Name: intravital video microscopy measurements of retinal blood flow in mice
Uploaded: 2013-12-26T14:15:00
Description: Watch this Scientific Journal Video about Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice at JoVE.com

Funded by NIH EY017599 (NRH).

The procedures involving the use of animals were reviewed and approved by the Institutional Animal Care and Use Committee of LSUHSC-S and performed according to the criteria outlined by the National Institutes of Health.
1. Preparation of Perfusion Solutions
<ol>
	<li>Sonicate a 1% (by weight) stock solution of 1.9&#160;&#181;m diameter microspheres.</li>
	<li>Withdraw ~25-30 x&#160;106 green fluorescent microspheres (that is, ~10 &#181;l of the stock solution) into a 300 &#181;l ...

<ol>
	<li>Wang, L., Fortune, B., Cull, G., McElwain, K. M., Cioffi, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microspheres+method+for+ocular+blood+flow+measurement+in+rats:+size+and+dose+optimization.">Microspheres method for ocular blood flow measurement in rats: size and dose optimization.</a> Exp. Eye Res. 84, 108-117 (2007).</li><li>Wang, L., Grant, C., Fortune, B., Cioffi, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+and+choroidal+vasoreactivity+to+altered+PaCO2+in+rat+measured+with+a+modified+microsphere+technique.">Retinal and choroidal vasoreactivity to altered PaCO2 in rat measured with a modified microsphere technique.</a> Exp. Eye Res. 86, 908-913 (2008).</li><li>Pouliot, M., Hetu, S., Lahjouji, K., Couture, R., Vaucher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+retinal+blood+flow+by+kinin+B(1)+receptor+in+Streptozotocin-diabetic+rats.">Modulation of retinal blood flow by kinin B(1) receptor in Streptozotocin-diabetic rats.</a> Exp. Eye Res. 92, 482-489 (2011).</li><li>Pouliot, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+and+regional+measurement+of+retinal+blood+flow+in+rats+using+N-isopropyl-p-[14C]-iodoamphetamine+([14C]-IMP).">Quantitative and regional measurement of retinal blood flow in rats using N-isopropyl-p-[14C]-iodoamphetamine ([14C]-IMP).</a> Exp. Eye Res. 89, 960-966 (2009).</li><li>Zhi, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volumetric+and+quantitative+imaging+of+retinal+blood+flow+in+rats+with+optical+microangiography.">Volumetric and quantitative imaging of retinal blood flow in rats with optical microangiography.</a> Biomed. Opti. Express. 2, 579-591 (2011).</li><li>Zhi, Z., Cepurna, W. O., Johnson, E. C., Morrison, J. C., Wang, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+intraocular+pressure+on+changes+of+blood+flow+in+the+retina,+choroid,+and+optic+nerve+head+in+rats+investigated+by+optical+microangiography.">Impact of intraocular pressure on changes of blood flow in the retina, choroid, and optic nerve head in rats investigated by optical microangiography.</a> Biomed. Opti. Express. 3, 2220-2233 (2012).</li><li>Li, G., De La Garza, B., Shih, Y. Y., Muir, E. R., Duong, T. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Layer-specific+blood-flow+MRI+of+retinitis+pigmentosa+in+RCS+rats.">Layer-specific blood-flow MRI of retinitis pigmentosa in RCS rats.</a> Exp. Eye Res. 101, 90-96 (2012).</li><li>Muir, E. R., Renteria, R. C., Duong, T. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+ocular+blood+flow+as+an+early+indicator+of+diabetic+retinopathy+in+a+mouse+model+of+diabetes.">Reduced ocular blood flow as an early indicator of diabetic retinopathy in a mouse model of diabetes.</a> Invest. Ophthalmol. Vis. Sci. 53, 6488-6494 (2012).</li><li>Wright, W. S., Harris, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ozagrel+attenuates+early+streptozotocin-induced+constriction+of+arterioles+in+the+mouse+retina.">Ozagrel attenuates early streptozotocin-induced constriction of arterioles in the mouse retina.</a> Exp. Eye Res. 86, 528-536 (2008).</li><li>Lee, S., Harris, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Losartan+and+ozagrel+reverse+retinal+arteriolar+constriction+in+non-obese+diabetic+mice.">Losartan and ozagrel reverse retinal arteriolar constriction in non-obese diabetic mice.</a> Microcirculation. 15, 379-387 (2008).</li><li>Wright, W. S., Messina, J. E., Harris, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attenuation+of+diabetes-induced+retinal+vasoconstriction+by+a+thromboxane+receptor+antagonist.">Attenuation of diabetes-induced retinal vasoconstriction by a thromboxane receptor antagonist.</a> Exp. Eye Res. 88, 106-112 (2009).</li><li>Wang, Z., Yadav, A. S., Leskova, W., Harris, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attenuation+of+streptozotocin-induced+microvascular+changes+in+the+mouse+retina+with+the+endothelin+receptor+A+antagonist+atrasentan.">Attenuation of streptozotocin-induced microvascular changes in the mouse retina with the endothelin receptor A antagonist atrasentan.</a> Exp. Eye Res. 91, 670-675 (2010).</li><li>Yadav, A. S., Harris, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+tempol+on+diabetes-induced+decreases+in+retinal+blood+flow+in+the+mouse.">Effect of tempol on diabetes-induced decreases in retinal blood flow in the mouse.</a> Curr. Eye Res. 36, 456-461 (2011).</li><li>Wang, Z., Yadav, A. S., Leskova, W., Harris, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+20-HETE+attenuates+diabetes-induced+decreases+in+retinal+hemodynamics.">Inhibition of 20-HETE attenuates diabetes-induced decreases in retinal hemodynamics.</a> Exp. Eye Res. 93, 108-113 (2011).</li><li>Wright, W. S., Yadav, A. S., McElhatten, R. M., Harris, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+blood+flow+abnormalities+following+six+months+of+hyperglycemia+in+the+Ins2(Akita)+mouse.">Retinal blood flow abnormalities following six months of hyperglycemia in the Ins2(Akita) mouse.</a> Exp. Eye Res. 98, 9-15 (2012).</li><li>Lee, S., Morgan, G. A., Harris, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ozagrel+reverses+streptozotocin-induced+constriction+of+arterioles+in+rat+retina.">Ozagrel reverses streptozotocin-induced constriction of arterioles in rat retina.</a> Microvasc. Res. 76, 217-223 (2008).</li><li>Rumbaut, R. E., Sial, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+phototoxicity+of+fluorescent+dye-labeled+albumin+conjugates.">Differential phototoxicity of fluorescent dye-labeled albumin conjugates.</a> Microcirculation. 6, 205-213 (1999).</li><li>Tadayoni, R., Paques, M., Gaudric, A., Vicaut, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erythrocyte+and+leukocyte+dynamics+in+the+retinal+capillaries+of+diabetic+mice.">Erythrocyte and leukocyte dynamics in the retinal capillaries of diabetic mice.</a> Exp. Eye Res. 77, 497-504 (2003).</li><li>Walsh, M. K., Quigley, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+time-lapse+fluorescence+imaging+of+individual+retinal+ganglion+cells+in+mice.">In vivo time-lapse fluorescence imaging of individual retinal ganglion cells in mice.</a> J. Neurosci. Methods. 169, 214-221 (2008).</li><li>Shahidi, M., Wanek, J., Blair, N. P., Mori, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+mapping+of+chorioretinal+vascular+oxygen+tension+in+the+rat.">Three-dimensional mapping of chorioretinal vascular oxygen tension in the rat.</a> Invest. Ophthalmol. Vis. Sci. 50, 820-825 (2009).</li><li>Hughes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+schematic+eye+for+the+rat.">A schematic eye for the rat.</a> Vis. Res. 19, 569-588 (1979).</li><li>Remtulla, S., Hallett, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+schematic+eye+for+the+mouse,+and+comparisons+with+the+rat.">A schematic eye for the mouse, and comparisons with the rat.</a> Vis. Res. 25, 21-31 (1985).</li><li>Schmucker, C., Schaeffel, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+biometry+in+the+mouse+eye+with+low+coherence+interferometry.">In vivo biometry in the mouse eye with low coherence interferometry.</a> Vis. Res. 44, 2445-2456 (2004).</li><li>Mulivor, A. W., Lipowsky, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation-+and+ischemia-induced+shedding+of+venular+glycocalyx.">Inflammation- and ischemia-induced shedding of venular glycocalyx.</a> Am. J. Physiol. Heart Circ. Physiol. 286, 1672-1680 (2004).</li><li>Harris, N. R., Whatley, J. R., Carter, P. R., Specian, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Venular+constriction+of+submucosal+arterioles+induced+by+dextran+sodium+sulfate.">Venular constriction of submucosal arterioles induced by dextran sodium sulfate.</a> Inflamm. Bowel Dis. 11, 806-813 (2005).</li></ol>

This technique of intravital video microscopy can be applied not only to mice, but also to rats. This protocol relies on only several assumptions, but is limited to use under anesthesia and with pupil dilation as we have described it. The other assumptions and limitations are as follows:
1. Assumption of minimal optical magnification error resulting from the refractive nature of the eye. As described by others18-20, the method of filling the space between the cornea ...

The retina is one of the most metabolically active tissues in the body, and consequently requires a generous blood supply. Two separate circulations meet this demand: the choroidal circulation for the outer portion of the retina, and the retinal circulation for the inner portion of the retina. Investigations of retinal perfusion are imperative for understanding the pathological mechanisms and consequences of diabetic retinopathy, oxygen-induced retinopathy, retinal artery or vein occlusion, and stroke. Several methods have been employed to quantify retinal blood flow, with each technique having its advantages, disadvantages, limitations, and assumptions. Among these techniques are infusion of 7-8 &#956;m diameter microspheres that lodge in precapillary arterioles1,2, quantitative autoradiography3,4, optical microangiopathy-optical coherence tomography5,6, magnetic resonance imaging7,8, and intravital video microscopy9-16. Advantages of the latter include direct live visualization of retinal vessels and flow, a dependence on only a few minor assumptions, and affordability for labs having a fluorescence microscope with an attached video camera. In previous studies of intravital video microscopy9-16, fluorescent dextran has been used as a plasma marker, and fluorescently labeled red blood cells (from a donor animal) have been used as velocity markers. In the current protocol, 1.9-&#956;m diameter fluorescently labeled microspheres, instead of red blood cells, are used to measure velocity, with this alteration negating the need for a blood cell donor.

<table border="0" cellpadding="0" cellspacing="0" width="1067">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Name of the Reagent/Equipment</td>
			<td style="width:317px;">Company</td>
			<td style="width:212px;">Catalog Number or Product</td>
			<td style="width:251px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorescent microspheres</td>
			<td>Bangs Laboratories</td>
			<td>FS04F/10584 (green)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">High molecular weight fluorescent dextran</td>
			<td>Molecular Probes</td>
			<td>D-7137 (green); D-7139 (red)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope system</td>
			<td>Nikon</td>
			<td>Eclipse E600FN + attachments</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4X objective</td>
			<td>Nikon</td>
			<td>Plan Fluor 4X</td>
			<td>numerical aperture 0.13; working distance 17.2 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10X objective</td>
			<td>Nikon</td>
			<td>Plan 10X</td>
			<td>numerical aperture 0.25; working distance 10.5 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tropicamide ophthalmic solution</td>
			<td>Bausch &#38; Lomb</td>
			<td>1% Tropicamide</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hypromellose ophthalmic solution</td>
			<td>HUB Pharmaceuticals</td>
			<td>2.5% Goniovisc</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Image processing software</td>
			<td>University of California San Francisco Vale Lab</td>
			<td>Micro-Manager</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digital video camera for microscopy</td>
			<td>Photometrics</td>
			<td>CoolSnap ES</td>
			<td>1392 x 1040 pixel resolution; pixel size 6.45 x 6.45 &#956;m</td>
		</tr>
	</tbody>
</table>

intravital video microscopy measurements of retinal blood flow in mice

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.

Figure 1 shows single frames of video from an experiment, with panels A-D and F-I showing fluorescent microsphere streaks captured with a 4X&#160;objective and 8 msec exposure time (and binning 2 x&#160;2 pixels to reduce the video file size). Figure 1E shows the orientation of the retinal vessels in the other panels of the figure. Not every frame of video will have a fluorescent streak in focus; however, some frames may have multiple streaks for use in the analysis (e.g.&#160;p...

Watch this Scientific Journal Video about Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice at JoVE.com

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Intravital microscopy can be used in animals to visualize and measure retinal vascular diameters, bloodstream velocities, and total retinal blood flow.

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered ...

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