Oxidative stress increases blood-brain barrier permeability and induces alterations in occludin during hypoxia-reoxygenation

Abstract

The blood-brain barrier (BBB) has a critical role in central nervous system homeostasis. Intercellular tight junction (TJ) protein complexes of the brain microvasculature limit paracellular diffusion of substances from the blood into the brain. Hypoxia and reoxygenation (HR) is a central component to numerous disease states and pathologic conditions. We have previously shown that HR can influence the permeability of the BBB as well as the critical TJ protein occludin. During HR, free radicals are produced, which may lead to oxidative stress. Using the free radical scavenger tempol (200 mg/kg, intraperitoneal), we show that oxidative stress produced during HR (6% O(2) for 1 h, followed by room air for 20 min) mediates an increase in BBB permeability in vivo using in situ brain perfusion. We also show that these changes are associated with alterations in the structure and localization of occludin. Our data indicate that oxidative stress is associated with movement of occludin away from the TJ. Furthermore, subcellular fractionation of cerebral microvessels reveals alterations in occludin oligomeric assemblies in TJ associated with plasma membrane lipid rafts. Our data suggest that pharmacological inhibition of disease states with an HR component may help preserve BBB functional integrity.

Figure 1

Hypoxia and reoxygenation (HR) induces hypoxia-inducible factor 1α (Hif-1α) translocation to the nucleus. Immunofluorescence of Hif-1α (green) and the nuclear dye TO-PRO-3 (blue) in brain microvessels of normoxic (Nx) plus saline (A), Nx plus tempol (B), HR plus saline (C), and HR plus tempol (D)-treated rats. Rats exposed to HR show an increase in translocation of Hif-1α to the nucleus (arrow). Immunofluorescence of the endothelial-specific marker von Willebrand factor (red) is shown in the inset.

Figure 2

Oxidative stress induces expression of heat shock protein (HSP-70). Immunofluorescence of HSP-70 (green) in brain microvessels of normoxic (Nx) plus saline (Nx+S) (A), Nx plus tempol (Nx+T) (B), hypoxia–reoxygenation (HR) plus S (HR+S) (C), and HR + T (D)-treated rats. Rats exposed to HR show an increase in HSP-70 immunofluorescence. Tempol attenuated this increase. Immunofluorescence of the endothelial-specific marker von Willebrand factor (red) is shown in the inset. (E) Mean fluorescence intensity of HSP-70 in all treatment groups expressed as a percent control. ^*P<0.05 versus HR+T and Nx+T. P<0.01 versus Nx+S (n=28 to 33 microvesels per treatment group).

Figure 3

Effects of hypoxia and reoxygenation (HR) and tempol treatment on BBB permeability to ¹⁴C-sucrose. After treatment, normoxia (Nx) plus saline (S) (Nx+S), Nx plus tempol (T) (Nx+T), HR+S, and HR+T rats were anesthetized and subjected to in situ brain perfusion for 10 mins with ¹⁴C-sucrose. The amount of radioactivity in the brain versus the perfusate was expressed as Rbr%. Results are expressed as mean±s.e. ^*P<0.05.

Figure 4

Oxidative stress induces changes in occludin localization. Immunofluorescence of occludin (green) and the endothelial-specific marker platelet-endothelial cell adhesion molecule 1 (PECAM-1) (red inset) in brain microvessels of normoxic (Nx) plus saline (A), Nx plus tempol (B), hypoxia–reoxygenation (HR) plus saline (C), and HR plus tempol (D). HR induces an increase in punctate staining of occludin (arrows), suggesting the movement of occludin away from the tight junction. Tempol prevents this increase, indicating that oxidative stress is involved in altering the cellular localization of occludin.

Figure 5

Density gradient fractionation of cerebral microvessels. (A) Profiles of protein concentrations and refractive indices (density) of cerebral microvessels after fractionation in normoxia (Nx) plus saline (S) (Nx+S), Nx plus tempol (T) (Nx+T), hypoxia–reoxygenation (HR) + S (HR+S), and HR+T. (B) Representative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot of occludin oligomers in fractions 7 and 8 under nonreducing (NR) and reducing (R) conditions. Fractions 7 and 8 have previously been shown to be associated with the tight junction in lipid rafts at the plasma membrane. (C) Western blot analysis of occludin oligomers in fractions 7 and 8 under reducing and nonreducing conditions. Data are presented as mean±s.e. and are representative of three separate experiments with three rats pooled in each treatment group. ^*P<0.001 versus Nx+S and Nx+T; P<0.01 versus HR+T in fraction 7. ^*P<0.01 versus Nx+S and Nx+T; P<0.05 versus HR+T in fraction 8.