The long term goal of this project is to identify blood-brain barrier (BBB) ion transporters that mediate ischemia-induced brain edema. During the early hours of ischemic stroke, edema forms in the presence of an intact BBB by a process involving BBB transport of Na and Cl from blood into brain. Our previous studies have shown that Na-K-Cl cotransport (NKCC) and Na/H exchange (NHE), present in the luminal BBB membrane, are stimulated by ischemic factors, including hypoxia, aglycemia and vasopressin (AVP) and that inhibiting BBB NKCC and/or NHE activity reduces brain edema in the rat middle cerebral artery occlusion (MCAO) model of stroke. Thus, BBB NKCC and NHE appear to be promising therapeutic targets for reducing ischemia- induced brain edema. Hyperglycemia, a common co-morbidity in stroke present in ~30% of patients, causes greater cerebral edema. However, the underlying mechanisms are poorly understood. In recent studies we have found that hyperglycemia increases expression and activity of BBB NKCC and NHE and augments their stimulation by ischemic factors. Further, we have found evidence that the hyperglycemia exacerbation of brain edema in MCAO is reduced by inhibition of BBB NKCC and NHE. Our hypothesis is that BBB NKCC and NHE participate in the hyperglycemia-induced augmentation of cerebral edema formation and constitute effective therapeutic targets in hyperglycemic stroke. We have recently also found evidence that a Na-HCO3 cotransporter (NBC) is a third prominent BBB Na transporter that is stimulated by ischemic factors and that hyperglycemia increases expression and activity of BBB NBC, suggesting the exciting possibility that BBB NBC may provide an additional therapeutic target for reducing edema formation in ischemic stroke, both normoglycemic and hyperglycemic. Our studies wil thus also examine this possibility.
The first aim i s to determine whether hyperglycemic conditions increase expression and activity of BBB NKCC and/or NHE and augment NKCC and NHE stimulation by ischemic factors. We will use Western blot, microspectrofluorometry and radioisotopic flux assays to test the efects of hyperglycemia on cerebral microvascular endothelial cell (CMEC) NKCC and NHE protein and activity. We will use immunoelectron microscopy to evaluate NKCC and NHE in BBB in situ of hyperglycemic rats.
The second aim i s to determine whether inhibition of BBB NKCC and/or NHE attenuates ischemia-induced edema in hyperglycemic animals. Here, we will examine the effects of BBB NKCC and NHE inhibition on ischemia-induced changes in rat brain Na and water, using nuclear magnetic resonance methods and rats with STZ-induced hyperglycemia. We will also assess the efficacy of NKCC and NHE for reduction of cerebral edema when administered after the onset of ischemia.
The third aim i s to determine whether an NBC protein is present at the luminal BBB membrane and is stimulated by ischemic factors. We will use Western blot and immunoEM to evaluate BBB NBC protein in CMEC and in BBB in situ, and microspectrofluorometry to assess ischemic factor and hyperglycemia effects on CMEC NBC activity.

Public Health Relevance

Brain edema (swelling) is a major cause of brain damage in stroke and a leading cause of death in the U.S. High blood sugar (hyperglycemia), a complicating factor present in about 30% of stroke patients, results in greater edema formation and worse stroke outcome, although the reasons for this are poorly understood. With the support of this grant we will test whether therapeutic approaches aimed at inhibiting activity of sodium transporters in brain microvessels will reduce edema and brain damage in hyperglycemic stroke patients.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
High Priority, Short Term Project Award (R56)
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Brain Injury and Neurovascular Pathologies Study Section (BINP)
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Bosetti, Francesca
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University of California Davis
Schools of Medicine
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Chen, Yi-Je; Wallace, Breanna K; Yuen, Natalie et al. (2015) Blood-brain barrier KCa3.1 channels: evidence for a role in brain Na uptake and edema in ischemic stroke. Stroke 46:237-44
Yuen, Natalie; Lam, Tina I; Wallace, Breanna K et al. (2014) Ischemic factor-induced increases in cerebral microvascular endothelial cell Na/H exchange activity and abundance: evidence for involvement of ERK1/2 MAP kinase. Am J Physiol Cell Physiol 306:C931-42
Wallace, Breanna K; Jelks, Karen A; O'Donnell, Martha E (2012) Ischemia-induced stimulation of cerebral microvascular endothelial cell Na-K-Cl cotransport involves p38 and JNK MAP kinases. Am J Physiol Cell Physiol 302:C505-17
Wallace, Breanna K; Foroutan, Shahin; O'Donnell, Martha E (2011) Ischemia-induced stimulation of Na-K-Cl cotransport in cerebral microvascular endothelial cells involves AMP kinase. Am J Physiol Cell Physiol 301:C316-26
Rutkowsky, Jennifer M; Wallace, Breanna K; Wise, Phyllis M et al. (2011) Effects of estradiol on ischemic factor-induced astrocyte swelling and AQP4 protein abundance. Am J Physiol Cell Physiol 301:C204-12
Lam, Tina I; Wise, Phyllis M; O'Donnell, Martha E (2009) Cerebral microvascular endothelial cell Na/H exchange: evidence for the presence of NHE1 and NHE2 isoforms and regulation by arginine vasopressin. Am J Physiol Cell Physiol 297:C278-89