Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Watts, Megan N.; Leskova, Wendy

doi:10.3791/51110

Cited by 11 publications

(12 citation statements)

References 26 publications

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“…Our value of mean diameter of primary vessels matches that of the in vivo slit lamp biomicroscope (Blair et al, 2016) study and is within the range reported by ex vivo scanning electron microscopy (Ninomiya & Inomata, 2006). However, the diameters reported by intravital microscopy studies (Harris et al, 2013; Lee & Harris, 2008; Z. Wang et al, 2010, 2011; Watts et al, 2013; Wright & Harris, 2008; Wright et al, 2009, 2012; Yadav & Harris, 2011) for the same vessel class are at least a factor of two greater than our values.…”

Section: Discussioncontrasting

confidence: 84%

“…While investigating the mouse literature for normative values of diameter, velocity and flow in retinal vessels, we found that there is a paucity of such studies. To the best of our knowledge, we have counted all of fifteen journal papers which measure retinal blood velocity and/or flow in the living mouse eye (Blair et al, 2016; Harris, Watts, & Leskova, 2013; Lee & Harris, 2008; Liu et al, 2017; Muir, Rentería, & Duong, 2012; Z. Wang, Yadav, Leskova, & Harris, 2010, 2011; Watts, Eshaq, Carter, & Harris, 2013; Wright & Harris, 2008; Wright, Messina, & Harris, 2009; Wright, Singh Yadav, McElhatten, & Harris, 2012; Yadav & Harris, 2011; Zawadzki et al, 2015; Zhi et al, 2014, 2012).…”

Section: Discussionmentioning

confidence: 99%

“…Using incorrect image magnification on the mouse retina is one possible source of the disparity in measured diameters. For mean velocities in primary vessels, some studies used fluorescent microspheres or labelled RBCs (Harris et al, 2013; Lee & Harris, 2008; Z. Wang et al, 2010, 2011; Watts et al, 2013; Wright et al, 2009, 2012; Yadav & Harris, 2011), with 10–30 measurements from sparse fluorescent streaks pooled to average out effects of pulsatile and cross–sectional profile variability.…”

Section: Discussionmentioning

confidence: 99%

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Imaging Single–Cell Blood Flow in the Smallest to Largest Vessels in the Living Retina

Joseph

Guevara–Torres

Schallek

2019

Preprint

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Impact Statement: Using a specialized camera that corrects for eye blur, millions of single blood cells are 9 imaged and their speed measured, as they travel through the largest-to-smallest vessels of the retina. 10 Abstract: Tissue light scatter limits the visualization of the microvascular network deep inside the living 11 mammal. The transparency of the mammalian eye provides a noninvasive view of the microvessels of the retina, a 12 part of the central nervous system. Despite its clarity, imperfections in the optics of the eye blur microscopic 13 retinal capillaries, and single blood cells flowing within. This limits early evaluation of microvascular diseases 14 that originate in capillaries. To break this barrier, we use adaptive optics to noninvasively measure single-cell 15 blood flow, in one of the most widely used research animals: the C57BL/6J mouse. Flow ranged four orders of 16 magnitude (0.0002-1.55 µL min -1 ) across the full spectrum of retinal vessel diameters (3.2-45.8 µm), without 17 requiring surgery or contrast dye. Here we describe the data collection approach using adaptive optics and provide 18 an analysis pipeline that can measure millions of blood cell speeds automatically. 19 93 noninvasively in the smallest to largest vessels of the mouse retinal circulation (lumen diameters: 3.2-45.8 µm); 94 without requiring exogenous contrast agents to facilitate measurement. Measured flow rate per vessel ranged four 95 orders of magnitude (0.0002-1.55 µL min -1 ) across the complete retinal vascular tree, from the largest vessel near 96 the optic disk to the smallest capillary. In addition to aiding basic science investigation, the non-invasive 97 approach provides a comprehensive array of bioreporters of microvascular perfusion, including: cell velocity, 98 lumen diameter, flow rate, single-cell flux in capillaries, pulsatile and laminar flow, and indices of intrinsic 99 temporal and spatial modulations in flow. To our knowledge, we provide the first measurement of cross-sectional 100 velocity profile and the first absolute measurement of pulsatile velocity in the mouse retina. We also provide the 101 4 first measures of mean (and pulsatile) velocity and flow rate in medium and small vessels (capillaries) of the 102 mouse retina. We found that there is a heterogenous distribution of velocity, flux and other hemodynamic 103 parameters in vessels of varying generation/size, showing that diameter alone is a poor predictor of 104 microcirculation hemodynamics in the central nervous system. Through the innovations presented in this work, 105 imaging the complete and intact perfusion system of the mammalian retina bridges an important gap in studying 106 perfusion dynamics in deep microvascular beds within the living body. And due to the central nervous system 107 origin of the retina, this method provides a powerful way to noninvasively study microvascular integrity and 108 kinetics in a portion of the living brain. 109 Methods 110 Animals 111 19 normal C57BL/6J mice (The Jackson Laboratory stock 000664...

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Section: Discussioncontrasting

confidence: 84%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Imaging Single–Cell Blood Flow in the Smallest to Largest Vessels in the Living Retina

Joseph

Guevara–Torres

Schallek

2019

Preprint

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“…In a protocol similar to that described previously, 13 a solution of fluorescent microspheres (1.9 µm diameter Dragon Green; Bangs Laboratories, Inc, Fishers, IN, USA) was infused (80 µg/rat) to measure blood velocity. Videos were recorded using a 4× objective through a Nikon fluorescein filter, with a video camera exposure time of 10 ms.…”

Section: Methodsmentioning

confidence: 99%

Altered Retinal Hemodynamics and Mean Circulation Time in Spontaneously Hypertensive Rats

Leskova

Warar

2020

Invest. Ophthalmol. Vis. Sci.

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Although it is known that the retinal arteriolar vasculature is constricted in hypertension, the details of retinal hemodynamics and perfusion of the retinal circulation have yet to be adequately characterized. METHODS. Male and female spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) controls were anesthetized before measurements of mean arterial blood pressure and preparation for intravital microscopy of the retinal microcirculation. Retinal vascular velocities were measured with the use of fluorescent microspheres, and diameters and mean circulation times were measured after the infusion of fluorescent dextran. Arteriolar and venular shear rates were calculated from the ratio of velocity to diameter. RESULTS. In the retinas of SHR, velocities were elevated (compared with control WKY) in arterioles, but not in venules. Both arteriolar and venular diameters were significantly smaller in SHR versus WKY, with substantial increases in shear rates. Despite a tendency toward lower retinal blood flow rates, the mean circulation time through the SHR retina was much faster than can be explained by the measured arteriolar and venular velocities. CONCLUSIONS. The pattern of hypertension-induced increases in blood velocity, dissipating from the arteriolar to venular side of the retinal circulation, indicates a potential transfer of the extra kinetic energy through the vasculature. The combination of elevated velocities through narrower retinal arterioles resulted in a markedly higher level of wall shear rate that may induce changes in the vessel wall. Finally, significantly more rapid transits through the hypertensive retina could be a result of altered blood flow distribution.

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“…The fluorescent MSs (6 × 10 7 ) were injected into each mouse. After systemic injection, the interactions of these MSs with the endothelium of normal and angiogenic vessels of live animals were studied by intravital video microscopy [22].…”

Section: Methodsmentioning

confidence: 99%

Notch Signaling Pathway Is Involved in bFGF-Induced Corneal Lymphangiogenesis and Hemangiogenesis

Xie

Zhang

Luo

et al. 2019

Journal of Ophthalmology

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Background. Notch/Dll4 involvement in cornea neovascularization (CRNV) and lymphangiogenesis is unclear. This study aimed to explore the role of notch signaling in basic fibroblast growth factor- (bFGF-) induced corneal lymphangiogenesis and hemangiogenesis. Methods. Corneal stroma of C57BL/6 mice was implanted with bFGF- or phosphate-buffered saline- (PBS-) soaked pellets. Corneal lymphangiogenesis and neovascularization were evaluated by immunofluorescence. Vascular endothelial growth factor-A (VEGF-A), Delta-like ligand 4 (Dll4), and Notch1 mRNA and protein expression were examined on days 1, 3, 7, and 14 by real-time polymerase chain reaction and western blot. Corneal cells were treated with ranibizumab, dexamethasone, and γ-secretase inhibitor (GSI). Microspheres were used to evaluate corneal hemangiogenesis in vivo. Results. Corneal hemangiogenesis reached its peak on day 7 after bFGF implantation, and corneal lymphangiogenesis was significantly higher on day 7 and 14, compared with PBS. mRNA and protein expression of VEGF-A, Dll4, and Notch1 were higher in bFGF-induced animal models compared with controls. Corneal hemangiogenesis and lymphangiogenesis decreased after 7 days of ranibizumab or dexamethasone treatment. After adding GSI for 24 h in bFGF-induced cells, the expression of Notch1 and Dll4 were downregulated compared with that in the control group whereas the expression level of VEGF-A was upregulated. Fluorescent particle number was higher in the GSI group. Ranibizumab and dexamethasone decreased the fluorescence signal. Conclusion. The notch signaling pathway plays a role in regulating VEGF expression, affecting corneal lymphangiogenesis and hemangiogenesis in mice. The molecular imaging probe technique can visualize the changes in the VEGF-A expression level of corneal limbus hemangiogenesis.

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Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Cited by 11 publications

References 26 publications

Imaging Single–Cell Blood Flow in the Smallest to Largest Vessels in the Living Retina

Imaging Single–Cell Blood Flow in the Smallest to Largest Vessels in the Living Retina

Altered Retinal Hemodynamics and Mean Circulation Time in Spontaneously Hypertensive Rats

Notch Signaling Pathway Is Involved in bFGF-Induced Corneal Lymphangiogenesis and Hemangiogenesis

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