Normobaric hyperoxia slows blood-brain barrier damage and expands the therapeutic time window for tissue-type plasminogen activator treatment in cerebral ischemia

Abstract

Background and purpose: Prolonged ischemia causes blood-brain barrier (BBB) damage and increases the incidence of neurovasculature complications secondary to reperfusion. Therefore, targeting ischemic BBB damage pathogenesis is critical to reducing neurovasculature complications and expanding the therapeutic time window of tissue-type plasminogen activator (tPA) thrombolysis. This study investigates whether increasing cerebral tissue PO2 through normobaric hyperoxia (NBO) treatment will slow the progression of BBB damage and, thus, improve the outcome of delayed tPA treatment after cerebral ischemia.

Methods: Rats were exposed to NBO (100% O2) or normoxia (21% O2) during 3-, 5-, or, 7-hour middle cerebral artery occlusion. Fifteen minutes before reperfusion, tPA was continuously infused to rats for 30 minutes. Neurological score, mortality rate, and BBB permeability were determined. Matrix metalloproteinase-9 was measured by gelatin zymography and tight junction proteins (occludin and cluadin-5) by Western blot in the isolated cerebral microvessels.

Results: NBO slowed the progression of ischemic BBB damage pathogenesis, evidenced by reduced Evan blue leakage, smaller edema, and hemorrhagic volume in NBO-treated rats. NBO treatment reduced matrix metalloproteinase-9 induction and the loss of tight junction proteins in ischemic cerebral microvessels. NBO-afforded BBB protection was maintained during tPA reperfusion, resulting in improved neurological functions, significant reductions in brain edema, hemorrhagic volume, and mortality rate, even when tPA was given after prolonged ischemia (7 hours).

Conclusions: Early NBO treatment slows ischemic BBB damage pathogenesis and significantly improves the outcome of delayed tPA treatment, providing new evidence supporting NBO as an effective adjunctive therapy to extend the time window of tPA thrombolysis for ischemic stroke.

Keywords: blood-brain barrier; ischemia; matrix metalloproteinase 9; tissue-type plasminogen activator.

© 2015 American Heart Association, Inc.

Figures

Figure 1. NBO treatment reduces Evan’s blue leakage in the ischemic brain after 3-h, 5-h or 7-h of MCAO with 2-h reperfusion

A. Schematic diagram of the overall experimental design. B. Representative brain slices showing EB leakage in the ischemic tissue. Quantification of EB extravasation is expressed as nanogram per gram of brain tissue (ng/g). *P<0.05 versus 3-h MCAO Normoxia-I; #P<0.05 versus 7-h MCAO Normoxia-I. Data are expressed as mean ± SEM (n = 5).

Figure 2. NBO reduces hemoglobin extravasation and brain edema in the ischemic brain after 3-h, 5-h or 7-h of MCAO with 2-h reperfusion

A. Representative brain slices showing hemoglobin extravasation and hemispheric enlargement and the quantitative analysis of hemoglobin leakage. *P<0.05 versus 3-h MCAO Normoxia-I; #P<0.05 versus the Normoxia-I with the same ischemia duration. (n=5) B. The ratio of ischemia-induced brain edema. #P<0.05 versus the Normoxia-treated with the same ischemia duration. Data are expressed as mean ± SEM (n=7).

Figure 3. NBO slows the process of MMP-9 induction and the loss of TJPs in ischemic microvessels after 3-h, 5-h or 7-h MCAO with 2-h reperfusion

A. Representative gelatin zymography showing MMP-9 activity in isolated microvessels from non-ischemic (Non-I) and ischemic (I) hemisphere, and the quantitative analysis results. STD: the standard of MMP-9. *P<0.05 versus the Normoxia-treated with the same ischemic duration. B. Representative western blots of occludin and claudin-5 and the quantitative analysis results. *P<0.05 versus the Normoxia-treated with the same ischemic duration. Data are expressed as mean±SEM (n=5).

Figure 4. NBO slows tPA-augmented MMP-9 gelatinolytic activity and tight junction proteins (occludin and claudin-5) loss

A. Gelatin zymography showing MMP-9 activity in the Non-I and I microvessels in tPA-treated rats and the quantitative analysis result. STD: the standard of MMP-9. *P<0.05 versus the tPA+normoxia with the same ischemic duration (n=4). B. Occludin and Claudin-5 protein levels in microvessel extracts. *P<0.05 versus the tPA+normoxia with the same ischemic duration. Data are expressed as mean±SEM (n=4).

Figure 5. NBO slows the Evan’s blue extravasation, hemoglobin extravasation and reduces brain edema in delayed tPA-treatment

A. Representative brain slices showing EB leakage and the quantitative analysis result in rats with 3, 5, or 7-h ischemia followed 2h reperfusion. *P<0.05 versus Normoxia/tPA+3h MCAO; #P<0.05 versus Normoxia-tPA-I+7h MCAO. Data are expressed as mean ± SEM (n=5). B. Representative brain slices showing hemoglobin extravasation and edema in tPA-treated rats, and the quantitative analysis results. *P<0.05 versus Normoxia/tPA+3h MCAO; #P<0.05 versus Normoxia+tPA-treated with the same ischemic duration (n=4). C. The ratio of edema. #P<0.05 versus the tPA-treated with the same ischemia duration (n=7).

Figure 6. NBO improves the outcome of stroke rats with delayed tPA treatment

A. Representative brain slices illustrating infarction in rats with 3-, 5- or 7-h MCAO plus 2-h reperfusion, and the quantitative analysis result. #P<0.05 versus normoxia+tPA with the same ischemia duration (n=5). B. Zea-Longa neurological scores. C. Ludmila Belayev neurological scores. #P<0.05 versus the normoxia+tPA with the same ischemia duration) (n=10 for normxia and NBO group; n=9 for Normoxia+tPA and tPA+NBO group). D. The mortality of rats at 24 h post ischemia onset. *P<0.05 versus 3h-Normoxia/tPA; #P<0.05 versus 7h-Normoxia/tPA.