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

Thom P. Santisakultarm, Nathan R. Cornelius, Nozomi Nishimura, Andrew I. Schafer, Richard T. Silver, Peter C. Doerschuk, William L. Olbricht and Chris B. Schaffer

American Journal of Physiology Heart and Circulatory Physiology (2012)

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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 electrocar- diogram 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 characteris- tics of RBC flow in the three-dimensional cortical vasculature, includ- ing 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.

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Notch4 Normalization Reduces Blood Vessel Size in Arteriovenous Malformations

Patrick A. Murphy, Tyson N. Kim, Gloria Lu, Andrew W. Bollen, Chris B. Schaffer, Rong A. Wang

Science Translational Medicine (2012)

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Abnormally enlarged blood vessels underlie many life-threatening disorders including arteriovenous (AV) malformations (AVMs). The core defect in AVMs is high-flow AV shunts, which connect arteries directly to veins, “stealing” blood from capillaries. Here, we studied mouse brain AV shunts caused by up-regulation of Notch signaling in endothelial cells (ECs) through transgenic expression of constitutively active Notch4 (Notch4*). Using four-dimensional two-photon imaging through a cranial window, we found that normalizing Notch signaling by repressing Notch4* expression converted large-caliber, high-flow AV shunts to capillary-like vessels. The structural regression of the high-flow AV shunts returned blood to capillaries, thus reversing tissue hypoxia. This regression was initiated by vessel narrowing without the loss of ECs and required restoration of EphB4 receptor expression by venous ECs. Normalization of Notch signaling resulting in regression of high-flow AV shunts, and a return to normal blood flow suggests that targeting the Notch pathway may be useful therapeutically for treating diseases such as AVMs.

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Real-Time Imaging of Perivascular Transport of Nanoparticles During Convection-Enhanced Delivery in the Rat Cortex

Conor P. Foley, Nozomi Nishimura, Keith B. Neeves, Chris B. Schaffer, and William L. Olbricht

Annals of Biomedical Engineering (2012)

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Convection-enhanced delivery (CED) is a promising technique for administering large therapeutics that do not readily cross the blood brain barrier to neural tissue. It is of vital importance to understand how large drug constructs move through neural tissue during CED to optimize construct and delivery parameters so that drugs are concentrated in the targeted tissue, with minimal leakage outside the targeted zone. Experiments have shown that liposomes, viral vectors, high molecular weight tracers, and nanoparticles infused into neural tissue localize in the perivascular spaces of blood vessels within the brain parenchyma. In this work, we used two-photon excited fluorescence microscopy to monitor the real-time distribution of nanoparticles infused in the cortex of live, anesthetized rats via CED. Fluorescent nanoparticles of 24 and 100 nm nominal diameters were infused into rat cortex through microfluidic probes. We found that perivascular spaces provide a high permeability path for rapid convective transport of large nanoparticles through tissue, and that the effects of perivascular spaces on transport are more significant for larger particles that undergo hindered transport through the extracellular matrix. This suggests that the vascular topology of the target tissue volume must be considered when delivering large therapeutic constructs via CED.

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

Andy Y Shih, Jonathan D Driscoll, Patrick J Drew, Nozomi Nishimura, Chris B Schaffer, and David Kleinfeld

Journal of Cerebral Blood Flow and Metabolism 32, 1277 (2012)

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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.

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Age-Related Intimal Stiffening Enhances Endothelial Permeability and Leukocyte Transmigration

John Huynh, Nozomi Nishimura, Kuldeepsinh Rana, John M. Peloquin, Joseph P. Califano, Christine R. Montague, Michael R. King, Chris B. Schaffer, Cynthia A. Reinhart-King

Science Translational Medicine (2011)

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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, endothe- lial 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 desta- bilizing 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.

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Cortical Microhemorrhages Cause Local Inflammation but Do Not Trigger Widespread Dendrite Degeneration

Nathanael L. Rosidi, Joan Zhou, Sanket Pattanaik, Peng Wang, Weiyang Jin, Morgan Brophy, William L. Olbricht, Nozomi Nishimura, Chris B. Schaffer

Public Library of Science (2011)

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Microhemorrhages are common in the aging brain, and their incidence is correlated with increased risk of neurodegenerative disease. Past work has shown that occlusion of individual cortical microvessels as well as large-scale hemorrhages can lead to degeneration of neurons and increased inflammation. Using two-photon excited fluorescence microscopy in anesthetized mice, we characterized the acute and chronic dynamics of vessel bleeding, tissue compression, blood flow change, neural degeneration, and inflammation following a microhemorrhage caused by rupturing a single penetrating arteriole with tightly-focused femtosecond laser pulses. We quantified the extravasation of red blood cells (RBCs) and blood plasma into the brain and determined that the bleeding was limited by clotting. The vascular bleeding formed a RBC-filled core that compressed the surrounding parenchymal tissue, but this compression was not sufficient to crush nearby brain capillaries, although blood flow speeds in these vessels was reduced by 20%. Imaging of cortical dendrites revealed no degeneration of the large-scale structure of the dendritic arbor up to 14 days after the microhemorrhage. Dendrites close to the RBC core were displaced by extravasating RBCs but began to relax back one day after the lesion. Finally, we observed a rapid inflammatory response characterized by morphology changes in microglia/ macrophages up to 200 mm from the microhemorrhage as well as extension of cellular processes into the RBC core. This inflammation persisted over seven days. Taken together, our data suggest that a cortical microhemorrhage does not directly cause significant neural pathology but does trigger a sustained, local inflammatory response.

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In Vivo Imaging of Myelin in the Vertebrate Central Nervous System Using Third Harmonic Generation Microscopy

Matthew J. Farrar, Frank W. Wise, Joseph R. Fetcho, and Chris B. Schaffer

Biophysical Journal (2011)

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Loss of myelin in the central nervous system (CNS) leads to debilitating neurological deficits. High-resolution optical imaging of myelin in the CNS of animal models is limited by a lack of in vivo myelin labeling strategies. We demonstrated that third harmonic generation (THG) microscopy—a coherent, nonlinear, dye-free imaging modality—provides micrometer resolution imaging of myelin in the mouse CNS. In fixed tissue, we found that THG signals arose from white matter tracts and were colocalized with two-photon excited fluorescence (2PEF) from a myelin-specific dye. In vivo, we used simultaneous THG and 2PEF imaging of the mouse spinal cord to resolve myelin sheaths surrounding individual fluorescently-labeled axons, and followed myelin disruption after spinal cord injury. Finally, we suggest optical mechanisms that underlie the myelin specificity of THG. These results establish THG microscopy as an ideal tool for the study of myelin loss and recovery.

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 Supplement 1

Occlusion of cortical ascending venules causes blood flow decreases, reversals in flow direction, and vessel dilation in upstream capillaries

John Nguyen, Nozomi Nishimura, Robert N Fetcho, Costantino Iadecola, and Chris B Schaffer

Journal of Cerebral Blood Flow and Metabolism 31, 2243 (2011)

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The accumulation of small strokes has been linked to cognitive dysfunction. Although most animal models have focused on the impact of arteriole occlusions, clinical evidence indicates that venule occlusions may also be important. We used two-photon excited fluorescence microscopy to quantify changes in blood flow and vessel diameter in capillaries after occlusion of single ascending or surface cortical venules as a function of the connectivity between the measured capillary and the occluded venule. Clotting was induced by injuring the target vessel wall with femtosecond laser pulses. After an ascending venule (AV) occlusion, upstream capillaries showed decreases in blood flow speed, high rates of reversal in flow direction, and increases in vessel diameter. Surface venule occlusions produced similar effects, unless a collateral venule provided a new drain. Finally, we showed that AVs and penetrating arterioles have different nearest-neighbor spacing but capillaries branching from them have similar topology, which together predicted the severity and spatial extent of blood flow reduction after occlusion of either one. These results provide detailed insights into the widespread hemodynamic changes produced by cortical venule occlusions and may help elucidate the role of venule occlusions in the development of cognitive disorders and other brain diseases.

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Preictal and Ictal Neurovascular and Metabolic Coupling Surrounding a Seizure Focus

Mingrui Zhao, John Nguyen, Hongtao Ma, Nozomi Nishimura, Chris B. Schaffer, and Theodore H. Schwartz

The Journal of Neuroscience (2011)

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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 neuro- vascular and neurometabolic coupling in the ictal surround using optical imaging of light scattering and cerebral blood volume, auto- fluorescence 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 metab- olism 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.

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Sub-surface, micrometer-scale incisions produced in rodent cortex using tightly-focused femtosecond laser pulses

John Nguyen, Jillian Ferdman, Mingrui Zhou, David Huland, Shatha Saqqa, Jan Ma, Nozomi Nishimura, Theodore H. Schwartz, and Chris B. Schaffer

Lasers in Surgery and Medicine (2011)

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Background and Objective: Techniques that allow tar- geted, micrometer-scale disruption in the depths of bio- logical tissue, without affecting overlying structures or causing significant collateral damage, could potentially lead to new surgical procedures. We describe an optical technique to make sub-surface incisions in in vivo rodent brain and characterize the relationship between the cut width and maximum depth of these optical transections as a function of laser energy. Materials and Methods: To produce cuts, high inten- sity, femtosecond laser pulses were tightly focused into and translated within the cortex, through a craniotomy, in anesthetized rodents. Imaging of stained brain slices was used to characterize cut width and maximum cutting depth. Results: Cut width decreased exponentially as a function of depth and increased as the cube root of laser energy, but showed about 50% variation at fixed depth and laser energy. For example, at a laser energy of 13 mJ, cut width decreased from 158 ` 43.1 mm (mean ` standard deviation) to 56 ` 33 mm over depths of approximately 200–800 mm, respectively. Maximal cut depth increased logarithmically with laser energy, with cut depths of up to 1 mm achieved with 13 mJ pulses. We further show- cased this technique by selectively cutting sub-surface cortical dendrites in a live, anesthetized transgenic mouse. Conclusions: Femtosecond laser pulses provide the nov- el capacity for precise, sub-surface, cellular-scale cuts for surgical applications in optically scattering tissues.

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