Name: visualization and quantification of the cell-free layer in arterioles of the rat cremaster muscle
Uploaded: 2016-10-19T13:00:00
Description: Watch this Scientific Journal Video about Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle at JoVE.com

This work was supported by National Medical Research Council (NMRC)/Cooperative Basic Research Grant (CBRG)/0078/2014.

This study is in accordance with the National University of Singapore Institutional Animal Care and Use Committee (approved protocol no. R15-0225).
1. Surgical Preparation of the Animal Model
<ol>
	<li>Vessel cannulations
	<ol>
		<li>Anesthetize a male Sprague-Dawley rats (6 - 7 weeks old) weighing (203 &#177; 20) g with ketamine (37.5 mg/ml) and xylazine (5 mg/ml ) cocktail through intraperitoneal (i.p.) injection (2 ml/kg). Do not recap the needle or remove it from the syr...

<ol>
	<li>Kim, S., Kong, R. L., Popel, A. S., Intaglietta, M., Johnson, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+and+spatial+variations+of+cell-free+layer+width+in+arterioles.">Temporal and spatial variations of cell-free layer width in arterioles.</a> Am J Physiol Heart Circ Physiol. 293 (3), H1526-H1535 (2007).</li><li>Ong, P. K., Namgung, B., Johnson, P. C., Kim, S. <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+erythrocyte+aggregation+and+flow+rate+on+cell-free+layer+formation+in+arterioles.">Effect of erythrocyte aggregation and flow rate on cell-free layer formation in arterioles.</a> Am J Physiol Heart Circ Physiol. 298 (6), H1870-H1878 (2010).</li><li>Namgung, B., Kim, S. <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+uneven+red+cell+influx+on+formation+of+cell-free+layer+in+small+venules.">Effect of uneven red cell influx on formation of cell-free layer in small venules.</a> Microvasc Res. 92, 19-24 (2014).</li><li>Goldsmith, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Microcirculatory+Society.">The Microcirculatory Society.</a> Eugene M. Landis Award Lecture. The Microrheology of Human-Blood. Microvasc Res. 31 (2), 121-142 (1986).</li><li>Buerk, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+We+Model+Nitric+Oxide+Biotransport?+A+Survey+of+Mathematical+Models+for+a+Simple+Diatomic+Molecule+with+Surprisingly+Complex+Biological+Activities.">Can We Model Nitric Oxide Biotransport? A Survey of Mathematical Models for a Simple Diatomic Molecule with Surprisingly Complex Biological Activities.</a> Annu Rev Biomed Eng. 3 (1), 109-143 (2001).</li><li>Tateishi, N., Suzuki, Y., Soutani, M., Maeda, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+dynamics+of+erythrocytes+in+microvessels+of+isolated+rabbit+mesentery:+cell-free+layer+and+flow+resistance.">Flow dynamics of erythrocytes in microvessels of isolated rabbit mesentery: cell-free layer and flow resistance.</a> J Biomech. 27 (9), 1119-1125 (1994).</li><li>Ong, P. K., Cho, S., Namgung, B., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+cell-free+layer+formation+on+NO/O2+bioavailability+in+small+arterioles.">Effects of cell-free layer formation on NO/O2 bioavailability in small arterioles.</a> Microvasc Res. 83 (2), 168-177 (2012).</li><li>Ong, P. K., Jain, S., Kim, S. <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+NO+bioavailability+by+temporal+variation+of+the+cell-free+layer+width+in+small+arterioles.">Modulation of NO bioavailability by temporal variation of the cell-free layer width in small arterioles.</a> Ann Biomed Eng. 39 (3), 1012-1023 (2011).</li><li>Park, S. W., Intaglietta, M., Tartakovsky, D. M. <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+stochastic+fluctuations+in+the+cell+free+layer+on+nitric+oxide+bioavailability.">Impact of stochastic fluctuations in the cell free layer on nitric oxide bioavailability.</a> Front Comput Neurosci. 9, 131 (2015).</li><li>Ng, Y. C., Namgung, B., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+transient+model+for+prediction+of+arteriolar+NO/O2+modulation+by+spatiotemporal+variations+in+cell-free+layer+width.">Two-dimensional transient model for prediction of arteriolar NO/O2 modulation by spatiotemporal variations in cell-free layer width.</a> Microvasc Res. 97, 88-97 (2015).</li><li>Sriram, K., 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=The+effect+of+small+changes+in+hematocrit+on+nitric+oxide+transport+in+arterioles.">The effect of small changes in hematocrit on nitric oxide transport in arterioles.</a> Antioxid Redox Sign. 14 (2), 175-185 (2011).</li><li>Hightower, C. 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=Integration+of+cardiovascular+regulation+by+the+blood/endothelium+cell-free+layer.">Integration of cardiovascular regulation by the blood/endothelium cell-free layer.</a> Wiley Interdiscip Rev Syst Biol Med. 3 (4), 458-470 (2011).</li><li>Ng, Y. C., Namgung, B., Leo, H. L., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erythrocyte+aggregation+may+promote+uneven+spatial+distribution+of+NO/O+in+the+downstream+vessel+of+arteriolar+bifurcations.">Erythrocyte aggregation may promote uneven spatial distribution of NO/O in the downstream vessel of arteriolar bifurcations.</a> J Biomech. , (2015).</li><li>Ong, P. K., Jain, S., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+variations+of+the+cell-free+layer+width+may+enhance+NO+bioavailability+in+small+arterioles:+Effects+of+erythrocyte+aggregation.">Temporal variations of the cell-free layer width may enhance NO bioavailability in small arterioles: Effects of erythrocyte aggregation.</a> Microvasc Res. 81 (3), 303-312 (2011).</li><li>Maeda, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erythrocyte+rheology+in+microcirculation.">Erythrocyte rheology in microcirculation.</a> Jpn J Physiol. 46 (1), 1-14 (1996).</li><li>Soutani, M., Suzuki, Y., Tateishi, N., Maeda, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Evaluation+of+Flow+Dynamics+of+Erythrocytes+in+Microvessels+-+Influence+of+Erythrocyte+Aggregation.">Quantitative Evaluation of Flow Dynamics of Erythrocytes in Microvessels - Influence of Erythrocyte Aggregation.</a> Am J Physiol Heart Circ Physiol. 268 (5), H1959-H1965 (1995).</li><li>Kim, S., Kong, R. L., Popel, A. S., Intaglietta, M., Johnson, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+computer-based+method+for+determination+of+the+cell-free+layer+width+in+microcirculation.">A computer-based method for determination of the cell-free layer width in microcirculation.</a> Microcirculation. 13 (3), 199-207 (2006).</li><li>Soutani, M., Suzuki, Y., Tateishi, N., Maeda, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Evaluation+of+Flow+Dynamics+of+Erythrocytes+in+Microvessels.">Quantitative Evaluation of Flow Dynamics of Erythrocytes in Microvessels.</a> Influence of Erythrocyte Aggregation. Am J Physiol Heart Circ Physiol. 268 (5), H1959-H1965 (1995).</li><li>Namgung, B., 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=A+comparative+study+of+histogram-based+thresholding+methods+for+the+determination+of+cell-free+layer+width+in+small+blood+vessels.">A comparative study of histogram-based thresholding methods for the determination of cell-free layer width in small blood vessels.</a> Physiol Meas. 31 (9), N61-N70 (2010).</li><li>Ong, P. K., 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=An+automated+method+for+cell-free+layer+width+determination+in+small+arterioles.">An automated method for cell-free layer width determination in small arterioles.</a> Physiol Meas. 32 (3), N1-N12 (2011).</li><li>Otsu, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Threshold+Selection+Method+from+Gray-Level+Histograms.">A Threshold Selection Method from Gray-Level Histograms.</a> IEEE Trans. Syst., Man, Cybern. 9 (1), 62-66 (1979).</li><li>Prewitt, J. M., Mendelsohn, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+analysis+of+cell+images.">The analysis of cell images.</a> Ann N Y Acad Sci. 128 (3), 1035-1053 (1966).</li><li>Ridler, T. W., Calvard, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Picture+Thresholding+Using+an+Iterative+Selection+Method.">Picture Thresholding Using an Iterative Selection Method.</a> IEEE Trans. Syst., Man, Cybern. 8 (8), 630-632 (1978).</li><li>Shanbhag, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilization+of+Information+Measure+as+a+Means+of+Image+Thresholding.">Utilization of Information Measure as a Means of Image Thresholding.</a> Cvgip-Graph Model Im. 56 (5), 414-419 (1994).</li><li>Bishop, J. J., Popel, A. S., Intaglietta, M., Johnson, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+erythrocyte+aggregation+and+venous+network+geometry+on+red+blood+cell+axial+migration.">Effects of erythrocyte aggregation and venous network geometry on red blood cell axial migration.</a> Am J Physiol Heart Circ Physiol. 281 (2), H939-H950 (2001).</li><li>Yamaguchi, S., Yamakawa, T., Niimi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+plasma+layer+in+cerebral+microvessels.">Cell-free plasma layer in cerebral microvessels.</a> Biorheology. 29 (2-3), 251-260 (1992).</li></ol>

The measurement of CFL width is essential for a better understanding of the hemodynamics in the microcirculation. In particular, the measurement of CFL widths has been performed in mesenteric6, spinotrapezius24 and cerebral25 microcirculations. Conventional measurement of in vivo CFL widths was restricted to estimations by manual inspection of the recorded video frames. The manual measurements required the averaging of several successive video frames before visually identifying t...

In vivo animal studies are instrumental to basic science for understanding human physiology and pathology. In particular, in vivo microhemodynamic studies can elucidate the potential impairment of microcirculatory functions altered by abnormal rheological conditions of blood. A number of previous microhemodynamic studies1 have used the rat cremaster muscle model for visualizing microvascular blood flow. The cremaster muscle is a thin layer of striated muscle surrounding the testes. Thus, the blood flow in the muscle can be visualized with a trans-illumination microscope by means of surgical exposure. This enables us to acquire the in vivo blood flow images without the use of any fluorescence or contrast agents. In addition, the entire blood perfusion of the muscle network can be controlled by reducing the upstream blood flow with abdominal aorta occlusion2. Owing to these advantages, the cremaster muscle model has been widely used to investigate the formation of cell-free layer (CFL) in microvessels1,3. 
The CFL width is a prominent hemodynamic parameter in microcirculation, which has been of great interest for its important roles in regulating microcirculatory functions. The CFL is formed by the shear-induced transverse inward migration of red blood cells (RBCs) towards the flow center4. Consequently, this migration leads to the depletion of RBCs near the vessel walls, eventually resulting in a cell-free plasma layer. Accordingly, the parietal CFL naturally becomes a diffusion barrier to oxygen (O2) delivery from the RBC core to the tissues, and to the scavenging of nitric oxide (NO) by the RBCs5,6. In addition, the production of NO can also be modulated by the dynamic variations of the CFL width7,8. Therefore, the roles of the CFL in both gas transport and the regulation of homeostasis in microcirculation need to be fully ascertained to better understand blood flow in microcirculation. Recent studies have focused on bridging the hemodynamics and gas transport functions of the CFL in the microcirculation9-12. Furthermore, a separate set of studies has also investigated how the pathological elevation in RBC aggregation modulates CFL formation and its effect on O2 and NO bioavailability in tissues13,14. 
The roles of the CFL become more significant in microcirculation where the relative size of the CFL width to the vessel diameter is prominent. This necessitates an effective approach of quantifying the CFL in in vivo blood flow. Particularly, image acquisition and image analysis are the two key components determining the accuracy of CFL width measurement. Successful visualization of tissue blood flow should be preceded by an appropriate surgical preparation of the animal model. Additionally, a proper image analysis technique is needed to overcome the limitations of conventional manual measurements that are mostly induced by human errors15,16. With advancements in optical instrumentation and computing power for digital image processing, it is now possible to achieve a more accurate and consistent measurement of the CFL width17-19. Nonetheless, the accuracy of these measurements, being image-based, still ultimately depends on the quality of the images.
Therefore, this study explores the factors influencing the measurement of the in vivo CFL width. We focused particularly on demonstrating the surgical preparation and digital image analysis for measurements of the CFL width in arterioles of the rat cremaster muscle.

<table border="0" cellpadding="0" cellspacing="0" width="788">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Intravital microscope</td>
			<td style="width:167px;">Olympus</td>
			<td style="width:119px;">BX51WI</td>
			<td style="width:253px;">Equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">High speed camera</td>
			<td style="width:167px;">Photron</td>
			<td style="width:119px;">1024PCI</td>
			<td>Equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Blue filter</td>
			<td style="width:167px;">HOYA</td>
			<td style="width:119px;">B390</td>
			<td>Equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Pressure sensor &#38;biopac system</td>
			<td style="width:167px;">Biopac system</td>
			<td style="width:119px;">TSD104A, MP100</td>
			<td>Equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Temperature controller</td>
			<td style="width:167px;">Shimaden</td>
			<td style="width:119px;">SR 1</td>
			<td>Equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Plasma Lyte A</td>
			<td style="width:167px;">Baxter</td>
			<td style="width:119px;">NDC:0338-0221</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Saline 0.9%</td>
			<td style="width:167px;">Braun</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Heparin (5000 IU/ml)</td>
			<td style="width:167px;">LEO</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">PE-10 polyethylene tube</td>
			<td style="width:167px;">Becton Dickinson</td>
			<td style="width:119px;">427400</td>
			<td>.024&#34; OD X .011&#34; ID&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">PE-50 polyethene tube</td>
			<td style="width:167px;">Becton Dickinson</td>
			<td style="width:119px;">427411</td>
			<td>.038&#34; OD X .023&#34; ID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">PE-205 polyethene tube</td>
			<td style="width:167px;">Becton Dickinson</td>
			<td style="width:119px;">427446</td>
			<td>.082&#34; OD X .062&#34; ID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">2-0 non-absorbable silk suture</td>
			<td style="width:167px;">Deknatel</td>
			<td style="width:119px;">113-S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">5-0 non-absorbable silk suture</td>
			<td style="width:167px;">Deknatel</td>
			<td style="width:119px;">106-S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Water circulating heating pad</td>
			<td style="width:167px;">Gaymar</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Water bath</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:119px;">Isotemp 205</td>
			<td>Equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Sterile Cotton Gauze&#160;</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:119px;">22-415-468</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Cotton-tipped applicators</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:119px;">23-400-124</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Dumont Forceps</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS14188</td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Micro Dissecting forceps</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS15915</td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Iris forceps 1x2 teeth</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS15917</td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Vessel cannulation forceps</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS500377</td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Micro scissor</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS14177</td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Iris scissor</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS14225</td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Vessel clip</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS14120</td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Gemini cautery system</td>
			<td style="width:167px;">Braintree Scientific</td>
			<td style="width:119px;">GEM 5917</td>
			<td>Surgical instrument</td>
		</tr>
	</tbody>
</table>

visualization and quantification of the cell-free layer in arterioles of the rat cremaster muscle

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo cell-free layer width and its spatiotemporal variations can provide a comprehensive understanding of hemodynamics in microcirculation. In this study, we used an intravital microscopic system coupled with a high-speed video camera to quantify the cell-free layer widths in arterioles in vivo. The cremaster muscle of Sprague-Dawley rats was surgically exteriorized to visualize the blood flow. A custom-built imaging script was also developed to automate the image processing and analysis of the cell-free layer width. This approach enables the quantification of spatiotemporal variations more consistently than previous manual measurements. The accuracy of the measurement, however, partly depends on the use of a blue filter and the selection of an appropriate thresholding algorithm. Specifically, we evaluated the contrast and quality of images acquired with and without the use of a blue filter. In addition, we compared five different image histogram-based thresholding algorithms (Otsu, minimum, intermode, iterative selection, and fuzzy entropic thresholding) and illustrated the differences in their determination of the cell-free layer width.

The visualization of the CFL in vivo is largely dependent on the surgical preparations of the animal. Excessive blood loss or extended surgery duration may subject the animal to shock and blood flow aberrations. Maintenance of tissue temperature using a heating pad as well as a customized platform during the surgery and experiment is also crucial for maintaining the physiological conditions of the rat. By using a 100 W halogen lamp in the microscope system, no discernible tissue damage was observed even at the e...

Watch this Scientific Journal Video about Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle at JoVE.com

Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle

This study demonstrates the surgical preparation of the rat cremaster muscle for the visualization of the in vivo cell-free layer. Considerable factors affecting the accuracy of the cell-free layer width measurement are discussed in this study.

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo ...

Research

JoVE Journal

Biology

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

Quantification of ÃƒÅ½Ã‚Â³H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

The Mouse Cremaster Muscle Preparation for Intravital Imaging of the Microcirculation

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic ...

Imaging of Estrogen Receptor-&alpha; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

Imaging of Estrogen Receptor-ÃƒÅ½Ã‚Â± in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

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

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...

Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy ...

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

Brown and beige adipocytes are now recognized as potential therapeutic targets for obesity and metabolic syndromes. Non-invasive molecular imaging methods ...