Normobaric Hyperoxia Reduces Blood Occludin Fragments in Rats and Patients With Acute Ischemic Stroke

Abstract

Background and purpose: Damage of the blood-brain barrier (BBB) increases the incidence of neurovascular complications, especially for cerebral hemorrhage after tPA (tissue-type plasminogen activator) therapy. Currently, there is no effective method to evaluate the extent of BBB damage to guide tPA use. Herein, we investigated whether blood levels of tight junction proteins could serve as biomarker of BBB damages in acute ischemic stroke (AIS) in both rats and patients. We examined whether this biomarker could reflect the extent of BBB permeability during cerebral ischemia/reperfusion and the effects of normobaric hyperoxia (NBO) on BBB damage.

Methods: Rats were exposed to NBO (100% O₂) or normoxia (21% O₂) during middle cerebral artery occlusion. BBB permeability was determined. Occludin and claudin-5 in blood and cerebromicrovessels were measured. Patients with AIS were assigned to oxygen therapy or room air for 4 hours, and blood occludin and claudin-5 were measured at different time points after stroke.

Results: Cerebral ischemia/reperfusion resulted in the degradation of occludin and claudin-5 in microvessels, leading to increased BBB permeability in rats. In blood samples, occludin increased with 4-hour ischemia and remained elevated during reperfusion, correlating well with its loss from ischemic cerebral microvessels. NBO treatment both prevented occludin degradation in microvessels and reduced occludin levels in blood, leading to improved neurological functions in rats. In patients with AIS receiving intravenous tPA thrombolysis, the blood occludin was already elevated when patients arrived at hospital (within 4.5 hours since symptoms appeared) and remained at a high level for 72 hours. NBO significantly lowered the level of blood occludin and improved neurological functions in patients with AIS.

Conclusions: Blood occludin may be a clinically viable biomarker for evaluating BBB damage during ischemia/reperfusion. NBO therapy has the potential to reduce blood occludin, protect BBB, and improve outcome in AIS patients with intravenous tPA thrombolysis.

Clinical trial registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT02974283.

Keywords: blood–brain barrier; humans; occludin; oxygen; stroke.

Figure 1. NBO-treated rats showed significantly less Evan’s blue leakage after 4-h of MCAO with 2-h reperfusion

A). Representative brain slices show EB leakage in ischemic hemisphere after 2-h or 4-h MCAO with 2-h reperfusion. B). Quantification of EB extravasation in brain tissue of ischemic hemisphere (I) and non-ischemic hemisphere (Non-I). N=5, *P<0.05 versus Normoxia-I in I-2h/R-2h group; ^#P<0.05 versus Normoxia-I in I-4h/R-2h group. Data are expressed as means ± SEM.

Figure 2. NBO reduced the loss of TJPs in isolated microvessels

A). Representative western blots and quantitative analysis of occludin in isolated microvessels following indicated ischemia/reperfusion (I/R) time. B). Representative western blots and the quantitative analysis of the level of claudin-5 in isolated microvessels. N=4, *p<0.05 versus sham group rats; ^#p<0.05 versus normoxic rats with same duration of I/R. Data are expressed as means ± SEM.

Figure 3. Changes of blood TJPs and effects of NBO following MCAO

A) Levels of occludin in blood of ischemic rats. 4-h MCAO induced a significant increase of blood occludin and remained high in subsequent 5min and 2h reperfusion; NBO-treatment reduced blood occludin. N=6, *p<0.05 versus I-0h in sham group, ^#p<0.05 versus the normoxia rats with the same ischemic duration. B). Levels of claudin-5 in blood. No significant difference of blood claudin-5 level was observed among three groups. Data were presented as means ± SEM.

Figure 4. NBO improved the neurological score and decreased 24-h mortality rate in ischemic stroke rats

A). Neurological scores were measured at 2 h (I-2h), 4h ischemia (I-4h) and 4h ischemia plus 2 h reperfusion (I-4h/R-2h). NBO treatment significantly improved neurological scores in I-4h and I-4h/R-2h groups. N=5. B). Morality rate at 24-h post reperfusion. NBO significantly reduced 24-h mortality. N=9. *p<0.05, versus I-2h in normoxic group; ^#p<0.05, versus normoxia rats with the same duration of I/R. Data were presented as means ± SEM.

Figure 5. Blood TJPs levels in AIS patients and healthy volunteers

A). Occludin level in blood of stroke patients at indicated time after stroke onset. B). Claudin-5 level in blood. N=8. *p<0.05 versus normal group. Data were presented as means ± SEM.

Figure 6. NBO reduced blood occludin and improved neurological functions in AIS patients

A). Blood occludin levels in normoxia and NBO-treated AIS patients. N=8. B). NIHSS scores of AIS patients. N=9. ^#P<0.05 versus normoxia group at the same time point. Data were presented as means ± SEM.