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Robust and fragile aspects of cortical blood flow in relation to the underlying angioarchitecture,

A.Y. Shih, C. Ruhlmann, P. Blinder, A. Devor, P. J. Drew, B. Friedman, P. M. Knutsen, P. D. Lyden, C. Mateo, L. Mellander, N. Nishimura, C. B. Schaffer, P. S. Tsai, and D. Kleinfeld

Microcirculation (2015)

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We review the organizational principles of the cortical vasculature and the underlying patterns of blood flow under normal conditions and in response to occlusion of single vessels. The cortex is sourced by a two-dimensional network of pial arterioles that feeds a three-dimensional network of subsurface microvessels in close proximity to neurons and glia. Blood flow within the surface and subsurface networks is largely insensitive to occlusion of a single vessel within either network. However, the penetrating arterioles that connect the pial network to the subsurface network are bottlenecks to flow; occlusion of even a single penetrating arteriole results in the death of a 500 μm diameter cylinder of cortical tissue despite the potential for collateral flow through microvessels. This pattern of flow is consistent with that calculated from a full reconstruction of the angioarchitecture. Conceptually, collateral flow is insufficient to compensate for the occlusion of a penetrating arteriole because penetrating venules act as shunts of blood that flows through collaterals. Future directions that stem from the analysis of the angioarchitecture concern cellular-level issues, in particular the regulation of blood flow within the subsurface microvascular network, and system-level issues, in particular the role of penetrating arteriole occlusions in human cognitive impairment.

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Use of Tethered Enzymes as a Platform Technology for Rapid Analyte Detection

R. Cohen, J. P. Lata, Y. Lee, J. C. Cruz-Hernandez, N. Nishimura, C. B. Schaffer, C. Mukai, J. L. Nelson, S. A. Brangman, Y. Agrawal, and A. J. Travis

Public Library of Science (2015)

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Background Rapid diagnosis for time-sensitive illnesses such as stroke, cardiac arrest, and septic shock is essential for successful treatment. Much attention has therefore focused on new strategies for rapid and objective diagnosis, such as Point-of-Care Tests (PoCT) for blood biomarkers. Here we use a biomimicry-based approach to demonstrate a new diagnostic platform, based on enzymes tethered to nanoparticles (NPs). As proof of principle, we use oriented immobilization of pyruvate kinase (PK) and luciferase (Luc) on silica NPs to achieve rapid and sensitive detection of neuron-specific enolase (NSE), a clinically relevant biomarker for multiple diseases ranging from acute brain injuries to lung cancer. We hypothesize that an approach capitalizing on the speed and catalytic nature of enzymatic reactions would enable fast and sensitive biomarker detection, suitable for PoCT devices. Methods and findings We performed in-vitro, animal model, and human subject studies. First, the efficiency of coupled enzyme activities when tethered to NPs versus when in solution was tested, demonstrating a highly sensitive and rapid detection of physiological and pathological concentrations of NSE. Next, in rat stroke models the enzyme-based assay was able in minutes to show a statistically significant increase in NSE levels in samples taken 1 hour before and 0, 1, 3 and 6 hours after occlusion of the distal middle cerebral artery. Finally, using the tethered enzyme assay for detection of NSE in samples from 20 geriatric human patients, we show that our data match well (r = 0.815) with the current gold standard for biomarker detection, ELISA—with a major difference being that we achieve detection in 10 minutes as opposed to the several hours required for traditional ELISA. Conclusions Oriented enzyme immobilization conferred more efficient coupled activity, and thus higher assay sensitivity, than non-tethered enzymes. Together, our findings provide proof of concept for using oriented immobilization of active enzymes on NPs as the basis for a highly rapid and sensitive biomarker detection platform. This addresses a key challenge in developing a PoCT platform for time sensitive and difficult to diagnose pathologies.

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A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord,

M. J. Farrar and C. B. Schaffer

Journal of Visualized Experiments (2014)

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Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

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Stalled cerebral capillary blood flow in mouse models of essential thrombocythemia and polycythemia vera revealed by in vivo two-photon imaging.

Santisakultarm TP, Paduano CQ, Stokol T, Southard TL, Nishimura N, Skoda RC, Olbricht WL, Schafer AI, Silver RT, Schaffer CB.

J Thromb Haemost. (2014)

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Background Essential thrombocythemia (ET) and polycythemia vera (PV) are myeloproliferative neoplasms (MPNs) that share the JAK2V617F mutation in hematopoietic stem cells, leading to excessive production of predominantly platelets in ET, and predominantly red blood cells (RBCs) in PV. The major cause of morbidity and mortality in PV and ET is thrombosis, including cerebrovascular occlusive disease. Objectives To identify the effect of excessive blood cells on cerebral microcirculation in ET and PV. Methods We used two-photon excited fluorescence microscopy to examine cerebral blood flow in transgenic mouse models that mimic MPNs. Results and conclusions We found that flow was ‘stalled’ in an elevated fraction of brain capillaries in ET (18%), PV (27%), mixed MPN (14%) and secondary (non-MPN) erythrocytosis (27%) mice, as compared with controls (3%). The fraction of capillaries with stalled flow increased when the hematocrit value exceeded 55% in PV mice, and the majority of stalled vessels contained only stationary RBCs. In contrast, the majority of stalls in ET mice were caused by platelet aggregates. Stalls had a median persistence time of 0.5 and 1 h in ET and PV mice, respectively. Our findings shed new light on potential mechanisms of neurological problems in patients with MPNs.

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Big Effects From Tiny Vessels: Imaging the Impact of Microvascular Clots and Hemorrhages on the Brain

Nozomi Nishimura and Chris B. Schaffer

Stroke 44, S90 (2013)

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Improvement in clinical imaging technologies has made it possible to resolve small, previously invisible lesions in the brains of elderly humans. Initially, these lesions were called silent strokes because they do not present with dramatic acute symptoms like major stroke. It was later shown that these small lesions have cognitive consequences and are a contributing factor to age-related mental decline and dementia.

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Optically induced occlusion of single blood vessels in rodent neocortex.

Shih AY, Nishimura N, Nguyen J, Friedman B, Lyden PD, Schaffer CB, Kleinfeld D.

Cold Spring Harbor (2013)

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The ability to form targeted vascular occlusions in small vessels of the brain is an important technique for studying the microscopic basis of cerebral ischemia. We describe two complementary methods that enable targeted occlusion of any single blood vessel within the upper 500 µm of adult rodent neocortex. Our goal is to generate highly localized regions of ischemia by blocking penetrating arterioles and ascending venules, which are bottlenecks of flow in the cortical angioarchitecture. One method, termed photothrombosis, makes use of linear optical absorption by a photosensitizer, transiently circulated in the blood stream, to induce a clot in a surface or near-surface segment of a vessel. The second method, termed plasma-mediated ablation, makes use of nonlinear optical interactions, without the need to introduce an exogenous absorber, to induce clots in subsurface segments of penetrating vessels, as well as subsurface microvessels and capillaries. The choice of the method for occlusion of individual vessels depends on the location of the vessels being studied and the objectives of the study. Here we describe concurrent high resolution in vivo imaging and auxiliary laser setups, occlusion protocols, and post hoc histological procedures.

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Optoporation and Genetic Manipulation of Cells Using Femtosecond Laser Pulses

Andrew A. Davis, Matthew J. Farrar, Nozomi Nishimura, Moonsoo M. Jin, and Chris B. Schaffer

Biophysical Journal 105, 862 (2013)

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Femtosecond laser optoporation is a powerful technique to introduce membrane-impermeable molecules, such as DNA plasmids, into targeted cells in culture, yet only a narrow range of laser regimes have been explored. In addition, the dynamics of the laser-produced membrane pores and the effect of pore behavior on cell viability and transfection efficiency remain poorly elucidated. We studied optoporation in cultured cells using tightly focused femtosecond laser pulses in two irra- diation regimes: millions of low-energy pulses and two higher-energy pulses. We quantified the pore radius and resealing time as a function of incident laser energy and determined cell viability and transfection efficiency for both irradiation regimes. These data showed that pore size was the governing factor in cell viability, independently of the laser irradiation regime. For viable cells, larger pores resealed more quickly than smaller pores, ruling out a passive resealing mechanism. Based on the pore size and resealing time, we predict that few DNA plasmids enter the cell via diffusion, suggesting an alternative mechanism for cell trans- fection. Indeed, we observed fluorescently labeled DNA plasmid adhering to the irradiated patch of the cell membrane, suggest- ing that plasmids may enter the cell by adhering to the membrane and then being translocated.

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Stimulus-Evoked Calcium Transients in Somatosensory Cortex Are Temporarily Inhibited by a Nearby Microhemorrhage

Flor A. Cianchetti, Dong Hwan Kim, Sally Dimiduk, Nozomi Nishimura, Chris B. Schaffer

Public Library of Science ONE 8, e65663 (2013)

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Although microhemorrhages are common in the brain of the elderly, the direct impact of these lesions on neural function remains unclear. In this work, we used femtosecond laser irradiation to rupture the wall of single arterioles in the brain of anesthetized rodents, producing a hematoma of ,100-mm diameter. Our objective was to study the impact of these microhemorrhages on cortical activity using cell-resolved two-photon imaging of bulk-loaded calcium-sensitive dye. We monitored peripheral sensory stimulus-induced calcium transients from individual neuronal cell bodies, regions of neuropil, and astrocytes at different distances from the microhemorrhage before and 0.5, 2, and 4 hours after the creation of the lesion. We found that immediately after the hemorrhage the average amplitude of the stimulus-induced calcium response was reduced to about half within 150 mm from the hematoma. Beyond 300 mm, there was little effect on cell response, with a smooth increase in response amplitude from 150 mm to 300 mm from the lesion. Cortical function gradually improved with time and by four hours after the lesion the response from neurons and astrocytes had recovered to baseline everywhere but within 150 mm from the hematoma. To assess whether the cells closest to the microhemorrhage recovered over a longer timeframe, we developed a re-openable chronic cranial window preparation that allowed reinjection of calcium-sensitive fluorescent dye. We found that the response largely recovered by one day after the microhemorrhage even within 150 mm from the hematoma. This work suggests that neuronal and astrocyte function is transiently lost near a microhemorrhage, but recovers within one day after the lesion.

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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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