Name: Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice (Video) | JoVE Journal
Uploaded: 2013-12-26T14:15:00.000+00:00
Duration: 10 min 16 s
Description: Learn intravital video microscopy to measure retinal blood flow in mice using fluorescent tracers. This method enables accurate quantification of vessel diameters and blood velocities for studying retinal pathologies.

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5 minutes

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 syringe. Dilute the microspheres by withdrawing an additional 240 &#181;l of saline into the syringe.</li>
	<li>Prepare a 50&#160;&#181;l solution of 2 x&#160;106 molecular weight fluorescent dextran dissolved in sterile saline, with the injected dose in the range of 3-10 mg/kg. Keep the dextran covered in aluminum foil until time for its infusion.</li>
</ol>
2. Animal Anesthesia and Vascular Cannulation
<ol>
	<li>Anesthetize the animal for surgical insertion of vascular cannulas and for stationary positioning under a microscope objective. Many choices of anesthetic protocols are available, with various advantages and disadvantages, and potential effects on systemic parameters. Among the many different choices are a combination of&#160;ketamine (100-150 mg/kg), xylazine (10 mg/kg), and acepromazine (2 mg/kg), or a combination of ketamine (50 mg/kg) and pentobarbital (50 mg/kg).&#160; The described procedures are acute and conclude with euthanasia induced by an overdose of pentobarbital (150 mg/kg) followed by a thoracotomy; inclusion of an analgesic is not necessary for the terminal procedure.</li>
	<li>Maintain a heating pad underneath the animal for the remainder of the experiment.</li>
	<li>Keep the eyes moist with phosphate buffered saline during the subsequent surgical insertions of vascular cannulas.</li>
	<li>Prepare a vascular cannula for subsequent infusions of fluorescent tracers. A 20&#160;cm length of polyethylene tubing (for example, 0.28 mm inside diameter; 0.61 mm outside diameter) is placed on the tip of a syringe needle, with the sharp end of the needle broken off beforehand by bending back and forth with a hemostat.</li>
	<li>Fill a syringe and tubing with heparinized (25 U/ml) saline.</li>
	<li>Shave one side of the lower abdomen and make an incision to expose the femoral vein.</li>
	<li>To cannulate the femoral vein, tie off the vessel with a 5.0 suture, make a partial incision of the vessel (approximately one-half the vessel diameter), and insert the tubing into the vessel, securing the tubing with the suture tied around the cannulated section.</li>
</ol>
3. Preparation for Intravital Microscopy
<ol>
	<li>Dilate the pupil of the eye with a drop of 1% tropicamide ophthalmic solution followed by a drop of 2.5% hypromellose ophthalmic solution.</li>
	<li>Cover the eye with a 5&#160;mm glass coverslip.</li>
	<li>Place the animal on a plexiglass board, which will be positioned underneath the microscope objective of an upright microscope. Strategically place surgical gauze underneath the head to allow a direct axis through the objective to the central retina and optic disk.</li>
	<li>Focus through the microscope on the optic disk, finding the disk with a low power 4X&#160;objective and using a fluorescein filter.</li>
</ol>
4. Infusion of Fluorescent Microspheres to Measure Velocities
<ol>
	<li>Using a 4X&#160;objective, focus on the retina with the optic disk in the center of the field of view.</li>
	<li>Infuse the fluorescent microsphere solution at a rate of 250 &#181;l/min/kg.</li>
	<li>Video-record the viewable retina around the optic disk, using a camera exposure time of ~8 msec.</li>
	<li>Stop the microsphere infusion when a sufficient duration of video has been recorded (1-2 min); some microsphere solution likely will remain unused.</li>
</ol>
5. Infusion of a Fluorescent Plasma Marker to Measure Diameters
<ol>
	<li>Using a 4X&#160;objective, focus on the retina with the optic disk in the center of the field of view.</li>
	<li>Infuse the prepared solution of fluorescent dextran into the femoral vein as a bolus over ~5 sec, while video-recording. This video will help determine the identity of the arterioles vs venules, with the arterioles being perfused first. Perform this step with the camera exposure time set to ~8 msec, although the precise exposure time is not critical.</li>
	<li>Switch to a 10X&#160;objective and 20-40 msec exposure time for improved resolution to video each of the 4-7 arterioles and 4-7 venules in the superficial retina. Focus on (and video-record) approximately one quadrant of the viewable retina at a time, with the center of the optic disk in one corner of the field of view. An attempt should be made to minimize the time that the microscope light illuminates the tissue to reduce the possibility of light/dye-induced phototoxicity17.</li>
</ol>
6. Video Analysis
<ol>
	<li>Using a micrometer scale, calibrate the video system for a conversion of &#181;m/pixel.</li>
	<li>With image processing software, play back the recorded video.</li>
	<li>From the video of the bolus infusion of fluorescent dextran, identify arterioles vs venules.</li>
	<li>For each of the 4-7 arterioles and 4-7 venules recorded with the 10X&#160;objective, measure the diameters filled with fluorescent dextran, averaging 5 measures of diameter per vessel along the viewable length.</li>
	<li>For each of the same arterioles and venules, measure the streak length of the fluorescent microspheres, using 10 successive microsphere streaks in each vessel to avoid selection bias. Divide the streak lengths by the exposure time to calculate the microsphere velocity, for example, 160 &#181;m/8 msec = 20 &#181;m/msec = 2 cm/sec. Average the 10 velocities per vessel.</li>
	<li>Calculate the volumetric flow rate for each vessel with the following equation: flow = mean velocity &#215; &#960; &#215; diameter2/4.</li>
	<li>Sum the arteriolar flow rates, and separately the venular flow rates, to obtain total retinal blood flow.</li>
</ol>

<ol><li>Wang, L., Fortune, B., Cull, G., McElwain, K. M., Cioffi, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microspheres+method+for+ocular+blood+flow+measurement+in+rats%3A+size+and+dose+optimization%5BTitle%5D)+AND+%22Exp.+Eye+Res%22%5BJournal%5D)">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/pubmed?term=((Retinal+and+choroidal+vasoreactivity+to+altered+PaCO2+in+rat+measured+with+a+modified+microsphere+technique%5BTitle%5D)+AND+%22Exp.+Eye+Res%22%5BJournal%5D)">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/pubmed?term=((Modulation+of+retinal+blood+flow+by+kinin+B%281%29+receptor+in+Streptozotocin-diabetic+rats%5BTitle%5D)+AND+%22Exp.+Eye+Res%22%5BJournal%5D)">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/pubmed?term=((Quantitative+and+regional+measurement+of+retinal+blood+flow+in+rats+using+N-isopropyl-p-%5B14C%5D-iodoamphetamine+%28%5B14C%5D-IMP%29%5BTitle%5D)+AND+%22Exp.+Eye+Res%22%5BJournal%5D)">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/pubmed?term=((Volumetric+and+quantitative+imaging+of+retinal+blood+flow+in+rats+with+optical+microangiography%5BTitle%5D)+AND+%22Biomed.+Opti.+Express%22%5BJournal%5D)">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/pubmed?term=((Impact+of+intraocular+pressure+on+changes+of+blood+flow+in+the+retina%2C+choroid%2C+and+optic+nerve+head+in+rats+investigated+by+optical+microangiography%5BTitle%5D)+AND+%22Biomed.+Opti.+Express%22%5BJournal%5D)">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/pubmed?term=((Layer-specific+blood-flow+MRI+of+retinitis+pigmentosa+in+RCS+rats%5BTitle%5D)+AND+%22Exp.+Eye+Res%22%5BJournal%5D)">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/pubmed?term=((Reduced+ocular+blood+flow+as+an+early+indicator+of+diabetic+retinopathy+in+a+mouse+model+of+diabetes%5BTitle%5D)+AND+%22Invest.+Ophthalmol.+Vis.+Sci%22%5BJournal%5D)">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/pubmed?term=((Ozagrel+attenuates+early+streptozotocin-induced+constriction+of+arterioles+in+the+mouse+retina%5BTitle%5D)+AND+%22Exp.+Eye+Res%22%5BJournal%5D)">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/pubmed?term=((Losartan+and+ozagrel+reverse+retinal+arteriolar+constriction+in+non-obese+diabetic+mice%5BTitle%5D)+AND+%22Microcirculation%22%5BJournal%5D)">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/pubmed?term=((Attenuation+of+diabetes-induced+retinal+vasoconstriction+by+a+thromboxane+receptor+antagonist%5BTitle%5D)+AND+%22Exp.+Eye+Res%22%5BJournal%5D)">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/pubmed?term=((Attenuation+of+streptozotocin-induced+microvascular+changes+in+the+mouse+retina+with+the+endothelin+receptor+A+antagonist+atrasentan%5BTitle%5D)+AND+%22Exp.+Eye+Res%22%5BJournal%5D)">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/pubmed?term=((Effect+of+tempol+on+diabetes-induced+decreases+in+retinal+blood+flow+in+the+mouse%5BTitle%5D)+AND+%22Curr.+Eye+Res%22%5BJournal%5D)">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/pubmed?term=((Inhibition+of+20-HETE+attenuates+diabetes-induced+decreases+in+retinal+hemodynamics%5BTitle%5D)+AND+%22Exp.+Eye+Res%22%5BJournal%5D)">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/pubmed?term=((Retinal+blood+flow+abnormalities+following+six+months+of+hyperglycemia+in+the+Ins2%28Akita%29+mouse%5BTitle%5D)+AND+%22Exp.+Eye+Res%22%5BJournal%5D)">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/pubmed?term=((Ozagrel+reverses+streptozotocin-induced+constriction+of+arterioles+in+rat+retina%5BTitle%5D)+AND+%22Microvasc.+Res%22%5BJournal%5D)">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/pubmed?term=((Differential+phototoxicity+of+fluorescent+dye-labeled+albumin+conjugates%5BTitle%5D)+AND+%22Microcirculation%22%5BJournal%5D)">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/pubmed?term=((Erythrocyte+and+leukocyte+dynamics+in+the+retinal+capillaries+of+diabetic+mice%5BTitle%5D)+AND+%22Exp.+Eye+Res%22%5BJournal%5D)">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/pubmed?term=((In+vivo+time-lapse+fluorescence+imaging+of+individual+retinal+ganglion+cells+in+mice%5BTitle%5D)+AND+%22J.+Neurosci.+Methods%22%5BJournal%5D)">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/pubmed?term=((Three-dimensional+mapping+of+chorioretinal+vascular+oxygen+tension+in+the+rat%5BTitle%5D)+AND+%22Invest.+Ophthalmol.+Vis.+Sci%22%5BJournal%5D)">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/pubmed?term=((A+schematic+eye+for+the+rat%5BTitle%5D)+AND+%22Vis.+Res%22%5BJournal%5D)">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/pubmed?term=((A+schematic+eye+for+the+mouse%2C+and+comparisons+with+the+rat%5BTitle%5D)+AND+%22Vis.+Res%22%5BJournal%5D)">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/pubmed?term=((In+vivo+biometry+in+the+mouse+eye+with+low+coherence+interferometry%5BTitle%5D)+AND+%22Vis.+Res%22%5BJournal%5D)">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/pubmed?term=((Inflammation-+and+ischemia-induced+shedding+of+venular+glycocalyx%5BTitle%5D)+AND+%22Am.+J.+Physiol.+Heart+Circ.+Physiol%22%5BJournal%5D)">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/pubmed?term=((Venular+constriction+of+submucosal+arterioles+induced+by+dextran+sodium+sulfate%5BTitle%5D)+AND+%22Inflamm.+Bowel+Dis%22%5BJournal%5D)">Venular constriction of submucosal arterioles induced by dextran sodium sulfate.</a> Inflamm. Bowel Dis. 11, 806-813 (2005).</li>
</ol>

The authors have nothing to disclose.

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><tbody><tr><td>Fluorescent microspheres</td><td>Bangs Laboratories</td><td>FS04F/10584 (green)</td><td></td></tr><tr><td>High molecular weight fluorescent dextran</td><td>Molecular Probes</td><td>D-7137 (green); D-7139 (red)</td><td></td></tr><tr><td>Microscope system</td><td>Nikon</td><td>Eclipse E600FN + attachments</td><td></td></tr><tr><td>4X objective</td><td>Nikon</td><td>Plan Fluor 4X</td><td>numerical aperture 0.13; working distance 17.2 mm</td></tr><tr><td>10X objective</td><td>Nikon</td><td>Plan 10X</td><td>numerical aperture 0.25; working distance 10.5 mm</td></tr><tr><td>Tropicamide ophthalmic solution</td><td>Bausch Lomb</td><td>1% Tropicamide</td><td></td></tr><tr><td>Hypromellose ophthalmic solution</td><td>HUB Pharmaceuticals</td><td>2.5% Goniovisc</td><td></td></tr><tr><td>Image processing software</td><td>University of California San Francisco Vale Lab</td><td>Micro-Manager</td><td></td></tr><tr><td>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 &amp;mu;m</td></tr></tbody></table>

intravital video microscopy measurements of retinal blood flow in mice

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 objective and 8 msec exposure time (and binning 2 x 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. panels C, D, G, ...

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 ...

The overall goal of this procedure is to measure retinal blood flow in mice. This is accomplished by first infusing fluorescent microspheres through the cannulated femoral vein of an anesthetized mouse. The second step is to video record the microspheres flowing through the mouse's retinal vessels.Next, infuse fluorescent dextran through the femoral vein and capture video as it flows through the retinal vessels. The final step is to measure microsphere velocities and vessel diameters from the recorded videos for a calculation of retinal blood flow. A primary advantage of this technique is the ability to accurately measure the retina blood flow using a standard fluorescence microscope.This method can help answer key questions in the retina field, such as the extent to which changes in blood flow contribute to diabetic retinopathy. Although we will be demonstrating this technique in the retina of a mouse. The general method can be used in other tissues and also in rats.Sonicate a 1%stock solution of 1.9 micron diameter microspheres withdraw 10 microliters of the stock solution into a 300 microliter syringe, then dilute the microspheres by drawing 240 microliters of sterile saline into the syringe. Next, prepare a 50 microliter solution of fluorescent dextran, dissolved in enough sterile saline so that the injected dose will be five milligrams per kilogram. Then cover the dextran with aluminum foil until time for its fusion.After anesthetizing the animal and testing for adequate anesthesia by a tow pinch, place a heating pad underneath the animal. Keep the eyes moist with phosphate buffered saline. Next, break off the sharp tip of a syringe needle by bending it back and forth with the hemostat.Then place a 20 centimeter length of polyethylene tubing on the blunted tip of the needle and fill the tubing with the heparinized saline. Now prepare a 25 units per milliliter heparinized saline solution. Fill the syringe with the heparinized saline after shaving one side of the lower abdomen, make a one centimeter incision at the transition between the abdomen and leg to expose the femoral vein.Then tie off the vessel just proximal to the widening of the femoral vein with a five T suture. Next, make a partial incision of one half the vessel diameter and insert the tubing into the vessel along its axis at a 20 to 30 degree angle above horizontal. Secure the tubing with the suture tied around the cannulated section.To prepare the animal for microscopy, dilate the pupil of the eye with one drop of 1%tropic aide ophthalmic solution, followed by a drop of 2.5%hyper melos ophthalmic solution. Then cover the eye with a five millimeter glass cover slip. After placing the animal on a plexiglass board, position the board underneath the microscope objective of an upright microscope.Place surgical gauze underneath the head to allow a direct axis through the objective to the central retina and optic disc. Using the Forex objective and a fluorescein filter, focus on the optic disc, keeping it in the center of the field of view. Next, infuse the fluorescent microsphere solution into the femoral vein at a rate of 250 microliters per minute per kilogram.Using a camera exposure time of eight milliseconds, video record the viewable retina around the optic disc. Stop the microsphere infusion. After three minutes, some microsphere solution may remain unused.Infuse the prepared solution of fluorescent dextran into the femoral vein as a bolus over five seconds. To begin measuring the diameters of the retinal vessels, maintain focus on the optic disc with the Forex objective. Then switch to a 10 x objective and 40 milliseconds exposure.Time to improve resolution video four to seven arterials and four to seven venues in the superficial retina. Focus on one quadrant of the viewable retina at a time keeping the center of the optic disc in one corner of the field of view. Record each quadrant for only 10 to 15 seconds to minimize the possibility of phototoxicity.Now, analyze the video to determine flow rates and total retinal blood flow. Using a micrometer scale, calibrate the video system for a conversion of microns to pixels With image processing software. Play back the recorded video from the video of the bolus infusion of fluorescent dextran.Identify arterials and vees the arterials perfuse before the vees. Measure the diameters of each of the arterials and vees filled with fluorescent dextran. Averaging five measures of diameter per vessel along the viewable length.Then for each of the same arterials and vees, measure the streak length of the fluorescent microspheres using 10 successive microsphere streaks in each vessel to avoid selection bias. Now divide the streak lengths by the exposure time to calculate the microsphere velocity and average the 10 velocities per vessel. Next, calculate the volumetric flow rate for each vessel.The flow equals the mean velocity times pi times the diameter squared divided by four. Finally sum the arteriolar flow rates and separately the ular flow rates to obtain the total retinal blood flow.Shown. Here are single frames of video showing 1.9 micron fluorescent microspheres appearing as streaks as they move through the retinal vessels.The middle image shows the orientation of the retinal vessels in the other panels of the figure. Note that some frames have multiple streaks. In this experiment, most of the fluorescent streaks are in the range of 120 to 170 microns in length, which corresponds to velocities of 1.5 to 2.1 centimeters per second.These next images are from the bolus infusion of fluorescent dextran. In the first frames, arterials are seen filling with dextran. The vees fill after the dye makes its transit through the capillary beds.Shown here is one of the 10 x images used to measure vessel diameters. The arteriolar diameters ranged from 47 to 55 microns and the venal diameters ranged from 50 to 65 microns. This figure shows flow measurements from a previous experiment in which both fluorescent microspheres and fluorescent red blood cells were injected into the same mouse to determine if similar vessel velocities and flows would be found.As this figure indicates, the measured velocities and flows were very similar Once mastered, this technique can be done in less than one hour if it is performed properly. While performing this procedure, it is important to remember to reduce the fluorescent excitation of the dyes by limiting the time the microscope shutter is open. After watching this video, you should have a good understanding of how fluorescent dyes can be used to measure retinal blood flow in mice.

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Research

JoVE Journal

Medicine

Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

This article reports on a detailed method for the dynamic measurement and quantification of blood flow velocity within individual blood vessels of the mouse liver vasculature using intravital microscopy imaging in combination with a specific methodology for image acquisition and analysis.

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

Live-cell imaging of the labeled blood cells in ocular circulation can provide information about inflammation and ischemia in diabetic retinopathy and age-related macular degeneration. A protocol to label blood cells and image the labeled cells in the retinal circulation is described.

Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation

Many vision-threatening ocular diseases are associated with dysfunctional retinal microvessels. Therefore, the measurement of retinal arteriole responses is important to investigate the underlying pathophysiological mechanisms. This article describes a detailed protocol for mouse retinal arteriole isolation and preparation to assess the effects of vasoactive substances on vascular diameter.

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

An open vessel-window approach using fluorescent tracers provides sufficient resolution for cochlear blood flow (CoBF) measurement. The method facilitates the study of structural and functional changes in CoBF in mouse under normal and pathological conditions.

Measurement of Strial Blood Flow in Mouse Cochlea Utilizing an Open Vessel-Window and Intravital Fluorescence Microscopy

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Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

This protocol describes a detailed method for the preparation and immunofluorescence staining of mice retinal flat mounts and analysis. The use of fluorescein fundus angiography (FFA) for mice pups and image processing are described in detail as well.

Monitoring Dynamic Growth of Retinal Vessels in Oxygen-Induced Retinopathy Mouse Model

This article describes a well-established and reproducible lectin stain assay for the whole mount retinal preparations and the protocols required for the quantitative measurement of vascular parameters frequently altered in proliferative and non-proliferative retinopathies.

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

Here, we present three data analysis protocols for fluorescein angiography (FA) and optical coherence tomography (OCT) images in the study of Retinal Vein Occlusion (RVO).

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Mouse retinal vasculature is particularly interesting in understanding the mechanisms of vascular pattern formation. This protocol automatically measures the diameter of mouse retinal vessels from fluorescent angiography fundus images at a fixed distance from the optic disk.

Measuring Retinal Vessel Diameter from Mouse Fluorescent Angiography Images

Learn intravital video microscopy to measure retinal blood flow in mice using fluorescent tracers. This method enables accurate quantification of vessel diameters and blood velocities for studying retinal pathologies.

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

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