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/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. 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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 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 ...

intravital-video-microscopy-measurements-of-retinal-blood-flow-in-mice

Research

JoVE Journal

Medicine

5.6K vistas.  Louisiana State University Health Sciences Center. Intravital microscopy can be used in animals to visualize and measure retinal vascular diameters, bloodstream velocities, and total retinal blood flow.

Video: Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

In vitro Measurements of Tracheal Constriction Using Mice

Transgenic and knockout mice have been powerful tools for the investigation of the physiology and pathophysiology of airways1,2. In vitro tensometry of isolated tracheal preparations has proven to be a useful assay of airway smooth muscle (ASM) contractile response in genetically modified mice. These in vitro tracheal preparations are relatively simple, provide a robust response, and retain both functional cholinergic nerve endings and muscle responses, even after long incubations.
Tracheal tensometry also provides a functional assay to study a variety of second messenger signaling pathways that affect contraction of smooth muscle. Contraction in trachea is primarily mediated by parasympathetic, cholinergic nerves that release acetylcholine onto ASM (Figure 1). The major ASM acetylcholine receptors are muscarinic M2 and M3 which are Gi/o and Gq coupled receptors, respectively3,4,5. M3 receptors evoke contraction by coupling to Gq to activate phospholipase C, increase IP3 production and IP3-mediated calcium release from the sarcoplasmic reticulum3,6,7. M2/Gi/o signaling is believed to enhance contractions by inhibition of adenylate cyclase leading to a decrease in cAMP levels5,8,9,10. These pathways constitute the so called "pharmaco-contraction coupling" of airway smooth muscle11. In addition, cholinergic signaling through M2 receptors (and modulated by M3 signaling) involves pathways that depolarize the ASM which in turn activate L-type, voltage-dependent calcium channels (Figure 1) and calcium influx (so called "excitation-contraction coupling")4,7. More detailed reviews on signaling pathways controlling airway constriction can be found4,12. The above pathways appear to be conserved between mice and other species. However, mouse tracheas differ from other species in some signaling pathways. Most prominent is their lack of contractile response to histamine and adenosine13,14, both well-known ASM modulators in humans and other species5,15.
Here we present protocols for the isolation of murine tracheal rings and the in vitro measurement of their contractile output. Included are descriptions of the equipment configuration, trachea ring isolation and contractile measurements. Examples are given for evoking contractions indirectly using high potassium stimulation of nerves and directly by depolarization of ASM muscle to activate voltage-dependent calcium influx (1. high K+, Figure 1). In addition, methods are presented for stimulations of nerves alone using electric field stimulation (2. EFS, Figure 1), or for direct stimulation of ASM muscle using exogenous neurotransmitter applied to the bath (3. exogenous ACH, Figure 1). This flexibility and ease of preparation renders the isolated trachea ring model a robust and functional assay for a number of signaling cascades involved in airway smooth muscle contraction.

In vitro Measurements of Tracheal Constriction Using Mice

Transgenic mice have been extremely useful in ascribing physiological function to genes. As such, research in general, and functional studies of airway, in particular, have undergone a remarkable shift toward murine models. Here we provide protocols for in vitro trachea constriction studies to evaluate smooth muscle function in murine airway.

Mediciones in vitro de la constricción de la tráquea Utilizando ratones

Transgenic and knockout mice have been powerful tools for the investigation of the physiology and pathophysiology of airways1,2. In vitro tensometry of ...

Investigación

Medicina

Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. However, very few methods exist to date to investigate lung metastases in a dynamic fashion at the microcirculatory level, due to the difficulty to access the lung with a microscope. Here, an intravital microscopy method is presented to functionally image and quantify the microcirculation of superficial pulmonary metastases in rats, using a closed-chest pulmonary window and automated analysis of blood flow velocity and direction. The utility of this method is demonstrated to measure increases in blood flow velocity in response to pharmacological intervention, and to image the well-known tortuous vasculature of solid tumors. This is the first demonstration of intravital microscopy on pulmonary metastases in a closed-chest model. Because of its minimized invasiveness, as well as due to its relative ease and practicality, this technology has the potential to experience widespread use in laboratories that specialize on pulmonary tumor research.

A method is presented to measure microcirculatory blood flow velocity in pulmonary cancer metastases of the pleural surface in rats in an automated fashion, using closed-chest pulmonary intravital microscopy. This model has potential to be used as a widespread tool to perform physiologic research on pulmonary metastases in rodents.

Medición Automatizada de la microcirculación de la sangre Velocidad del caudal en metástasis pulmonar de ratas

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. ...

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.
First we describe how to generate subcutaneous tumors in mice, using antigen-negative cell lines as negative controls and antigen-positive cells as positive controls in the same mice for intraindividual comparison. We outline how to administer intravenously near-infrared fluorophore labelled (AlexaFluor680) antigen-specific nanobodies and conventional antibodies. In vivo imaging was performed with a small-animal NIRF-Imaging system. After the in vivo imaging experiments the mice were sacrificed. We then describe how to prepare the tumors for parallel ex vivo analyses by flow cytometry and fluorescence microscopy to validate in vivo imaging results.
The use of the near-infrared fluorophore labelled nanobodies allows for non-invasive same day imaging in vivo. Our protocols describe the ex vivo quantification of the specific labeling efficiency of tumor cells by flow cytometry and analysis of the distribution of the antibody constructs within the tumors by fluorescence microscopy. Using near-infrared fluorophore labelled probes allows for non-invasive, economical in vivo imaging with the unique ability to exploit the same probe without further secondary labelling for ex vivo validation experiments using flow cytometry and fluorescence microscopy.

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.

Validación de nanocuerpo y basados ​​en anticuerpos<em&gt; En Vivo</em&gt; Experimentos tumor de xenoinjerto NIRF-de formación de imágenes en ratones utilizando<em&gt; Ex Vivo</em&gt; Citometría de flujo y microscopía

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice ...

An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An inflammatory factor, histamine, is demonstrated to compromise vessel integrity in the cultured retina through positive staining of IgG outside of the blood vessels. The effects of histamine itself and those of candidate drugs for potential treatments, such as lipoxin A4, are assessed using three parameters: blood vessel leakage via IgG immunostaining, activation of M&#252;ller cells via GFAP staining and change in neuronal dendrites through staining for MAP2. Furthermore, the layered organization of the retina allows a detailed analysis of the processes of M&#252;ller and ganglion cells, such as changes in width and continuity. While the data presented is with swine retinal culture, the system is applicable to multiple species. Thus, the model provides a reliable tool to investigate the early effects of compromised retinal vessel integrity on different cell types and also to evaluate potential drug candidates for treatment.

A low-cost, easy-to-use and powerful system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach induced by histamine. Blood vessel leakage, M&#252;ller cell activation and the continuity of neuronal processes are utilized to assess the damage response and its reversal with a potential drug, lipoxin A4.

Un modelo agudo de retina para evaluar sanguíneos de la retina barrera violación, así como potenciales fármacos para el tratamiento

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An ...

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

La microscopia intravital de la utilización de la vasculatura asociada a Tumor avanzado Dorsal cámaras de ventana pliegues cutáneos en ratones transgénicos fluorescentes

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static ...

Intravital Microscopy and Thrombus Induction in the Earlobe of a Hairless Mouse

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions between cellular and non-cellular blood components during thrombus formation, reliable studies of the physiology and pathophysiology of thrombosis can only be performed in vivo. Therefore, this article presents an ear model in hairless mice and focuses on the in vivo analysis of microcirculation, thrombus formation, and thrombus evolution. By using intravital fluorescence microscopy and the intravenous (iv) application of the respective fluorescent dyes, a repetitive analysis of microcirculation in the auricle can easily be performed, without the need for surgical preparation. Furthermore, this model can be adapted for in vivo studies of different issues, including wound healing, reperfusion injury, or angiogenesis. In summary, the ear of hairless mice is an ideal model for the in vivo study of skin microcirculation in physiological or pathophysiological conditions and for the evaluation of its reaction to different systemic or topical treatments.

The ear model of the hairless SKH1-Hrhr mouse enables intravital fluorescence microscopy of microcirculation and phototoxic thrombus induction without prior surgical preparation in the examined microvascular bed. Therefore, the ear of the hairless mouse is an excellent in vivo model to study the complex interactions during microvascular thrombus formation, thrombus evolution, and thrombolysis.

Microscopia intravital y trombos de inducción en el lóbulo de la oreja de un ratón sin pelo

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions ...

Quantitative Analysis of Cellular Composition in Advanced Atherosclerotic Lesions of Smooth Muscle Cell Lineage-Tracing Mice

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel interventions have remained elusive. This is likely due, in part, to a reliance on preclinical prevention models investigating the effects of genetic manipulations or pharmacological treatments on atherosclerosis development rather than the established disease. Also, results of these studies are often confounding because of the use of superficial lesion analyses and a lack of characterization of lesion cell populations. To help overcome these translational hurdles, we propose an increased reliance on intervention models that employ investigation of changes in cellular composition at a single cell level by immunofluorescent staining and confocal microscopy. To this end, we describe a protocol for testing a putative therapeutic agent in a murine intervention model including a systematic approach for animal dissection, embedding, sectioning, staining, and quantification of brachiocephalic artery lesions. In addition, due to the phenotypic diversity of cells within late-stage atherosclerotic lesions, we describe the importance of using cell-specific, inducible lineage tracing mouse systems and how this can be leveraged for unbiased characterization of atherosclerotic lesion cell populations. Together, these strategies may assist vascular biologists to more accurately model therapeutic interventions and analyze atherosclerotic disease and will hopefully translate into a higher rate of success in clinical trials.

We propose a standardized protocol to characterize the cellular composition of late-stage murine atherosclerotic lesions including systematic methods of animal dissection, tissue embedding, sectioning, staining, and analysis of brachiocephalic arteries from atheroprone smooth muscle cell lineage tracing mice.

Análisis cuantitativo de la composición celular en las lesiones ateroscleróticas avanzadas de músculo liso células linaje trazado ratones

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel ...

Blood Collection Through Subclavian Vein Puncture in Mice

The mouse is the foremost mammalian model for studying human disease and human health. However, blood sample collection from mice is challenging in research work. Tail blood collection is a popular method when a small amount of blood sample is needed. Orbital artery could be considered if a large amount of blood is needed but this blood collection method has ethical issues. Formerly, we demonstrated the feasibility and safety of blood sample collection through subclavian vein puncture in rats, and here we investigate whether this method could be used in mice. We report that this method is safe and practical for blood collection in mice. Blood collection through the subclavian vein puncture in mice can be a convenient method in daily research works.

Here, we present a protocol to take blood samples from the subclavian vein of mice.

Recolección de sangre a través de punción venosa Subclaviana en ratones

The mouse is the foremost mammalian model for studying human disease and human health. However, blood sample collection from mice is challenging in ...

Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central nervous system (CNS). However, further optimization of these technologies would benefit from methods allowing rapid and streamline determination of the extent of CNS and cell-specific targeting upon administration of viral vectors or nanoparticles in the body. Here, we present a protocol that takes advantage of the high throughput and multiplexing capabilities of flow cytometry to allow a straightforward quantification of different cell subtypes isolated from mouse brain or spinal cord, namely microglia/macrophages, lymphocytes, astrocytes, oligodendrocytes, neurons and endothelial cells. We apply this approach to highlight critical differences between two tissue homogenization methods in terms of cell yield, viability and composition. This could instruct the user to choose the best method depending on the cell type(s) of interest and the specific application. This method is not suited for analysis of anatomical distribution, since the tissue is homogenized to generate a single-cell suspension. However, it allows to work with viable cells and it can be combined with cell-sorting, opening the way for several applications that could expand the repertoire of tools in the hands of the neuroscientist, ranging from establishment of primary cultures derived from pure cell populations, to gene-expression analyses and biochemical or functional assays on well-defined cell subtypes in the context of neurodegenerative diseases, upon pharmacological treatment or gene therapy.

We present a flow cytometry method to identify simultaneously different cell types retrieved from mouse brain or spinal cord. This method could be exploited to isolate or characterize pure cell populations in neurodegenerative diseases or to quantify the extent of cell targeting upon in vivo administration of viral vectors or nanoparticles.

Caracterización citométrica de flujo simultáneo de múltiples tipos de células recuperadas del cerebro del ratón/cordón espinal a través de diferentes métodos de homogeneización

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central ...

Hemocompatibility Testing of Blood-Contacting Implants in a Flow Loop Model Mimicking Human Blood Flow

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the body&#39;s circulatory system demands a reliable and multiparametric approach that evaluates the possible hematologic complications caused by these devices (i.e., activation and destruction of blood components). Comprehensive in vitro hemocompatibility testing of blood-contacting implants is the first step towards successful in vivo implementation. Therefore, extensive analysis according to the International Organization for Standardization 10993-4 (ISO 10993-4) is mandatory prior to clinical application. The presented flow loop describes a sensitive model to analyze the hemostatic performance of stents (in this case, neurovascular) and reveal adverse effects. The use of fresh human whole blood and gentle blood sampling are essential to avoid the preactivation of blood. The blood is perfused through a heparinized tubing containing the test specimen by using a peristaltic pump at a rate of 150 mL/min at 37 &#176;C for 60 min. Before and after perfusion, hematologic markers (i.e., blood cell count, hemoglobin, hematocrit, and plasmatic markers) indicating the activation of leukocytes (polymorphonuclear [PMN]-elastase), platelets (&#946;-thromboglobulin [&#946;-TG]), the coagulation system (thombin-antithrombin III [TAT]), and the complement cascade (SC5b-9) are analyzed. In conclusion, we present an essential and reliable model for extensive hemocompatibility testing of stents and other blood-contacting devices prior to clinical application.

This protocol describes a comprehensive hemocompatibility evaluation of blood-contacting devices using laser-cut neurovascular implants. A flow loop model with fresh, heparinized human blood is applied to mimic blood flow. After perfusion, various hematologic markers are analyzed and compared to the values gained directly after blood collection for hemocompatibility evaluation of the tested devices.