University of Vermont (P30 GM103498)
"Center for Neuroscience Excellence"

Pilot Project 1: "Endothelial Ca2+ Signals and Blood Brain Barrier Permeability Following Traumatic Brain Injury"
Investigator: Kalev Freeman, M.D., Ph.D.

Traumatic brain injury (TBI) affects more than 1.7 million Americans each year, and survivors may experience long-term neurocognitive problems. Disruption of the blood brain barrier (BBB) may play a role in outcomes after traumatic brain injury (TBI), due to resulting cerebral edema and vasospasm. The fundamental mechanisms that alter BBB function after TBI are unknown. The BBB consists of a matrix of tight junctions and interconnecting endothelial cells (EC) that are responsible for BBB integrity. ECs also play a key role in vasodilatory pathways, which are activated by EC Ca2+ signals to oppose vasoconstriction. Vasogenic brain edema can result from the opening of the endothelial barrier due to endothelial contractions activated by controlled cortical injury. Experimental brain injury also leads to increased production of reactive oxygen species (ROS), which can disrupt endothelial tight junctions and interfere with BBB electrical resistance and permeability. Paradoxically, the BBB may act as a target for ROS-mediated cellular damage, but it also acts to impede transport of potentially therapeutic antioxidant compounds. Reversible changes in BBB permeability have also been linked to mobilization of extracellular Ca2+ in cerebral ECs, but the role of EC Ca2+ in BBB permeability is unknown.

 In a rodent model of TBI, we found that mesenteric arteries exhibit increased myogenic tone and impaired endothelium-mediated dilations, similar to the reported effects of hypertension. Contrary to expectation, we found that Ca2+ signals were profoundly elevated in arteries from TBI animals. This suggested that Ca2+ signals are uncoupled from vasodilatory responses. Mechanisms responsible for EC Ca2+ signals include the vanilloid transient receptor potential cation-channel (TRPV4) and intracellular inositol trisphosphate receptors (IP3Rs). TRPV4 opening causes local Ca2+ entry from the extracellular environment, whereas IP3R activation causes release of intracellular Ca2+ stores as propagating waves or discrete pulsars in endothelial projections. Using high-speed confocal microscopy, Ca2+ imaging produces patterns unique to each molecular signaling pathway, which can be analyzed to determine the channel involved in creating the signal. TRPV4 signals are of particular interest, because moderate level of TRPV4 channel activation causes vasodilation and higher activity may affect barrier function in other vascular tissue beds. The applicant's K08 project seeks to understand the molecular basis and functional consequences of endothelial Ca2+ signals in mesenteric resistance vessels after TBI. We now propose a completely novel, yet complementary project that exploits the availability of cerebral vessels from the same animals to address a fundamental neuroscience question: how does acute neural injury affect the blood brain barrier?

 The goal of this project is to understand the changes in cerebrovascular endothelial Ca2+ signals, vasodilation, and BBB permeability after acute brain injury. We hypothesize that TBI causes alterations in cerebral EC Ca2+ signaling that produce functional consequences on vasodilation and BBB electrical resistance. If an acute brain injury can cause long-term changes in the endothelium of a vascular bed far removed from the brain, as demonstrated by our pilot data, it is likely that endothelial cells in the cerebral vasculature are also affected. Our rational for focusing on TRPV4 channel is that activation of this Ca2+ channel has been linked to altered barrier function in other tissues. If our hypothesis is correct, the results expected from this pilot project will support a novel mechanism that could explain the neuropathology, including edema, vasospasm, and altered neurovascular coupling, that follows acute brain injury.