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First published online January 12, 2006
doi: 10.1242/10.1242/jcs.02734

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Nitric oxide mediates neurodegeneration and breakdown of the blood-brain barrier in tPA-dependent excitotoxic injury in mice

¹ Program in Molecular and Cellular Biology, Department of Pharmacological Sciences, University Medical Center at Stony Brook, Stony Brook, NY 11794-8651, USA
² Program in Molecular and Cellular Pharmacology, Department of Pharmacological Sciences, University Medical Center at Stony Brook, Stony Brook, NY 11794-8651, USA

View larger version (69K): [in a new window]   Fig. 1. (A) NOS activity in hippocampal extracts from wild-type (wt, black), tPA–/– (mid-gray) and tPA–/– mice treated with 1 µg/µl tPA (light gray), with or without KA. NOS activity was assessed by quantitation of [3H]L-arginine converted to [3H]L-citrulline. One unit enzyme activity produces 1 nmole NO per minute at 37°C. Statistical comparison was conducted comparing individual time points to their corresponding non-treated controls using a two-tailed t test. **P<0.01, ***P <0.001. Statistical comparison comparing all treatment groups within time points was conducting using one-way ANOVA followed by a Bonferroni-Dunn test for multiple comparisons. P<0.05; n=6-7 per treatment group. (B) NOS activity in wild-type mice treated with tPA Stop and MK-801 as well as NOS activity in tPA–/– mice infused with the catalytically inactive tPA mutant S481A. Statistical comparison was conducted as in A. (C) Western blot analysis for anti-nTyr in extracts from wild-type and tPA–/– hippocampi injected with KA and sacrificed at day 5. Arrows indicate a band that exhibits increased staining on the injected side in wild-type mice. Equal loading of protein was verified using anti-actin. n=6. (D) Coronal sections of wild-type and tPA–/– mice injected with KA and sacrificed at day 5 were stained with Cresyl Violet to assess neurodegeneration (a,b, asterisks). Immunohistochemical staining for nTyr identified scattered positive cells in all hippocampal regions, and a strongly immunoreactive focus that coincided with degenerating pyramidal layer neurons on the injected side of wild-type mice only (arrows indicate region of interest, c-g). Insets show high magnification of nTyr-stained neurons in the CA1 region of the hippocampus. Bar, 50 µm.

View larger version (59K): [in a new window]   Fig. 2. Scavenging ONOO– with 2 µM FeTMPyP or 10 µM FeTPPS protects against KA-mediated excitotoxicity and oxidative stress. (A) Wild-type and wild-type mice infused with either 2 µM FeTMPyP or 10 µM FeTPPS were injected with KA and sacrificed at day 5 (left). tPA–/– mice were infused with 2 µM FeTMPyP prior to injection with KA with or without co-injection of 5 µg/µl tPA (right). Sections were stained with Cresyl Violet to assess neuronal survival. Bars, 50 µm. (B) To quantify the injury-derived oxidative stress, mice were injected with 1 mg/ml DHE intraperitoneally 2 hours prior to sacrifice. Wild-type mice and tPA–/– mice injected with tPA, but not tPA–/– mice, FeTMPyP- or FeTPPS-treated mice, exhibit increased DHE oxidation in CA1 and CA3. Fluorescent images were converted to pseudocolor using the Scion software. A scale for quantitation is shown on the side. (C) Quantification of neuronal survival was performed as described in the Materials and Methods. Statistical analysis was performed using a two-tailed t test as compared with wild-type KA-treated mice. **P<0.01. There was no significant difference between FeTMPyP, FeTPPS and tPA–/– mice. (D) Quantification of fluorescence was determined using Scion Image and represents fluorescent intensity of the injected side after subtracting the background of the uninjected side. Statistical analysis using one-way ANOVA followed by a Bonferroni-Dunn test for multiple comparisons was used to compare fluorescent intensities between groups of the CA1 and CA3 subregions of the hippocampus. ***P<0.001 indicates significant increase in fluorescent intensity; n.s. indicates no significance between groups. n=6 per treatment group.

View larger version (71K): [in a new window]   Fig. 3. Wild-type C57 mice are protected from KA-induced excitotoxicity by the NOS blocker NMMA. (A,B) Two days prior to injection, wild-type or tPA–/– mice were infused with the indicated concentrations of NMMA. The mice were sacrificed five days after the injection of KA with or without a co-injection of tPA and neuronal survival visualized as described in Fig. 1. Infusion of NMMA followed by injection of PBS as a control had no effect on neuronal viability. (C,D) Quantification of the neurodegeneration was performed using Scion Image beta 4.02 and is presented as percentage of surviving neurons in the total hippocampal region on the ipsilateral (injected) side in comparison with the contralateral (non-injected) side. Statistical analysis was performed using one-way ANOVA followed by a Bonferroni-Dunn test for multiple comparisons. Infusion of 5, 100, 200 and 400 µM NMMA prior to KA injection protected neurons from degeneration. *P<0.05; **P<0.01; ***P<0.001; n.s. indicates no significance between groups. n=6 per concentration of NMMA. The experiment was performed three times and the data pooled for statistical comparison. The arrow in A depicts the site of KA injection.

View larger version (134K): [in a new window]   Fig. 4. Injection of NO donor NOC-18 prior to KA injection exacerbates excitotoxicity in wild-type mice. Wild-type mice were injected with either PBS or 1 mM NOC-18 immediately prior to injection with the indicated concentrations of KA or with PBS and sacrificed at day 1. Cresyl Violet staining indicated that 1 mM NOC-18 alone is not toxic to neurons. However, injection of 1 mM NOC-18 immediately prior to injection of low doses of KA potentiates neuronal cell death. n=6 per treatment.

View larger version (88K): [in a new window]   Fig. 5. Injection of NOC-18 restores the toxic effects of KA in tPA–/– mice. Wild-type and tPA–/– mice were injected with either KA alone, 1 mM NOC-18 or both. At day 1, mice were sacrificed and treated as in Fig. 1. Cresyl Violet staining confirmed that tPA–/– mice are resistant to KA-induced excitotoxicity, whereas wild-type mice are not. Injection of 1 mM NOC-18 alone has no affect on tPA–/– mice. However, injection of 1 mM NOC-18 prior to KA injection restores the toxic effects of KA. n=9. Infusion of FeTMPyP prior to KA and NOC-18 injection is neuroprotective to hippocampal neurons (n=6).

View larger version (54K): [in a new window]   Fig. 6. NO and tPA contribute to BBB breakdown. (A) Wild-type and tPA–/– mice infused or not with tPA (1 µg/µl) were injected with 2% Evans Blue intraperitoneally immediately after intrahippocampal KA injection. At day 1, the mice were sacrificed, their hippocampi dissected, weighed and homogenized. The amount of BBB breakdown was assessed as the amount of Evans Blue that leaked into the brain parenchyma quantified at 620 nm minus the background and divided by the wet weight of each hemisphere. Each experimental group is represented by the percentage of the total signal for each individual experiment. *Indicates a significant difference as compared with the respective uninjected side (P<0.05) by a two-tailed t test; # indicates a significant difference (P<0.05) between the 400 µM NMMA ipsilateral side as compared with wild-type, PBS-infused, ipsilateral side. n=7. (B) Infusion of NMMA and 300 µM PTIO, but not 2 µM FeTMPyP or 10 µM FeTPPS, prior to KA injection partially attenuates vascular permeability, indicating a role for NO in BBB breakdown. (C) Injection of 1 mM NOC-18 alone does not mediated BBB breakdown in wild-type or tPA–/– mice. However, co-injection of NOC-18 and KA in tPA–/– mice restores the ability of KA to disrupt the BBB. (D) The specificity of BBB breakdown was assessed by immunofluorescent staining of the tight junction protein occludin. Only the KA-injected side of wild-type mice and wild-type mice treated with FeTMPyP showed diminished staining with the anti-occludin antibody, indicating a breakdown in the BBB. n=7.

View larger version (39K): [in a new window]   Fig. 7. NO plays multiple roles during excitotoxicity. NO is produced both in neurons (blue) and in microglia (green). NO can diffuse across the membrane, and either interacts with superoxide species to create ONOO– or disrupts the BBB (pink). Extracellular ONOO– can cause lipid peroxidation or cause nTyr formation on cell membrane proteins. Alternatively, ONOO– can be formed intracellularly in neurons, by activation of the NMDAR through glutamate, where it can act as a potent oxidant. tPA can directly mediate disruption of the BBB and/or activate microglia by interaction with extracellular annexin II. Once activated, microglia can produce additional NO to be converted to ONOO– or interact with the BBB.