Effects of hypoxia-reoxygenation on rat blood-brain barrier permeability and tight junctional protein expression

Abstract

Cerebral microvessel endothelial cells that form the blood-brain barrier (BBB) have tight junctions (TJs) that are critical for maintaining brain homeostasis. The effects of initial reoxygenation after a hypoxic insult (H/R) on functional and molecular properties of the BBB and TJs remain unclear. In situ brain perfusion and Western blot analyses were performed to assess in vivo BBB integrity on reoxygenation after a hypoxic insult of 6% O2 for 1 h. Model conditions [blood pressure, blood gas chemistries, cerebral blood flow (CBF), and brain ATP concentration] were also assessed to ensure consistent levels and criteria for insult. In situ brain perfusion revealed that initial reoxygenation (10 min) significantly increased the uptake of [14C]sucrose into brain parenchyma. Capillary depletion and CBF analyses indicated the perturbations were due to increased paracellular permeability rather than vascular volume changes. Hypoxia with reoxygenation (10 min) produced an increase in BBB permeability with associated alterations in tight junctional protein expression. These results suggest that H/R leads to reorganization of TJs and increased paracellular diffusion at the BBB, which is not a result of increased CBF, vascular volume change, or endothelial uptake of marker. Additionally, the tight junctional protein occludin had a shift in bands that correlated with functional changes (i.e., increased permeability) without significant change in expression of claudin-3, zonula occludens-1, or actin. H/R-induced changes in the BBB may result in edema and/or associated pathological outcomes.

Protein Kinase C Activation Modulates Reversible Increase in Cortical Blood–Brain Barrier Permeability and Tight Junction Protein Expression during Hypoxia and Posthypoxic Reoxygenation

“…Our results are consistent with other studies, which have shown specific brain regions to be especially vulnerable, whereas other regions are more resistant to global cerebral ischemia/Hx (Pulsinelli et al, 1982;Himeda et al, 2005). Previous studies have shown that Hxwith HR-induced increased paracellular [ 14 C]-sucrose (molecular weight B342) diffusion across a range of Hx conditions (6% O 2 30 minutes to 18% O 2 60 minutes) (Witt et al, 2003). However, vascular permeability was measured after a period of HR ( > 10 minutes).…”

“…Previous studies in our laboratory have shown increased [ 14 C]-sucrose permeability and edema formation with changes in occludin protein expression in cerebral microvessels after Hx and HR exposure (Witt et al, 2003(Witt et al, , 2008. Total PKC activity in isolated cerebral microvessels was also increased, whereas HR (10 minutes) attenuated the increased total PKC activity.…”

“…It could be argued that autoregulation may reduce cerebral blood flow and blood pressure resulting in ischemic conditions. However, using this Hx model, Witt et al (2003) reported increased CBF and blood pressure during the first 10 to 20 minutes of Hx insult and the initial period of reoxygenation. Thus, ischemia is unlikely to be a component in this study.…”

“…Studies show that specific brain regions are vulnerable to global cerebral ischemia/Hx, especially the hippocampal CA1 pyramidal neurons and cortical neurons, whereas other regions are more resistant (Pulsinelli et al, 1982;Himeda et al, 2005). During the ischemic, and subsequent reperfusion phase of stroke, the integrity of the blood-brain barrier (BBB) is lost (Mark and Davis, 2002;Witt et al, 2003Witt et al, , 2008. Elucidating the mechanisms of Hx and reoxygenation (HR)-induced changes in BBB integrity remains a considerable task.…”

Activation of PKC modulates blood-brain barrier endothelial cell permeability changes induced by hypoxia and posthypoxic reoxygenation

“…It has been determined that the lack of oxygen (hypoxia, H) followed by reperfusion (posthypoxic reoxygenation, H/R) during stroke contributes to both neuronal and vascular damage. Both H and H/R cause increases in cerebrovascular permeability with concomitant increases in vasogenic cerebral edema (1,36,47).Previous studies (36, 47) have demonstrated that H and H/R cause changes in paracellular permeability to [ 14 C]sucrose in cerebral vascular endothelial cells. These permeability changes have been correlated with alterations in the expression and localization of several TJ proteins (36).…”