Dmitriy N. Atochin

Through-skull fluorescence imaging of the brain in a new near-infrared window

To date, brain imaging has largely relied on X-ray computed tomography and magnetic resonance angiography with limited spatial resolution and long scanning times. Fluorescence-based brain imaging in the visible and traditional near-infrared regions (400–900 nm) is an alternative but currently requires craniotomy, cranial windows and skull thinning techniques, and the penetration depth is limited to 1–2 mm due to light scattering. Here, we report through-scalp and through-skull fluorescence imaging of mouse cerebral vasculature without craniotomy utilizing the intrinsic photoluminescence of single-walled carbon nanotubes in the 1.3–1.4 micrometre near-infrared window. Reduced photon scattering in this spectral region allows fluorescence imaging reaching a depth of >2 mm in mouse brain with sub-10 micrometre resolution. An imaging rate of ~5.3 frames/s allows for dynamic recording of blood perfusion in the cerebral vessels with sufficient temporal resolution, providing real-time assessment of blood flow anomaly in a mouse middle cerebral artery occlusion stroke model.

Tissue Plasminogen Activator Promotes Matrix Metalloproteinase-9 Upregulation After Focal Cerebral Ischemia

Background and Purpose— Thrombolytic therapy with tissue plasminogen activator (tPA) in ischemic stroke is limited by increased risks of cerebral hemorrhage and brain injury. In part, these phenomena may be related to neurovascular proteolysis mediated by matrix metalloproteinases (MMPs). Here, we used a combination of pharmacological and genetic approaches to show that tPA promotes MMP-9 levels in stroke in vivo. Methods— In the first experiment, spontaneously hypertensive rats were subjected to 3 hours of transient focal cerebral ischemia. The effects of tPA (10 mg/kg IV) on ischemic brain MMP-9 levels were assessed by zymography. In the second experiment, wild-type (WT) and tPA knockout mice were subjected to 2 hours of transient focal cerebral ischemia, and MMP-9 levels and brain edema during reperfusion were assessed. Phenotype rescue was performed by administering tPA to the tPA knockout mice. Results— In the first experiment, exogenous tPA did not change infarct size but amplified MMP-9 levels in ischemic rat brain at 24 hours. Coinfusion of the plasmin inhibitor tranexamic acid (300 mg/kg) did not ameliorate this effect, suggesting that it was independent of plasmin. In the second experiment, ischemic MMP-9 levels, infarct size, and brain edema in tPA knockouts were significantly lower than WT mice. Administration of exogenous tPA (10 mg/kg IV) did not alter infarction but reinstated the ischemic MMP-9 response back up to WT levels and correspondingly worsened edema. Conclusions— These data demonstrate that tPA upregulates brain MMP-9 levels in stroke in vivo, and suggest that combination therapies targeting MMPs may improve tPA therapy.

The phosphorylation state of eNOS modulates vascular reactivity and outcome of cerebral ischemia in vivo

NO plays critical roles in vascular function. We show that modulation of the eNOS serine 1179 (S1179) phosphorylation site affects vascular reactivity and determines stroke size in vivo. Transgenic mice expressing only a phosphomimetic (S1179D) form of eNOS show greater vascular reactivity, develop less severe strokes, and have improved cerebral blood flow in a middle cerebral artery occlusion model than mice expressing an unphosphorylatable (S1179A) form. These results provide a molecular mechanism by which multiple diverse cardiovascular risks, such as diabetes and obesity, may be centrally integrated by eNOS phosphorylation in vivo to influence blood flow and cardiovascular disease. They also demonstrate the in vivo relevance of posttranslational modification of eNOS in vascular function.

Effects of Neuroglobin Overexpression on Acute Brain Injury and Long-Term Outcomes After Focal Cerebral Ischemia

Background and Purpose— Emerging data suggest that neuroglobin (Ngb) may protect against hypoxic/ischemic neuronal insults. However, the underlying mechanisms in vivo and implications for long-term outcomes are still not well understood. Methods— Using our newly created Ngb overexpressing transgenic (Ngb-Tg) mice, we measured brain infarction on day 1 and day 14 after transient focal cerebral ischemia and performed neurobehavioral assessments in sensorimotor deficits on days 1, 3, 7, and 14. To test the hypothesis that Ngb may play a role in reducing oxidative stress after stroke, intracellular malondialdehyde levels were measured and compared in Ngb-Tg and wild-type mice. Results— Increased Ngb mRNA and protein levels were identified in Ngb-Tg brains. Malondialdehyde levels in ischemic hemispheres of Ngb-Tg were significantly reduced compared with wild-type controls at 8 hours and 22 hours after transient focal cerebral ischemia. Compared with wild-type controls, brain infarction volumes 1 day and 14 days after transient focal cerebral ischemia were significantly reduced in Ngb-Tg mice. However, there were no significant improvements in sensorimotor deficits for up to 14 days after stroke in Ngb-Tg mice compared with wild-type controls. Conclusions— Ngb reduces tissue infarction and markers of oxidative stress after stroke. Tissue protection by overexpressing Ngb can be sustained for up to 2 weeks.

Rapid Cerebral Ischemic Preconditioning in Mice Deficient in Endothelial and Neuronal Nitric Oxide Synthases

Background and Purpose— The purpose of this study was to test the hypothesis that nitric oxide is required for preconditioning in an intact animal model of focal ischemia using neuronal and endothelial nitric oxide synthase (nNOS and eNOS) knockout mice. Methods— Cerebral blood flow was measured in wild-type, nNOS knockout, and eNOS knockout mice by hydrogen clearance (absolute) and laser Doppler flowmetry (relative). Mice were preconditioned by three 5-minute episodes of transient middle cerebral artery occlusion (MCAO) and subjected to permanent MCAO. Neurological deficit and infarct size were determined 24 hours later. Results— Although wild-type mice showed protection from ischemic preconditioning, neither eNOS nor nNOS knockout mice showed protection. Laser Doppler measurements indicated that the relative blood flow decreases in core ischemic areas were the same in all groups. Conclusions— Neither eNOS nor nNOS knockout mice show protection from rapid ischemic preconditioning, suggesting that nitric oxide may play a role in the molecular mechanisms of protection.

Contributions of endothelial and neuronal nitric oxide synthases to cerebrovascular responses to hyperoxia.

Hyperoxia causes a transient decrease in CBF, followed by a later rise. The mediators of these effects are not known. We used mice lacking endothelial or neuronal nitric oxide synthase (NOS) isoforms (eNOS−/− and nNOS−/− mice) to study the roles of the NOS isoforms in mediating changes in cerebral vascular tone in response to hyperoxia. Resting regional cerebral blood flow (rCBF) did not differ between wild type (WT), eNOS−/− mice, and nNOS−/− mice. eNOS−/− mice showed decreased cerebrovascular reactivities to NG-nitro-L-arginine methyl ester (L-NAME), PAPA NONOate, acetylcholine (Ach), and SOD1. In response to hyperbaric oxygen (HBO2) at 5 ATA, WT and nNOS−/− mice showed decreases in rCBF over 30 minutes, but eNOS−/− mice did not. After 60 minutes HBO2, rCBF increased more in WT mice than in eNOS−/− or nNOS−/− mice. Brain NO-metabolites (NOx) decreased in WT and eNOS−/− mice within 30 minutes of HBO2, but after 45 minutes, NOx rose above control levels, whereas they did not change in nNOS−/− mice. Brain 3NT increased during HBO2 in WT and eNOS−/− but did not change in nNOS−/− mice. These results suggest that modulation of eNOS-derived NO by HBO2 is responsible for the early vasoconstriction responses, whereas late HBO2-induced vasodilation depends upon both eNOS and nNOS.

Endothelial nitric oxide synthase transgenic models of endothelial dysfunction

Endothelial production of nitric oxide is critical to the regulation of vascular responses, including vascular tone and regional blood flow, leukocyte–endothelial interactions, platelet adhesion and aggregation, and vascular smooth muscle cell proliferation. A relative deficiency in the amount of bioavailable vascular NO results in endothelial dysfunction, with conditions that are conducive to the development of atherosclerosis: thrombosis, inflammation, neointimal proliferation, and vasoconstriction. This review focuses on mouse models of endothelial dysfunction caused by direct genetic modification of the endothelial nitric oxide synthase (eNOS) gene. We first describe the cardiovascular phenotypes of eNOS knockout mice, which are a model of total eNOS gene deficiency and thus the ultimate model of endothelial dysfunction. We then describe S1177A and S1177D eNOS mutant mice as mouse models with altered eNOS phosphorylation and therefore varying degrees of endothelial dysfunction. These include transgenic mice that carry the eNOS S1177A and S1177D transgenes, as well as knockin mice in which the endogenous eNOS gene has been mutated to carry the S1177A and S1177D mutations. Together, eNOS knockout mice and eNOS S1177 mutant mice are useful tools to study the effects of total genetic deficiency of eNOS as well as varying degrees of endothelial dysfunction caused by eNOS S1177 phosphorylation.

Oxygen seizure latency and peroxynitrite formation in mice lacking neuronal or endothelial nitric oxide synthases.

Nitric oxide (NO) from endothelial or neuronal NO synthases (eNOS or nNOS) may contribute both to the cerebrovascular responses to oxygen and potentially to the peroxynitrite-mediated toxic effects of hyperbaric oxygen (HBO(2)) on the central nervous system (CNS O(2) toxicity). In mice lacking eNOS or nNOS (-/-), regional cerebral blood flow (rCBF) and 3-nitrotyrosine (3-NT), a biochemical marker for peroxynitrite (ONOO(-)) formation, were measured in the brain during HBO(2) exposure. These variables were then correlated with EEG spiking activity related to CNS O(2) toxicity. In wild-type (WT) mice, HBO(2) exposure transiently reduced rCBF, but by 60 min rCBF was restored to baseline levels and above, followed by EEG spikes. Mice lacking nNOS also showed initial depression of rCBF followed by hyperemia but the delay in the onset of EEG discharges was greater. In contrast, in eNOS-deficient mice rCBF did not decrease and hyperemia was less pronounced during HBO(2). EEG spike latency was longer in eNOS(-/-) compared to WT or nNOS(-/-) mice. 3-NT gradually increased in all strains during HBO(2) but accumulation was slower in nNOS(-/-) mice, consistent with less ONOO(-) production. These results indicate that NOS-deficient mice have different cerebrovascular responses and tolerance to HBO(2) depending on which enzyme isoform is affected. The data suggest a key role for eNOS-dependent NO production in cerebral vasoconstriction and in the development of hyperoxic hyperemia preceding O(2) seizures, whereas neuronal NO may mediate toxic effects of HBO(2) mainly by its reaction with superoxide to generate the stronger oxidant, peroxynitrite.

Optical coherence tomography for the quantitative study of cerebrovascular physiology

Doppler optical coherence tomography (DOCT) and OCT angiography are novel methods to investigate cerebrovascular physiology. In the rodent cortex, DOCT flow displays features characteristic of cerebral blood flow, including conservation along nonbranching vascular segments and at branch points. Moreover, DOCT flow values correlate with hydrogen clearance flow values when both are measured simultaneously. These data validate DOCT as a noninvasive quantitative method to measure tissue perfusion over a physiologic range.

Cerebrovascular Thromboprophylaxis in Mice by Erythrocyte-Coupled Tissue-Type Plasminogen Activator

Background— Cerebrovascular thrombosis is a major source of morbidity and mortality after surgery, but thromboprophylaxis in this setting is limited because of the formidable risk of perioperative bleeding. Thrombolytics (eg, tissue-type plasminogen activator [tPA]) cannot be used prophylactically in this high-risk setting because of their short duration of action and risk of causing hemorrhage and central nervous system damage. We found that coupling tPA to carrier red blood cells (RBCs) prolongs and localizes tPA activity within the bloodstream and converts it into a thromboprophylactic agent, RBC/tPA. To evaluate the utility of this new approach for preventing cerebrovascular thrombosis, we examined the effect of RBC/tPA in animal models of cerebrovascular thromboembolism and ischemia. Methods and Results— Preformed fibrin microemboli were injected into the middle carotid artery of mice, occluding downstream perfusion and causing severe infarction and 50% mortality within 48 hours. Preinjected RBC/tPA rapidly lysed nascent cerebral thromboemboli, providing rapid, durable reperfusion and reducing morbidity and mortality. These beneficial effects were not achieved by preinjection of tPA, even at a 10-fold higher dose, which increased mortality from 50% to 90% by 10 hours after embolization. RBC/tPA injected 10 minutes after tail amputation to simulate postsurgical hemostasis did not cause bleeding from the wound, whereas soluble tPA caused profuse bleeding. RBC/tPA neither aggravated brain damage caused by focal ischemia in a filament model of middle carotid artery occlusion nor caused postthrombotic hemorrhage in hypertensive rats. Conclusions— These results suggest a potential RBC/tPA utility as thromboprophylaxis in patients who are at risk for acute cerebrovascular thromboembolism.