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>

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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 and coverslip with an ophthalmic solution fluid layer counteracts the refractive power of the cornea. Any unaccounted magnification would affect measurements of length and diameter.
2. Limitation of optical resolution. We typically use a 10X&#160;objective (working distance = 1.05 cm) for our diameter measurements, with this objective having a numerical aperture of 0.25 and a nominal resolution of ~1.2 &#181;m (approximately the uncertainty in the measurement). The overall resolution is not limited by the camera/image processor resolution at this magnification, equal to 0.47 &#181;m/pixel with no pixel binning and equal to 0.94 &#181;m/pixel with 2 x&#160;2 binning. Higher numerical aperture objectives can be used for this protocol to improve resolution, as long as the working distances are sufficient: the axial lengths of the mouse and rat eyes are ~3.4 mm and ~6.3 mm, respectively21-23.
3. Assumption of dextran filling the entire vascular lumen. The endothelial glycocalyx may largely exclude the 2 x&#160;106 molecular weight dextran over the short time frame involved in the measurement protocol. Therefore, this technique likely defines the inner vessel diameter as beginning at the lumen edge of the glycocalyx, which is typically estimated to extend 0.1-0.5 &#181;m from the endothelial cell membrane24. This thickness is smaller than the optical resolution of the video collection system, but contributes to an uncertainty in the diameter measurement.
4. Assumption of right circular cylindrical vessels. Previous measurements of retinal blood flow using intravital video microscopy have found slightly higher values of venular flow compared to arteriolar flow11-13,15, with this discrepancy likely due to an elliptical shape of the venular diameter. The venular diameter may have its longer radius perpendicular to the optical axis, and therefore calculations of venular flow may be slightly overestimated. We have previously documented similar geometrical effects on intravital microscopic measurements of flow in intestinal submucosal venules of mice25.
5. Assumption that the sampled velocities represent the mean. The microspheres flowing closer to the vessel wall will travel more slowly that those in the centerline. Our assumption in past studies (using fluorescently labeled RBCs) is that 10 velocity measurements are sufficient to obtain a reasonably accurate distribution of velocities whose mean approximates the true average red blood cell velocity. However, measuring more than 10 velocities per vessel is likely to provide even more precision in the average.
This protocol was written specifically for use in the retina; however, the procedures can be applied to other tissues that are accessible via intravital microscopy. The protocol requires minimal troubleshooting, with the main step that requires some practice being the alignment of the mouse&#39;s head in the correct position to allow a direct optical path through the objective to the center of the optic disk. If the retina is not easily seen through the iris, visually confirm that the pupil has been sufficiently dilated by the tropicamide solution. In addition, the arteriolar and venular microspheres will not always be in focus simultaneously, as the arterioles and venules are slightly offset from each other: therefore, collecting the video requires some adjustments up and down in the focus. Several steps in the procedure can be modified to variations in the available equipment. For example, a digital camera (although helpful) is not required: we have used a similar protocol with a nondigital camera that used standard frame rates, and with our previous method of using fluorescently labeled red blood cells or platelets from donor animals instead of microspheres10,16. The critical steps are simply to be able to record the diameters of the fluorescently filled blood vessels and to record the velocity of fluorescently labeled bloodstream markers.

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;panels C, D, G, H, and I). In this experiment, most of the fluorescent streaks (captured during an 8 msec exposure time) were in the range of 120-170 &#181;m in length, which corresponds to velocities of 1.5-2.1 cm/sec.
Figure 2 shows nine frames of video from the bolus infusion of 2 x&#160;106 molecular weight fluorescein isothiocyanate-labeled dextran, with 0.071 sec (twice the frame capture rate) between the selected frames. The length of time between the first and final panels of Figure 2 is ~0.57 sec. The vessels filling first with the fluorescent dextran (shown in panels A-D) are arterioles, in this case corresponding to &#8220;clock&#8221; positions of ~1:30, 4:30, 7:00, and 10:00. After a short delay, the venules (in positions of ~12:00, 3:30, 5:30, and 9:00) are filled after the dye makes its transit through the capillary beds.
Figure 3 shows a 10X&#160;view of two of the vessels (in the 9:00 and 10:00 positions), with this and similar pictures of the other vessels used to measure diameters. In this experiment, the arteriolar diameters were measured to be in the range of 47-55 &#181;m, and the venules measured to be 50-65 &#181;m in diameter.
The velocities and diameters are inserted into a volumetric flow calculation, flow = mean velocity &#215; &#960; &#215; diameter2/4. For example, 2.0 cm/sec &#215; &#960; &#215; (55&#215;10-4 cm)2/4 = 4.8&#215;10-5 cm3/sec = 48 nl/sec. Summing the 4 arterioles (or the 4 venules) together provides a calculation of the total retinal blood flow rate. In the mouse eye of this example experiment, the sum of arteriolar flow was 138 nl/sec and the sum of venular flow was 172 nl/sec.
Figure 4 shows flow measurements from an experiment in which both fluorescent microspheres and fluorescent red blood cells (as described in our previous studies9-16) were injected into the same mouse, to determine if similar vessel velocities and flows would be found. In this experiment, the mouse retina had 6 arterioles and 6 venules, with measured velocities and flows very similar (p=0.75 for arterioles; p=0.38 for venules).
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51110/51110fig1highres.jpg" src="/files/ftp_upload/51110/51110fig1.jpg" /> 
Figure 1.&#160;Video frames captured during an intravital microscopy experiment. Fluorescent microspheres 1.9 &#181;m in diameter flow through retinal vessels (panels A-D and F-I), with the appearance of the microspheres as streaks during the camera exposure time of 8 msec. Panel E shows the orientation of the blood vessels in the superficial layer of the retina. The scale bar in panel E corresponds to all images in Figure 1.
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51110/51110fig1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51110/51110fig2highres.jpg" src="/files/ftp_upload/51110/51110fig2.jpg" /> 
Figure 2.&#160;Infusion of high molecular weight dextran through the retinal circulation. Bolus infusion of fluorescent dextran, with 0.071 sec between the selected frames. The vessels filling first with the fluorescent dextran (panels A-D) are arterioles, with the venules filled after the dye makes its transit through the capillary beds. The scale bar in panel E corresponds to all images in Figure 2.
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51110/51110fig2highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/51110/51110fig3highres.jpg" src="/files/ftp_upload/51110/51110fig3.jpg" /> 
Figure 3.&#160;A retinal arteriole and venule at higher magnification. A 10X&#160;objective was used to capture images of the superficial retinal vessels for measurements of diameter.
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51110/51110fig3highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/51110/51110fig4highres.jpg" src="/files/ftp_upload/51110/51110fig4.jpg" /> 
Figure 4.&#160;Retinal microvessel flows measured with the use of fluorescent microspheres and fluorescent red blood cells. No statistical differences between the two techniques were found (using a paired t-test). In this experiment, the flows were measured in 6 arterioles and 6 venules of a mouse retina.
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51110/51110fig4highres.jpg" target="_blank">Click here to view larger image</a>.

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

Nanyang Technological University

Research

JoVE Journal

Medicine

5.6K ビュー.  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.

ビデオ: 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.

マウスを用いた気管狭窄のin vitroでの測定で

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

医学

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.

ラットの肺転移における微小循環血流速度の自動測定

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.

ナノボディ及び抗体ベースの検証<em&gt;インビボ</em使用してマウスにおける&gt;腫瘍異種移植NIRFイメージング実験<em&gt;エクスビボ</em&gt;フローサイトメトリーおよび顕微鏡

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.

治療のために血液網膜関門の違反と潜在的な薬を評価するための急性網膜モデル

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.

腫瘍関連血管系を使用しての生体顕微鏡が背高度な蛍光トランスジェニック マウスの皮下脂肪のウィンドウの部屋

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.

ヘアレスマウスの耳たぶにおける生体顕微鏡および血栓誘導

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

Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other vessels), and describes techniques for evaluating tissue perfusion using Laser Doppler Flowmetry (LDF) and microvessel density utilizing fluorescently labeled Griffonia simplicifolia (GS1) lectin. Current methods for studying isolated resistance arteries at transmural pressures encountered in vivo and in the absence of parenchymal cell influences provide a critical link between in vivo studies and information gained from molecular reductionist approaches that provide limited insight into integrative responses at the whole animal level. LDF and techniques to selectively identify arterioles and capillaries with fluorescently-labeled GS1 lectin provide practical solutions to enable investigators to extend the knowledge gained from studies of isolated resistance arteries. This paper describes the application of these techniques to gain fundamental knowledge of vascular physiology and pathology in the rat as a general experimental model, and in a variety of specialized genetically engineered &#34;designer&#34; rat strains that can provide important insight into the influence of specific genes on important vascular phenotypes. Utilizing these valuable experimental approaches in rat strains developed by selective breeding strategies and new technologies for producing gene knockout models in the rat, will expand the rigor of scientific premises developed in knockout mouse models and extend that knowledge to a more relevant animal model, with a well understood physiological background and suitability for physiological studies because of its larger size.

This manuscript describes in vitro video microscopy protocols for evaluating vascular function in rat cerebral resistance arteries. The manuscript also describes techniques for evaluating microvessel density with fluorescently labeled lectin and tissue perfusion using Laser Doppler Flowmetry.

分離抵抗性ラットの動脈のビデオ顕微鏡を用いた血管制御機構の評価

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other ...

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.

平滑筋細胞系譜トレース マウスの高度な動脈硬化病変における細胞構成の定量的解析

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.

マウスの鎖骨下静脈穿刺による血液採取

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.

異なる均質化法を通じてマウス脳/脊髄から取り出された複数の細胞型の同時フロー細胞特性評価

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