Haute-Normandie-INSERM ERI-28, Institute for Research and Innovation in Biomedicine of Rouen University, Rouen, France.
PLoS One. 2013 Aug 6;8(8):e71263. doi: 10.1371/journal.pone.0071263. Print 2013.
Hypoxia-ischemia (HI) and excitotoxicity are validated causes of neonatal brain injuries and tissue plasminogen activator (t-PA) participates in the processes through proteolytic and receptor-mediated pathways. Brain microvascular endothelial cells from neonates in culture, contain and release more t-PA and gelatinases upon glutamate challenge than adult cells. We have studied t-PA to gelatinase (MMP-2 and MMP-9) activity links in HI and excitotoxicity lesion models in 5 day-old pups in wild type and in t-PA or its inhibitor (PAI-1) genes inactivated mice. Gelatinolytic activities were detected in SDS-PAGE zymograms and by in situ fluorescent DQ-gelatin microscopic zymographies. HI was achieved by unilateral carotid ligature followed by a 40 min hypoxia (8%O₂). Excitotoxic lesions were produced by intra parenchymal cortical (i.c.) injections of 10 µg ibotenate (Ibo). Gel zymograms in WT cortex revealed progressive extinction of MMP-2 and MMP-9 activities near day 15 or day 8 respectively. MMP-2 expression was the same in all strains while MMP-9 activity was barely detectable in t-PA⁻/⁻ and enhanced in PAI-1⁻/⁻ mice. HI or Ibo produced activation of MMP-2 activities 6 hours post-insult, in cortices of WT mice but not in t-PA⁻/⁻ mice. In PAI-1⁻/⁻ mice, HI or vehicle i.c. injection increased MMP-2 and MMP-9 activities. In situ zymograms using DQ-gelatin revealed vessel associated gelatinolytic activity in lesioned areas in PAI-1⁻/⁻ and in WT mice. In WT brain slices incubated ex vivo, glutamate (200 µM) induced DQ-gelatin activation in vessels. The effect was not detected in t-PA⁻/⁻ mice, but was restored by concomitant exposure to recombinant t-PA (20 µg/mL). In summary, neonatal brain lesion paradigms and ex vivo excitotoxic glutamate evoked t-PA-dependent gelatinases activation in vessels. Both MMP-2 and MMP-9 activities appeared t-PA-dependent. The data suggest that vascular directed protease inhibition may have neuroprotection potential against neonatal brain injuries.
缺氧缺血（HI）和兴奋性毒性是新生儿脑损伤的公认原因，组织型纤溶酶原激活物（t-PA）通过蛋白水解和受体介导的途径参与这些过程。在培养的来自新生儿的脑微血管内皮细胞中，谷氨酸刺激后比成年细胞释放更多的 t-PA 和明胶酶。我们已经在野生型和 t-PA 或其抑制剂（PAI-1）基因失活的小鼠中，在 5 天大的幼鼠的 HI 和兴奋性毒性损伤模型中研究了 t-PA 到明胶酶（MMP-2 和 MMP-9）活性连接。在 SDS-PAGE 酶谱和原位荧光 DQ-明胶酶显微镜酶谱中检测到明胶酶活性。HI 通过单侧颈总动脉结扎，然后进行 40 分钟的缺氧（8%O₂）来实现。兴奋性毒性损伤通过皮质内（i.c.）注射 10µg 异博定（Ibo）来产生。在 WT 皮质的凝胶酶谱中，MMP-2 和 MMP-9 的活性分别在第 15 天或第 8 天逐渐消失。所有品系的 MMP-2 表达相同，而 t-PA⁻/⁻小鼠中 MMP-9 活性几乎检测不到，而 PAI-1⁻/⁻小鼠中增强。HI 或 Ibo 在 WT 小鼠皮质中引起 MMP-2 活性在损伤后 6 小时激活，但在 t-PA⁻/⁻小鼠中没有。在 PAI-1⁻/⁻小鼠中，HI 或 vehicle i.c. 注射增加了 MMP-2 和 MMP-9 的活性。使用 DQ-明胶的原位酶谱显示在 PAI-1⁻/⁻和 WT 小鼠的损伤区域存在与血管相关的明胶酶活性。在 WT 脑切片体外孵育时，谷氨酸（200µM）诱导血管中 DQ-明胶的激活。在 t-PA⁻/⁻小鼠中未检测到这种效应，但通过同时暴露于重组 t-PA（20µg/mL）恢复。总之，新生儿脑损伤模型和体外兴奋性毒性谷氨酸诱发的 t-PA 依赖性明胶酶激活与血管有关。MMP-2 和 MMP-9 的活性似乎都依赖于 t-PA。该数据表明，针对血管的蛋白酶抑制剂可能具有针对新生儿脑损伤的神经保护潜力。
