Nozomi Nishimura

Deep tissue multiphoton microscopy using longer wavelength excitation

We compare the maximal two-photon fluorescence microscopy (TPM) imaging depth achieved with 775-nm excitation to that achieved with 1280-nm excitation through in vivo and ex vivo TPM of fluorescently-labeled blood vessels in mouse brain. We achieved high contrast imaging of blood vessels at approximately twice the depth with 1280-nm excitation as with 775-nm excitation. An imaging depth of 1 mm can be achieved in in vivo imaging of adult mouse brains at 1280 nm with approximately 1-nJ pulse energy at the sample surface. Blood flow speed measurements at a depth of 900 mum are performed.

Suppressed neuronal activity and concurrent arteriolar vasoconstriction may explain negative blood oxygenation level-dependent signal.

Synaptic transmission initiates a cascade of signal transduction events that couple neuronal activity to local changes in blood flow and oxygenation. Although a number of vasoactive molecules and specific cell types have been implicated, the transformation of stimulus-induced activation of neuronal circuits to hemodynamic changes is still unclear. We use somatosensory stimulation and a suite of in vivo imaging tools to study neurovascular coupling in rat primary somatosensory cortex. Our stimulus evoked a central region of net neuronal depolarization surrounded by net hyperpolarization. Hemodynamic measurements revealed that predominant depolarization corresponded to an increase in oxygenation, whereas predominant hyperpolarization corresponded to a decrease in oxygenation. On the microscopic level of single surface arterioles, the response was composed of a combination of dilatory and constrictive phases. Critically, the relative strength of vasoconstriction covaried with the relative strength of oxygenation decrease and neuronal hyperpolarization. These results suggest that a neuronal inhibition and concurrent arteriolar vasoconstriction correspond to a decrease in blood oxygenation, which would be consistent with a negative blood oxygenation level-dependent functional magnetic resonance imaging signal.

Targeted insult to subsurface cortical blood vessels using ultrashort laser pulses: three models of stroke.

We present a method to produce vascular disruptions within rat brain parenchyma that targets single microvessels. We used two-photon microscopy to image vascular architecture, to select a vessel for injury and to measure blood-flow dynamics. We irradiated the vessel with high-fluence, ultrashort laser pulses and achieved three forms of vascular insult. (i) Vessel rupture was induced at the highest optical energies; this provides a model for hemorrhage. (ii) Extravasation of blood components was induced near the lowest energies and was accompanied by maintained flow in the target vessel. (iii) An intravascular clot evolved when an extravasated vessel was further irradiated. Such clots dramatically impaired blood flow in downstream vessels, in which speeds dropped to as low as ∼10% of baseline values. This demonstrates that a single blockage to a microvessel can lead to local cortical ischemia. Lastly, we show that hemodilution leads to a restoration of flow in secondary downstream vessels.

Penetrating arterioles are a bottleneck in the perfusion of neocortex

Penetrating arterioles bridge the mesh of communicating arterioles on the surface of cortex with the subsurface microvascular bed that feeds the underlying neural tissue. We tested the conjecture that penetrating arterioles, which are positioned to regulate the delivery of blood, are loci of severe ischemia in the event of occlusion. Focal photothrombosis was used to occlude single penetrating arterioles in rat parietal cortex, and the resultant changes in flow of red blood cells were measured with two-photon laser-scanning microscopy in individual subsurface microvessels that surround the occlusion. We observed that the average flow of red blood cells nearly stalls adjacent to the occlusion and remains within 30% of its baseline value in vessels as far as 10 branch points downstream from the occlusion. Preservation of average flow emerges 350 μm away; this length scale is consistent with the spatial distribution of penetrating arterioles. We conclude that penetrating arterioles are a bottleneck in the supply of blood to neocortex, at least to superficial layers.

Dynamics of femtosecond laser-induced breakdown in water from femtoseconds to microseconds

Using time-resolved imaging and scattering techniques, we directly and indirectly monitor the breakdown dynamics induced in water by femtosecond laser pulses over eight orders of magnitude in time. We resolve, for the first time, the picosecond plasma dynamics and observe a 20 ps delay before the laser-produced plasma expands. We attribute this delay to the electron-ion energy transfer time.

Two-photon microscopy as a tool to study blood flow and neurovascular coupling in the rodent brain

The cerebral vascular system services the constant demand for energy during neuronal activity in the brain. Attempts to delineate the logic of neurovascular coupling have been greatly aided by the advent of two-photon laser scanning microscopy to image both blood flow and the activity of individual cells below the surface of the brain. Here we provide a technical guide to imaging cerebral blood flow in rodents. We describe in detail the surgical procedures required to generate cranial windows for optical access to the cortex of both rats and mice and the use of two-photon microscopy to accurately measure blood flow in individual cortical vessels concurrent with local cellular activity. We further provide examples on how these techniques can be applied to the study of local blood flow regulation and vascular pathologies such as small-scale stroke.

Age-Related Intimal Stiffening Enhances Endothelial Permeability and Leukocyte Transmigration

Inhibiting endothelial cell contractility reverses the deleterious effects of age-related matrix stiffening on normal cell function, which could help prevent the development of atherosclerosis. Rock Your Heart Out According to novelist Thomas Bailey Aldrich, “To keep the heart unwrinkled, to be hopeful, kindly, cheerful, reverent, is to triumph over old age” (from Ponkapoag Papers). Unfortunately, despite a positive attitude, aging is accompanied by several changes of heart, at least at the cellular level. One age-related “wrinkle” is stiffening of the extracellular matrix that lines the blood vessels, a change that has been linked to atherosclerosis; yet, the cellular and mechanical features that couple the two conditions have remained elusive. Now, using a clever combination of biomaterials, cells, aortas, and mice, Huynh and colleagues have demystified the correlation between aging and atherosclerosis, showing that cell contractility is at the heart of it all. The authors first developed an in vitro system that mimicked the basic structures of both young and old blood vessels. Synthetic hydrogel matrices of varying stiffnesses were seeded with bovine aortic endothelial cells. By administering a solution of fluorescently labeled molecules to the cell-gel system and watching how the dye moved across the cell layer, Huynh et al. determined that permeability increased as a function of matrix stiffness, suggesting that age alone was a disruptive factor. These results were confirmed ex vivo by performing atomic force microscopy with decellularized thoracic aortas from both young (~10 weeks) and old (~92 weeks) mice. In both of these systems, the enhanced vessel permeability resulted from an increase in the distance—or junction—between neighboring cells. This increase in the so-called gap junction width also permitted the passage of leukocytes through the endothelial cell monolayer; along with leaky vasculature, cellular transmigration is a hallmark of atherosclerosis progression. Because the Rho signaling pathway is linked to the cellular cytoskeleton and, in turn, contractility, Huynh et al. hypothesized that they could reverse the effects of age-related intimal stiffening by inhibiting Rho-associated kinase (ROCK). By administering a pharmacological ROCK inhibitor (Y-27632) to their in vitro setup and to old mice, the authors showed that gap junction widths and endothelial cellular forces decreased. In vitro, the inhibitor also prevented leukocyte transmigration. These observations suggest that directly interfering with Rho signaling is a viable treatment option for age-related atherosclerosis. And because inhibitors of Rho signaling, such as fasudil, are already available in the clinic, one might say that physicians and researchers are ready to rock. Age is the most significant risk factor for atherosclerosis; however, the link between age and atherosclerosis is poorly understood. During both aging and atherosclerosis progression, the blood vessel wall stiffens owing to alterations in the extracellular matrix. Using in vitro and ex vivo models of vessel wall stiffness and aging, we show that stiffening of extracellular matrix within the intima promotes endothelial cell permeability—a hallmark of atherogenesis. When cultured on hydrogels fabricated to match the elasticity of young and aging intima, endothelial monolayers exhibit increased permeability and disrupted cell-cell junctions on stiffer matrices. In parallel experiments, we showed a corresponding increase in cell-cell junction width with age in ex vivo aortas from young (10 weeks) and old (21 to 25 months) healthy mice. To investigate the mechanism by which matrix stiffening alters monolayer integrity, we found that cell contractility increases with increased matrix stiffness, mechanically destabilizing cell-cell junctions. This increase in endothelial permeability results in increased leukocyte extravasation, which is a critical step in atherosclerotic plaque formation. Mild inhibition of Rho-dependent cell contractility using Y-27632, an inhibitor of Rho-associated kinase, or small interfering RNA restored monolayer integrity in vitro and in vivo. Our results suggest that extracellular matrix stiffening alone, which occurs during aging, can lead to endothelial monolayer disruption and atherosclerosis pathogenesis. Because previous therapeutics designed to decrease vascular stiffness have been met with limited success, our findings could be the basis for the design of therapeutics that target the Rho-dependent cellular contractile response to matrix stiffening, rather than stiffness itself, to more effectively prevent atherosclerosis progression.

Limitations of collateral flow after occlusion of a single cortical penetrating arteriole

Occlusions of penetrating arterioles, which plunge into cortex and feed capillary beds, cause severe decreases in blood flow and are potential causes of ischemic microlesions. However, surrounding arterioles and capillary beds remain flowing and might provide collateral flow around the occlusion. We used femtosecond laser ablation to trigger clotting in single penetrating arterioles in rat cortex and two-photon microscopy to measure changes in microvessel diameter and red blood cell speed after the clot. We found that after occlusion of a single penetrating arteriole, nearby penetrating and surface arterioles did not dilate, suggesting that alternate blood flow routes are not actively recruited. In contrast, capillaries showed two types of reactions. Capillaries directly downstream from the occluded arteriole dilated after the clot, but other capillaries in the same vicinity did not dilate. This heterogeneity in capillary response suggests that signals for vasodilation are vascular rather than parenchymal in origin. Although both neighboring arterioles and capillaries dilated in response to topically applied acetylcholine after the occlusion, the flow in the territory of the occluded arteriole did not improve. Collateral flow from neighboring penetrating arterioles is neither actively recruited nor effective in improving blood flow after the occlusion of a single penetrating arteriole.

In vivo two-photon excited fluorescence microscopy reveals cardiac- and respiration-dependent pulsatile blood flow in cortical blood vessels in mice

Subtle alterations in cerebral blood flow can impact the health and function of brain cells and are linked to cognitive decline and dementia. To understand hemodynamics in the three-dimensional vascular network of the cerebral cortex, we applied two-photon excited fluorescence microscopy to measure the motion of red blood cells (RBCs) in individual microvessels throughout the vascular hierarchy in anesthetized mice. To resolve heartbeat- and respiration-dependent flow dynamics, we simultaneously recorded the electrocardiogram and respiratory waveform. We found that centerline RBC speed decreased with decreasing vessel diameter in arterioles, slowed further through the capillary bed, and then increased with increasing vessel diameter in venules. RBC flow was pulsatile in nearly all cortical vessels, including capillaries and venules. Heartbeat-induced speed modulation decreased through the vascular network, while the delay between heartbeat and the time of maximum speed increased. Capillary tube hematocrit was 0.21 and did not vary with centerline RBC speed or topological position. Spatial RBC flow profiles in surface vessels were blunted compared with a parabola and could be measured at vascular junctions. Finally, we observed a transient decrease in RBC speed in surface vessels before inspiration. In conclusion, we developed an approach to study detailed characteristics of RBC flow in the three-dimensional cortical vasculature, including quantification of fluctuations in centerline RBC speed due to cardiac and respiratory rhythms and flow profile measurements. These methods and the quantitative data on basal cerebral hemodynamics open the door to studies of the normal and diseased-state cerebral microcirculation.

Preictal and Ictal Neurovascular and Metabolic Coupling Surrounding a Seizure Focus

Epileptic events initiate a large focal increase in metabolism and cerebral blood flow (CBF) to the ictal focus. In contrast, decreases in CBF have been demonstrated surrounding the focus, the etiology of which is unknown (i.e., arising either from active shunting of blood or passive steal). The relationship between these events and neuronal activity and metabolism are also unknown. We investigated neurovascular and neurometabolic coupling in the ictal surround using optical imaging of light scattering and cerebral blood volume, autofluorescence flavoprotein imaging (AFI), direct measurements of the cortical metabolic rate of oxygen and two-photon imaging of blood vessel diameter in a rat model of ictal events elicited with focal injection of 4-aminopyridine. We discovered a novel phenomenon, in which ictal events are preceded by preictal vasoconstriction of blood vessels in the surround, occurring 1–5 s before seizure onset, which may serve to actively shunt oxygenated blood to the imminently hypermetabolic focus or may be due to small local decreases in metabolism in the surround. Early ictal hypometabolism, transient decreases in cell swelling and cerebral blood volume in the surround are consistent with early ictal surround inhibition as a precipitating event in seizure onset as well as shaping the evolving propagating ictal wavefront, although the exact mechanism of these cerebrovascular and metabolic changes is currently unknown. AFI was extremely sensitive to the ictal onset zone and may be a useful mapping technique with clinical applications.