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)
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.
Arne Lauer, Flor A. Cianchetti, Elizabeth M. Van Cott, Frieder Schlunk, Elena Schulz, Waltraud Pfeilschifter, Helmuth Steinmetz, Chris B. Schaffer, Eng H. Lo, Christian Foerch
The direct thrombin inhibitor dabigatran etexilate (DE) may constitute a future replacement of vitamin K antagonists for long-term anticoagulation. Whereas warfarin pretreatment is associated with greater hematoma expansion after intracerebral hemorrhage (ICH), it remains unclear what effect direct thrombin inhibitors would have. Using different experimental models of ICH, this study compared hematoma volume among DE-treated mice, warfarin-treated mice, and controls. Methods and Results—CD-1 mice were fed with DE or warfarin. Sham-treated mice served as controls. At the time point of ICH induction, DE mice revealed an increased activated partial thromboplastin time compared with controls (mean+/-SD 46.1+/-5.0 versus 18.0+/-1.5 seconds; P=0.022), whereas warfarin pretreatment resulted in a prothrombin time prolongation (51.4+/-17.9 versus 10.4+/-0.3 seconds; P<0.001). Twenty-four hours after collagenase-induced ICH formation, hematoma volume was 3.8+/-2.9 µL in controls, 4.8+/-2.7 µL in DE mice, and 14.5+/-11.8 µL in warfarin mice (n=16; Welch ANOVA between-group differences P0.007; posthoc analysis with the Dunnett method: DE versus controls, P=0.899; warfarin versus controls, P<0.001; DE versus warfarin, P=0.001). In addition, a model of laser-induced cerebral microhemorrhage was applied, and the distances that red blood cells and blood plasma were pushed into the brain were quantified. Warfarin mice showed enlarged red blood cell and blood plasma diameters compared to controls, but no difference was found between DE mice and controls. Conclusions—In contrast with warfarin, pretreatment with DE did not increase hematoma volume in 2 different experimental models of ICH. In terms of safety, this observation may represent a potential advantage of anticoagulation with DE over warfarin.
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)
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.
Matthew J. Farrar, Frank W. Wise, Joseph R. Fetcho, and Chris B. Schaffer
Biophysical Journal (2011)
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.
John Nguyen, Nozomi Nishimura, Robert N Fetcho, Costantino Iadecola, and Chris B Schaffer
Journal of Cerebral Blood Flow and Metabolism 31, 2243 (2011)
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.
Thom P Santisakultarm and Chris B Schaffer
Journal of Cerebral Blood Flow and Metabolism (2011)
In vivo imaging allows detailed studies of cerebral blood flow and brain metabolism in both normal and pathological states. Since the 1980s, several methods to measure tissue perfusion in the brain have been introduced. Magnetic resonance imaging (MRI) tech- niques such as blood oxygen level dependent MRI (BOLD MRI) (Sorensen et al, 1995) and arterial spin labeling (Buxton and Frank 1997) as well as micro- positron emission tomography (Heiss et al, 1994) have enabled noninvasive chronic imaging of both regional blood flow changes and absolute perfusion in both humans and animal models. These methods, however, suffer from poor spatial resolution and are mainly useful when studying regional blood flow in the brain. Intrinsic optical imaging (Ts’o et al, 1990) enables higher spatial resolution mapping of changes in blood volume. Laser Doppler spectroscopy (Eyre et al, 1988) enables measurement of relative changes in blood flow speeds averaged over B1-mm2 cortical areas, while laser speckle contrast imaging (Boas and Dunn, 2010) enables these relative blood flow changes to be imaged with a spatial resolution of < 100 mm. None of these optical techniques, however, can accurately resolve changes in flow in individual microvessels or determine the absolute perfusion of cortical tissue. When absolute speed measurement of individual vessels is required, two-photon excited fluorescence (2PEF) microscopy has been the tool of choice (Schaffer et al, 2006). It is, however, limited to measurement of flow speed in vessels that are oriented parallel to the imaging plane, and measure- ments need to be made one vessel at a time, which is a time-consuming process. Currently lacking is an imaging technique that enables absolute blood flow speed measurements in multiple individual vessels at once. In this issue of JCBFM, Srinivasan et al (2011) introduce the use of Doppler optical coherence tomography (DOCT) (Chen et al, 1997) to fill this gap.
Mingrui Zhao, John Nguyen, Hongtao Ma, Nozomi Nishimura, Chris B. Schaffer, and Theodore H. Schwartz
The Journal of Neuroscience (2011)
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.
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)
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.