Traumatic Brain Injury Disrupts Cerebrovascular Tone Through Endothelial Inducible Nitric Oxide Synthase Expression and Nitric Oxide Gain of Function

Abstract

BackgroundTraumatic brain injury (TBI) has been reported to increase the concentration of nitric oxide (NO) in the brain and can lead to loss of cerebrovascular tone; however, the sources, amounts, and consequences of excess NO on the cerebral vasculature are unknown. Our objective was to elucidate the mechanism of decreased cerebral artery tone after TBI.Methods and ResultsCerebral arteries were isolated from rats 24 hours after moderate fluid‐percussion TBI. Pressure‐induced increases in vasoconstriction (myogenic tone) and smooth muscle Ca2+ were severely blunted in cerebral arteries after TBI. However, myogenic tone and smooth muscle Ca2+ were restored by inhibition of NO synthesis or endothelium removal, suggesting that TBI increased endothelial NO levels. Live native cell NO, indexed by 4,5‐diaminofluorescein (DAF‐2 DA) fluorescence, was increased in endothelium and smooth muscle of cerebral arteries after TBI. Clamped concentrations of 20 to 30 nmol/L NO were required to simulate the loss of myogenic tone and increased (DAF‐2T) fluorescence observed following TBI. In comparison, basal NO in control arteries was estimated as 0.4 nmol/L. Consistent with TBI causing enhanced NO‐mediated vasodilation, inhibitors of guanylyl cyclase, protein kinase G, and large‐conductance Ca2+‐activated potassium (BK) channel restored function of arteries from animals with TBI. Expression of the inducible isoform of NO synthase was upregulated in cerebral arteries isolated from animals with TBI, and the inducible isoform of NO synthase inhibitor 1400W restored myogenic responses following TBI.ConclusionsThe mechanism of profound cerebral artery vasodilation after TBI is a gain of function in vascular NO production by 60‐fold over controls, resulting from upregulation of the inducible isoform of NO synthase in the endothelium.

Nitric Oxide–Dependent Feedback Loop Regulates Transient Receptor Potential Vanilloid 4 (TRPV4) Channel Cooperativity and Endothelial Function in Small Pulmonary Arteries

“…NO levels were assessed using 5 μmol/L DAF‐FM (4‐amino‐5 methylamino‐2′,7′‐difluorofluorescenin diacetate) prepared in HEPES PSS with 0.02% pluronic acid 29. DAF‐FM forms a fluorescent triazole compound after binding to NO.…”

Traumatic Brain Injury Causes Endothelial Dysfunction in the Systemic Microcirculation through Arginase-1–Dependent Uncoupling of Endothelial Nitric Oxide Synthase

“…29,30 A fluid percussion injury was induced to a target pressure of <70 pounds per square inch (psi) over a 500 msec period; the pressure was transduced and measured in each surgery. This level allows >90% recovery, defined as ability to maintain upright posture, ambulate, and take oral hydration, and produces a highly reproducible outcome of moderate brain injury severity in those surviving animals.…”

“…This level allows >90% recovery, defined as ability to maintain upright posture, ambulate, and take oral hydration, and produces a highly reproducible outcome of moderate brain injury severity in those surviving animals. These experimental animals have measurable deficits in sensorimotor coordination, 30 along with significant cardiovascular 30 and cerebrovascular 29 effects. 29,30 Control animals were subjected to scalp incisions but without the percussion injury.…”

“…These experimental animals have measurable deficits in sensorimotor coordination, 30 along with significant cardiovascular 30 and cerebrovascular 29 effects. 29,30 Control animals were subjected to scalp incisions but without the percussion injury. At 24 h after recovery from surgery, animals were euthanized and tissues were collected for experiments.…”

“…Images were acquired at 30 frames/sec using an Andor Technology Nipkow spinning-disc confocal system coupled to a Nikon Eclipse E600 FN upright microscope equipped with a 60· water-dipping objective (numerical aperture 1.0) and an electron-multiplying charge-coupled device camera, as we have described previously. 29,34 DAF-2T fluorescence was detected using an excitation wavelength of 488 nm, and emitted fluorescence was collected using a 527-549-nm band-pass filter. Fluorescence in collected images was assessed offline by measuring the average fluorescence of 10 images from the same field using custom-designed software (A. Bonev, University of Vermont, Burlington, VT).…”