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. In preliminary studies we have now found evidence that a BBB Na-HCO3 cotransporter (NBC) constitutes a 3rd prominent BBB Na transporter that is also stimulated during ischemia to substantially participate in cerebral edema formation. This gives us an important additional therapeutic target for reducing the highly injurious early edema formation in ischemic stroke. While our studies indicate that NKCC, NHE and NBC appear to be promising therapeutic targets for reducing edema in otherwise healthy stroke patients, it must be recognized that hyperglycemia, a common co-morbidity in stroke present in ~30% of patients, causes greater cerebral edema and worsened outcome. The underlying mechanisms for this are poorly understood. However, in recent studies we have found that hyperglycemia increases expression and activity of BBB NKCC, NHE and NBC and augments their stimulation by ischemic factors. Further, we have also found evidence that the hyperglycemia exacerbation of brain edema in MCAO is reduced by inhibition of BBB NKCC, NHE and NBC. Our hypothesis is that BBB NKCC, NHE and/or NBC participate in the hyperglycemia-induced augmentation of cerebral edema formation and constitute effective therapeutic targets in hyperglycemic stroke.
The first aim i s to determine whether 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 effects on CMEC NBC activity.
The second aim i s to determine whether hyperglycemic conditions increase expression and activity of BBB NKCC, NHE and/or NBC and augment stimulation of these Na transporters by ischemic factors. We will use Western blot, microspectrofluorometry and radioisotopic flux assays to test the effects of hyperglycemia on CMEC NKCC, NHE and NBC protein and activity. We will also use immunoEM to evaluate NKCC, NHE and NBC in BBB in situ of hyperglycemic rats.
The third aim i s to determine whether inhibition of BBB NKCC, NHE and/or NBC attenuates ischemia-induced edema in hyperglycemic animals. Here, we will examine the effects of inhibiting BBB NKCC, NHE and NBC 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, NHE and NBC inhibitors for reduction of edema when administered after the onset of ischemia.

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS039953-10
Application #
8720068
Study Section
Brain Injury and Neurovascular Pathologies Study Section (BINP)
Program Officer
Bosetti, Francesca
Project Start
2000-04-01
Project End
2017-05-31
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
10
Fiscal Year
2014
Total Cost
$327,022
Indirect Cost
$110,460
Name
University of California Davis
Department
Physiology
Type
Schools of Medicine
DUNS #
047120084
City
Davis
State
CA
Country
United States
Zip Code
95618
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
O'Donnell, Martha E; Chen, Yi-Je; Lam, Tina I et al. (2013) Intravenous HOE-642 reduces brain edema and Na uptake in the rat permanent middle cerebral artery occlusion model of stroke: evidence for participation of the blood-brain barrier Na/H exchanger. J Cereb Blood Flow Metab 33:225-34
O'Donnell, Martha E; Lam, Tina I; Tran, Lien Q et al. (2006) Estradiol reduces activity of the blood-brain barrier Na-K-Cl cotransporter and decreases edema formation in permanent middle cerebral artery occlusion. J Cereb Blood Flow Metab 26:1234-49
Pedersen, S F; O'Donnell, M E; Anderson, S E et al. (2006) Physiology and pathophysiology of Na+/H+ exchange and Na+ -K+ -2Cl- cotransport in the heart, brain, and blood. Am J Physiol Regul Integr Comp Physiol 291:R1-25
Foroutan, Shahin; Brillault, Julien; Forbush, Biff et al. (2005) Moderate-to-severe ischemic conditions increase activity and phosphorylation of the cerebral microvascular endothelial cell Na+-K+-Cl- cotransporter. Am J Physiol Cell Physiol 289:C1492-501
O'Donnell, Martha E; Duong, Vicki; Suvatne, Jimmy et al. (2005) Arginine vasopressin stimulation of cerebral microvascular endothelial cell Na-K-Cl cotransporter activity is V1 receptor and [Ca] dependent. Am J Physiol Cell Physiol 289:C283-92
O'Donnell, Martha E; Tran, Lien; Lam, Tina I et al. (2004) Bumetanide inhibition of the blood-brain barrier Na-K-Cl cotransporter reduces edema formation in the rat middle cerebral artery occlusion model of stroke. J Cereb Blood Flow Metab 24:1046-56