, 48 and 72 h after injection of microspheres, and the striatum and cortical regions of the ipsilateral hemisphere were dissected out for immunoblotting analysis.

Drug treatments

DY-9760e was dissolved in 100% dimethylsulfoxide (DMSO) and diluted in a 5% glucose solution to a final concentration of 1% DMSO. In each case, DY-9760e was administered twice, 30 min and 6 h after microsphere injection (12.5 mg/kg or 25 mg/kg, i.p.). Vehicle-treated animals were administered the same volume of 1% DMSO in 5% glucose 30 min and 6 h after ME. The dose of DY-9760e used did not affect the mean arterial blood pressure monitored 30 min after DY-9760e administration (data not shown).

Protein extraction

After decapitation, brains were removed and rinsed once with cold 0.32 m sucrose. Coronal sections 2 mm thick were prepared using a brain slicer, and ipsilateral hemispheres including cortex and striatum were dissected and stored at − 80°C. Frozen brain tissues were homogenized in buffer containing 50 mm Tris-HCl (pH 7.4), 0.5% Triton X-100, 4 mm EGTA, 10 mm EDTA, 1 mm Na3VO4, 30 mm sodium pyrophosphate, 50 mm NaF, 50 µg/mL leupeptin, 25 µg/mL pepstatin A, 50 µg/mL trypsin inhibitor and 1 mm dithiothreitol (DTT). Soluble cytosolic fraction was obtained by a 10 min centrifugation at 15 000 g. The pellet was suspended in nuclei extraction buffer (50 mm Tris-HCl, 0.5% Triton X-100, 4 mm EGTA, 10 mm EDTA, 0.5 m NaCl, 1 mm Na3VO4, 30 mm sodium pyrophosphate, 50 mm NaF), vortexed, shaken for 30 min on ice and subsequently centrifuged at 14 000 g for 10 min in 4°C. The supernatant fluid contained the nuclear protein extract. Protein concentration in each fraction was determined using Bradford's method.

Electrophoresis and western blotting

Samples containing equivalent amounts of protein were applied to a 10% acrylamide denaturing gel [sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE)] (Laemmli 1970). Proteins were then transferred to an Immobilon polyvinylidene difluoride (PVDF) transfer membrane for 2 h at 70 V. Membranes were blocked in 20 mm Tris-HCl (pH 7.4), 150 mm NaCl and 0.1% Tween 20 (TBS-T) containing 5% fat-free milk powder, for 1 h at room temperature (28°C), and incubated with antibodies to nNOS (rabbit polyclonal antibody 1 : 5000; Sigma-Aldrich, St Louis, MO, USA), iNOS (rabbit polyclonal antibody 1 : 2000; Upstate Biotechnology, Lake Placid, NY, USA), eNOS (rabbit polyclonal antibody 1 : 500; Sigma-Aldrich), β-tubulin (mouse monoclonal antibody 1 : 10 000; Sigma-Aldrich) and poly(ADP-ribose) polymerase (PARP) (rabbit polyclonal antibody 1 : 500; BioMol, Plymouth Meeting, PA, USA), overnight, at 4°C. After washing, membranes were incubated for 60 min at room temperature (28°C) with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody diluted in TBS-T solution for 60 min at room temperature (28°C). Immunoreactive proteins were visualized with the enhanced chemiluminescence detection system (Amersham Life Sciences, Little Chalfont, UK). Images were scanned and analyzed semi-quantitatively using Image Gauge Software (Fuji film, Tokyo, Japan).

Immunohistochemical studies

At 24 h after ME, rats were anesthetized and transcardially perfusion-fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) as previously described (Kawano et al. 2002). Whole brains were immediately removed and post-fixed overnight at 4°C in the same fixative solution. Coronal brain sections at the co-ordinates of 1 mm posterior to bregma (35 µm thick) were prepared using a vibratome. Sections were incubated at room temperature (28°C) with 0.01% Triton-X100 in PBS for 30 min and for a further 1 h in 3% bovine serum albumin (BSA) in PBS. For immunolabeling, sections were incubated with anti-eNOS (rabbit polyclonal antibody 1 : 200) or anti-PARP-p85 (rabbit polyclonal antibody 1 : 200; Promega, Madison, WI, USA) with anti-nitrotyrosine (mouse monoclonal antibody 1 : 200; Upstate Biotechnology), overnight, at 4°C. After washing, sections were incubated with biotinylated anti-rabbit IgG (1 : 5000) in Tris-buffered NaCl blocking (TNB) buffer for 1 h, followed by both streptavidin-HRP (1 : 5000) and Alexa 594 anti-mouse IgG (Molecular Probes) in TNB buffer (1 : 400), and labeled for 2 h. Sections were then stained with tetramethylrhodamine tyramide for 10 min using the Tyramide signal amplification (TSA)-Detect kit (NEN Life Science Products, Boston, MA, USA). Immunofluorescent images were taken with a confocal laser scanning microscope (TCS SP, Leica Microsystems). To normalize immunoreactivity with anti-nitrotyrosine antibody, we analyzed cortical slices without changing the confocal laser intensity and laser aperture.

Evaluation of BBB damage

The loss of BBB integrity was assessed by leakage of Evans blue from microvessels after intravenous injection (Uyama et al. 1988). Evans blue solution (2% in saline, 4 mL/kg) was intravenously administrated via the tail vein at 22 h after onset of ME. Rats were then perfused transcardially with saline, under anesthesia with pentobarbital, 24 h after ME to clear the blood and intravascular Evans blue remaining in the vascular system. After decapitation, forebrains except cerebellum were removed and divided into ipsilateral and contralateral hemispheres. Each hemisphere was weighed and then homogenized in 50% trichloroacetic acid solution. Following centrifugation at 10 000 g for 10 min, supernatant fluids were diluted with ethanol (1 : 3) and fluorescence was measured at 620 nm to determine the amount of Evans blue, using a spectrophotometer. The tissue content of Evans blue was quantified from a standard curve derived from known amounts of the dye. Data are expressed as micrograms of Evans blue per gram of tissue.

The loss of BBB integrity was also verified by leakage of rabbit IgG from microvessels after intravenous injection. Rabbit IgG solution (0.1% in PBS, 4 mL/kg) was intravenously administered via the tail vein at 22 h after onset of ME. Rats were perfused transcardially with saline as above. After decapitation, brains were prepared according to the immunohistochemical methods, and rabbit IgG leakage was determined by immunofluoroscence staining.

Statistical analysis

Data were represented as means ± SD. The significance of differences between treated and control animals at each time point was assessed by the Student's t-test. Multiple comparisons between experimental groups were performed by one-way analysis of variance (anova), followed by a Dunnett's test. p < 0.05 was considered significant.


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References

Neuronal NOS and endothelial NOS protein are up-regulated following ME

We recently reported that ME caused a marked increase in NO production 24–48 h after microsphere injection in rats (Shirakura et al. 2005). We therefore determined which species of NOS mediates increased NO production in subacute NO production. Immunoblotting analysis with anti-nNOS, anti-iNOS and anti-eNOS antibodies revealed a slight increase in nNOS protein 2–48 h after ischemia, a marked increase in eNOS protein 24–48 h after ischemia, and no change in iNOS expression (Figs 1a–c). Notably, the more pronounced increase in eNOS protein was found 24 and 48 h after ischemia compared with 2 and 6 h (Fig. 1c).


Figure 1.  ME ischemia-induced expression of nitric oxide synthase expression in rats. Proteins were separated on SDS-PAGE and transferred for western blotting with anti-nNOS, anti-iNOS and anti-eNOS antibodies. (a) Time course of nNOS expression in ME-induced ischemic rats. Immunoblotting analysis with an anti-β-tubulin antibody showed an equal amount of loaded protein in each lane. (b) Western blot analysis of iNOS expression after ME at the indicated times. (c) ME-induced eNOS expression in ischemic rats at indicated times. The data shown are means ± SD (n = 4 in each group); *p < 0.05 compared with the sham-operated group, #p < 0.05 compared with 2 h and 6 h after ME.

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ME-induced eNOS expression and protein tyrosine nitration occurs predominantly in vascular endothelial cells

We next defined the immunohistochemical localization of increased eNOS and tyrosine-nitrated proteins. Interestingly, immunoreactivity of microvessels against the anti-eNOS antibody markedly increased in intensity in the ipsilateral forebrain (Fig. 2d). Although slightly increased eNOS expression was detected in hippocampal pyramidal neurons (data not shown), significant increases in eNOS expression in neurons of the forebrain were not observed. The markedly increased eNOS expression coincided with increased anti-nitrotyrosine immunoreactivity in microvessels (Figs 2d–f). These results suggest that increased eNOS expression mediates protein tyrosine nitration in vascular endothelial cells. We previously reported that DY-9760e, a novel calmodulin inhibitor, inhibits eNOS activity in vitro (Fukunaga et al. 2000) and attenuates ME-induced protein tyrosine nitration in brain extracts (Shirakura et al. 2005). To confirm whether DY-9760e treatment attenuates protein tyrosine nitration in endothelial cells in the ME brain, DY-9760e was administered twice, 30 min and 6 h after microsphere injection. Consistent with our previous observation using brain homogenates, DY-9760e treatment largely attenuated nitrotyrosine immunoreactivity in endothelial cells (Fig. 2h). However, ME-induced eNOS expression was not affected by DY-9760e treatment (Fig. 2g). These results are consistent with our previous observation using gerbil forebrain ischemia (Hashiguchi et al. 2003).


Figure 2.  Fluorescent immunohistochemical staining of nitrotyrosine and eNOS in the cortical microvessels of ME rats. Anti-nitrotyrosine (red) and eNOS (green) staining was performed 24 h after ME. eNOS staining is observed in microvessels of sham-operated groups (a) where nitrotyrosine staining is absent (b). ME induced both nitrotyrosine formation (e) and eNOS expression (d) in microvessels of the ipsilateral cortex, and co-localized in the same region as microvessels (f). DY-9760e significantly decreased nitrotyrosine immunopositivity (h) but not eNOS expression (g), compared with vehicle-treated rats. (Scale bar = 40 µm).

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Protein tyrosine nitration coincides with BBB disruption in endothelial cells

To confirm whether protein tyrosine nitration induces endothelial cell damage, we assessed leakage of rabbit IgG around nitrotyrosine-positive microvessels after peripheral injection of IgG. At 24 h after ME, forebrain slices were double-stained with anti-nitrotyrosine and anti-rabbit IgG antibodies. As expected, smeared and strong immunoreactivity coincided with nitrotyrosine immunoreactivity in injured endothelial cells (Figs 3d–f). In sham-operated rats, lack of nitrotyrosine immunoreactivity correlated with faint IgG immunoreactivity (Figs 3a and b). These findings clearly demonstrate that protein tyrosine nitration is associated with endothelial cell damage and likely causes BBB disruption. Significantly increased nitrotyrosine immunoreactivity was also observed in surrounding injured microvessels without staining with rabbit IgG. Faint immunoreactivity surrounding microvessels likely reflects nitrotyrosine formation in glial cells or neurons. These findings confirm that nitrotyrosine is found primarily in the endothelium of microvessels, where eNOS is up-regulated.


Figure 3.  Fluorescent immunohistochemical staining of injected-rabbit IgG and nitrotyrosine antibodies in the cortex of ME rats. Anti-nitrotyrosine (e)- and rabbit IgG (d)-positive staining is observed 24 h after ME (lower panel) but not in sham-operated groups (a–c, upper panel). Rabbit IgG staining is primarily localized around disrupted microvessels and anti-nitrotyrosine-stained endothelium of microvessels. (Scale bar = 100 µm).

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DY-9760e attenuates BBB disruption promoted by protein tyrosine nitration

As the temporal profile of BBB disruption has not been documented in microsphere embolism, we assessed it by Evans blue leakage (Fig. 4a) 2, 6, 12, 24, 48 and 72 h after ME by quantifying the amount of Evans blue that penetrated brain tissue (Fig. 4b). In the ipsilateral hemisphere, Evans blue extravasated from blood vessels into an area surrounding the brain parenchyma increased significantly at 6 h, and dramatically at 24–72 h, after ME (Fig. 4b). To confirm that protein tyrosine nitration caused BBB disruption, we tested the effect of DY-9760e on ME-induced BBB disruption. DY-9760e (50 mg/kg) treatment significantly reduced EB leakage 24 h after ME in the ipsilateral hemisphere (Fig. 4c), whereas DY-9760e (25 mg/kg) treatment failed to reduce Evans blue leakage significantly.


Figure 4.  Changes in BBB integrity in brain regions following ME ischemia. (a) Extensive Evans blue (EB) dye extravasation (dark areas) is prominent in the ipsilateral hemisphere of rat brain after ME. (Scale bar = 1 mm). (b) EB dye extravasation occurs between 2 and 6 h following ME, and more widespread increases in regional BBB permeability are observed from 24 to 72 h. (c) The effect of DY-9760e treatment on BBB permeability 24 h after ME. Extravasation of EB was expressed as µg/mg brain tissue. The data in (b) and (c) are shown as means ± SD (n = 4 in each group); *p < 0.05 compared with the right hemisphere of the same brain, #p < 0.05 compared with vehicle-treated group.

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ME-induced PARP cleavage is inhibited by DY-9760e treatment

To determine whether ME-induced eNOS expression and protein tyrosine nitration precede apoptosis in the vascular endothelium, we examined PARP cleavage. Caspase 3-mediated cleavage of PARP into 85 and 24 kDa fragments is considered indicative of caspase 3 activation and the apoptosis cascade. We found proteolytic cleavage of PARP, with the resulting 85 kDa fragment progressively increasing, 6–72 h after ME induction (Fig. 5a). Consistent with disruption of vascular endothelium, as shown in Fig. 4(b), DY-9760e (50 mg/kg) significantly reduced PARP cleavage into 85 kDa products (Fig. 5b). To confirm whether PARP activation coincides with protein tyrosine nitration in the vascular endothelium, the forebrain was double-stained with anti-PARP-p85 and anti-nitrotyrosine antibodies. In the ME forebrain, immunoreactivity for PARP-p85 was seen predominantly in microvessel endothelium of the ipsilateral side at 24 h and co-localized with nitrotyrosine immunoreactivity (Figs 6d–f). DY-9760e (50 mg/kg) treatment significantly suppressed ME-induced nitrotyrosine immunoreactivity (Fig. 6h) and PARP-p85 expression (Fig. 6g). Therefore, we conclude that ME induces apoptosis of endothelial cells and likely contributes to BBB disruption and, subsequently, to brain edema.


Figure 5.  ME ischemia induces PARP cleavage. (a) Representative image showing PARP-p85 expression following ME at indicated times. (b) Effects of DY-9760e on PARP-p85 expression 24 h after ME. Lane 1: sham; lane 2: vehicle; lanes 3 and 4: DY-9760e (25 mg/kg) and DY-9760e (50 mg/kg) treatment groups. The data show means ± SD (n = 4 in each group); *p < 0.05 compared with sham-operated rats, #p < 0.05 compared with vehicle-treated group.

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Figure 6.  Fluorescent immunohistochemical staining of nitrotyrosine and PARP-p85 in the cortical microvessels of ME rats. Anti-nitrotyrosine (e) and PARP-p85 (d) staining was performed 24 h after ME. No PARP-p85 (a)- and nitrotyrosine (b)-positive staining was observed in microvessels of sham-operated groups. ME-induced both nitrotyrosine formation (d) and PARP-p85 expression (e) in microvessels, and co-localized in the same region of microvessels (f). DY-9760e significantly decreased nitrotyrosine immunoreactivity (h) and PARP-p85 expression (g) compared with vehicle-treated rats. (Scale bar = 40 µm).

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  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References

The role of NO in the development of post-ischemic cerebral infarction has been extensively studied (Kumura et al. 1996; Coeroli et al. 1998; Onitsuka et al. 1998; Lei et al. 1999), but few studies have investigated its role in microvascular damage occurring after cerebral ischemia. Using a BBB disruption model in ME-induced ischemia, we report a relationship between BBB leakage and eNOS induction after ME-induced ischemia. The critical observation in this study is that induction of eNOS and, in turn, peroxynitrite formation in the microvascular endothelium precedes events of microvessel disruption in an ME model. Expression of eNOS in the microvascular endothelium is closely associated with increased protein tyrosine nitration and concomitant increased cleaved-PARP immunoreactivity, which implies induction of apoptosis in microvascular cells.

Previously, we found that eNOS expression was induced by forebrain ischemia in the gerbil (Hashiguchi et al. 2003). Like the ME rats analyzed in this study, increased eNOS expression in gerbils peaked 24–48 h after transient ischemia. However, increased eNOS expression in the gerbil hippocampus was not associated with increased protein tyrosine nitration, which was transient and returned to basal levels within 24 h. In contrast with the transient forebrain ischemia, a more pronounced increase in protein tyrosine nitration was observed at 24–48 h with concomitant eNOS overexpression in ME. Notably, under a lethal and permanent ischemic condition such as ME, eNOS overexpressed in the endothelial cells results in protein tyrosine nitration, thereby eliciting injury of microvascular endothelium. ME-induced permanent cerebral ischemia is known to cause more severe injury than ischemia-reperfusion models in brain (Miyake et al. 1993). Indeed, ME-induced cerebral ischemia showed a more pronounced and prolonged increase in protein tyrosine nitration (Shirakura et al. 2005). Co-localization of eNOS with anti-nitrotyrosine immunoreactivity in microvessels further confirms that eNOS overexpression in the endothelial cells is causative for injury of microvascular endothelium. In support of this idea, protein tyrosine nitration was predominant in microvascular endothelium immunoreactive with anti-PARP-p85. Thus, eNOS expression in the endothelium in permanent ischemia leads to aberrant protein tyrosine nitration and PARP activation. As peroxynitrite is formed through generation of both NO and superoxide, superoxide is likely generated by eNOS in endothelial cells. Notably, overexpression of eNOS accounts for superoxide generation as well as NO in the ME setting, because eNOS is known to produce superoxide in the absence of substrates such as tetrahydrobiopterin and arginine. Taken together, under lethal ischemia conditions, eNOS expression in endothelial cells accounts for increased protein tyrosine nitration, thereby leading to cell injury and PARP cleavage.

In addition to increased eNOS expression following ME, small but significant increases in nNOS expression were observed in the ipsilateral hemisphere without changes in iNOS expression. A slight increase in nNOS was observed within 2 h, similar to eNOS expression after ME. The change in nNOS was not significant in immunohistochemical studies (data not shown). Although a slight increase in immunoreactivity with an anti-nitrotyrosine antibody was also observed in cortical neurons, we could not conclude whether the increased nNOS had a detrimental effect on neurons or on the BBB in the present study. In transient ischemia in gerbils, we previously demonstrated increased protein tyrosine nitration in hippocampal pyramidal neurons, which precedes delayed neuronal death in the hippocampus (Hashiguchi et al. 2003). Similarly, inhibition of protein tyrosine nitration in pyramidal neurons by DY-9760e treatment largely attenuated delayed neuronal death in the hippocampus. Taken together, protein tyrosine nitration in cortical neurons likely has a detrimental effect on neurons. Up-regulation of both nNOS and eNOS with concomitant increases in protein tyrosine nitration was also reported in neonatal rat brain subjected to unilateral carotid artery occlusion (Van den Tweel et al. 2005). In neonatal brain ischemia, increased expression of both nNOS and eNOS was transient and returned to basal levels within 24 h of ischemia. Increased nitrotyrosine formation was also transient and seen only 30 min after ischemia. However, the authors of that study did not show that nitrotyrosine production was associated with neuronal damage. Similarly, Ochiai-Kanai et al. (1999) reported that increased immunoreactivity against nitrotyrosine was induced following hypoxia or NMDA treatment in neonatal rat cerebrocortical slices. The clinical significance of induction of NOSs and nitrotyrosine formation was also demonstrated in Alzheimer's brains using immunohistochemical methods (Luth et al. 2002). In that study, aberrant expression of nNOS, eNOS and iNOS in the Alzheimer's brain was correlated with increased protein nitration. Specifically, aberrant expression of nNOS co-localized with nitrotyrosine immunoreactivity in cortical pyramidal cells. On the other hand, both iNOS and eNOS were highly expressed in astrocytes with concomitant increased nitrotyrosine in the Alzheimer's brain. The authors concluded that increased expression of all NOS isoforms in astrocytes and neurons contributes to synthesis of peroxynitrite, thereby leading to nitrotyrosine generation. Similarly, calcium-dependent NOS activity and iNOS immunoreactivity increased in the cerebral cortex of amyloid-precursor protein Tg2576 transgenic mice (Rodrigo et al. 2004). Taken together, up-regulation of NOS isoforms expressed in the brain could trigger peroxynitrite formation in various cell types, thereby leading to cellular damage through protein tyrosine nitration. Free 3-nitrotyrosine released from nitrated proteins also accounts for vascular endothelial dysfunction and neurotoxicity through promotion of DNA damage (Mihm et al. 2000). That study showed that protein nitration also caused dysfunction of enzymes, including manganese superoxide dismutase, an enzyme that scavenges toxic superoxide (Mn-SOD). Other proteins critical for respiratory activity in mitochondria are nitrated during inflammatory events in the liver of lipopolysacchride-treated rats (Aulak et al. 2001). Although we cannot define the substance that stimulates increased NO production and protein tyrosine nitration in endothelial cells, several cytokines and neurotransmitters could trigger NO and peroxynitrite production following brain ischemia (Bredt 1999; Bachschmid et al. 2003; Ohtaki et al. 2003; Walford et al. 2004).

We showed here that PARP cleavage, an event triggering apoptosis, occurred in tyrosine nitration-positive endothelial cells following ME ischemia. Persistent cleavage of PARP after ME suggests that it is caused by caspases mediating apoptotic cell death in the microvasuclar endothelium. Anti-PARP-p85 immunoreactivity was predominantly co-localized with anti-nitrotyrosine immunoreactivity in endothelial cells. This result in consistent with a previous study indicating that apoptosis in brain vascular endothelial cells is associated with caspase activation and PARP cleavage (Meguro et al. 2001; Akin et al. 2002). Furthermore, co-localization of tyrosine nitration immunoreactivity with leaked rabbit IgG immunoreactivity also confirmed that protein tyrosine nitration in endothelial cells led to leakage of serum protein. However, it is unclear whether PARP cleavage in endothelial cells causes BBB disruption. Further study is required to define key molecules involved in BBB disruption.

We recently described a neuroprotective agent, DY-9760e, that inhibits NO generation and protein tyrosine nitration formation (Fukunaga et al. 2000; Hashiguchi et al. 2003; Shirakura et al. 2005). DY-9760e treatment inhibits BBB disruption in a rat middle cerebral occlusion model (Sato et al. 2003). Here, we found that DY-9760e preferentially inhibits protein tyrosine nitration induced by eNOS in endothelial cells. Its inhibition of BBB disruption was closely associated with inhibition of protein tyrosine nitration and PARP cleavage in the microvascular endothelium. DY-9760e did not, however, affect ME-induced eNOS expression in endothelial cells. Taken together, inhibition of protein tyrosine nitration predominantly contributes to the BBB protective action of DY-9760e. In addition, we recently demonstrated that DY-9760e inhibits calpain-induced proteolysis of structural proteins, such as fodrin/spectrin. Fodrin co-localizes with tight junction proteins including ZO-1 and connexin-43 in cardiomyocytes (Toyofuku et al. 1998). As the tight junction proteins in vascular endothelial cells are important for BBB integrity (Ballabh et al. 2004), DY-9760e-induced inhibition of fodrin breakdown likely mediates, in part, BBB integrity.

In conclusion, we have shown that induction of eNOS following ME-induced prolonged ischemia produced a marked increase in protein tyrosine nitration, predominantly in vascularendothelial cells, thereby triggering apoptotic pathways. Protein tyrosine nitration and/or calpain-induced fodrin breakdown likely accounts for ME-induced BBB disruption, thereby causing brain edema. A neuroprotective agent, DY-9760e, is an attractive therapeutic drug exhibiting a potent protective action on the BBB in brain ischemia.


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
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