Focal cerebral ischemic tolerance and change in blood-brain barrier permeability after repetitive pure oxygen exposure preconditioning in a rodent model

Abstract

OBJECTIVE The goal of this study was to demonstrate that repetitive pure oxygen exposure preconditioning (O₂PC) for 8 hours per day for 3 or 7 days, a practicable preconditioning for clinical use, is able to induce cerebral ischemic tolerance (IT) and further clarify the accompanying changes in the blood-brain barrier (BBB) that may be involved. METHODS A total of 68 adult male Sprague-Dawley rats and eight 1-day-old rat pups were used in this study. The adult rats were exposed to pure O₂ (38 rats) 8 hours a day for 3 or 7 days or to room air (in an identical setup) for 8 hours a day for 7 days as controls (30 rats). Arterial O₂ tension (PaO₂) was measured in 6 rats exposed to O₂ and 3 controls. Focal cerebral ischemia was elicited by middle cerebral artery occlusion (MCAO) in 37 rats, of which 21 had been exposed to pure O₂ for 3 or 7 days and 16 to room air for 7 days as controls. Neurological behavior was scored with the Garcia score in 15 MCAO rats, of which 10 had been exposed to pure O₂ for 3 or 7 days and 5 to room air for 7 days as controls, and cerebral infarct volumes were assessed with TTC (2,3,5-triphenyltetrazolium chloride) staining in 10 rats (5 from each group) after 7 days of exposure. Formamide-extraction method was used to detect leakage of Evans blue (EB) dye in 7 rats exposed to pure O₂ for 7 days and 7 exposed to room air for 7 days. Fluorescence microscopy was used to analyze the leaked EB in the nonischemic areas of 4 rats exposed to pure O₂ for 7 days and 4 exposed to room air for 7 days before MCAO and the brain of the rats that had not been subjected to MCAO. Astrocyte changes associated with O₂PC were evaluated by means of fluorescence microscopy and electron microscopy in 14 rats that were exposed to the same O₂ or control conditions as the MCAO rats but without MCAO. Astrocytes were also obtained from 8 rat pups and cultured; levels of AQP4 and VEGF were detected by Western blot and ELISA in cells with and without O₂ treatment. RESULTS A significant increase in PaO₂ was seen after O₂PC. The neurological score was significantly increased in the O₂PC groups (10.6 ± 0.6 in the 3-day O₂PC group, p < 0.05; 12 ± 0.84 in the 7-day O₂PC group, p < 0.05) compared with the control group (7 ± 0.55). The ratio of cerebral infarct volume to contralateral cerebral hemisphere volume was significantly lower in the O₂PC group than in the control group (0.204 ± 0.03 vs 0.48 ± 0.05, p < 0.05). The amount of leaked EB in the ischemic cerebral hemisphere was also lower in the O₂-treated rats than in controls (7.53 ± 1.4 vs 11.79 ± 3.3 μg EB/g brain weight, p < 0.05). However, fluorescence microscopy showed significantly greater BBB permeability in the nonischemic areas in the O₂PC group than in controls (p < 0.05). More red fluorescence could be observed in the nonischemic areas in both the ipsilateral and contralateral sides of the ischemic brain in the O₂PC animals than in the nonischemic areas in the corresponding sides of the controls. Further investigation of the effect of the O₂PC itself on the BBB of rats that were not subjected to MCAO showed that there was no EB leakage in the brain parenchyma in the rats exposed to room air, but some red fluorescence patches were noticed in the normal brain from the rats in the O₂PC group. Astrocytes, including those from areas around the BBB, were activated in the O₂PC group. Levels of both aquaporin 4 (AQP4) and vascular endothelial growth factor (VEGF) were significantly increased in cultured astrocytes after O₂PC. CONCLUSIONS These findings suggest that O₂PC is able to induce IT, which makes it a strong candidate for clinical use. Moreover, O₂PC can also promote BBB opening, which may contribute to the induction of IT as well as representing a possible strategy for promoting drug transportation into the CNS. Activated astrocytes are likely to be involved in these processes through astrocyte-derived factors, such as AQP4 and VEGF.

Keywords: AQP4 = aquaporin 4; BBB = blood-brain barrier; BSA = bovine serum albumin; EB = Evans blue; FBS = fetal bovine serum; GFAP = glial fibrillary acidic protein; HBA = hyperbaric air; HBO = hyperbaric oxygen; IP = intraperitoneally; IT = ischemic tolerance; MCAO = middle cerebral artery occlusion; O2PC = pure oxygen exposure preconditioning; PC = preconditioning; PaO2 = partial pressure of O2, arterial; TTC = 2,3,5-triphenyltetrazolium chloride; VEGF = vascular endothelial growth factor; astrocytes; blood-brain barrier; ischemic tolerance; preconditioning; repetitive pure oxygen exposure; vascular disorders.