This work was supported by the American Heart Association (EIA 0740129N) and NIH T32 HL90610.

<ol>
	<li>Goodwill, A. G., Frisbee, S. J., Stapleton, P. A., James, M. E., Frisbee, J. C. <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+Chronic+Anticholesterol+Therapy+on+Development+of+Microvascular+Rarefaction+in+the+Metabolic+Syndrome.">Impact of Chronic Anticholesterol Therapy on Development of Microvascular Rarefaction in the Metabolic Syndrome.</a> Microcirculation. , 1-18 (2009).</li><li>Goodwill, A. G., James, M. E., Frisbee, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+vascular+thromboxane+generation+impairs+dilation+of+skeletal+muscle+arterioles+of+obese+Zucker+rats+with+reduced+oxygen+tension.">Increased vascular thromboxane generation impairs dilation of skeletal muscle arterioles of obese Zucker rats with reduced oxygen tension.</a> Am. J. Physiol. Heart Circ. Physiol. 295, H1522-H1528 (2008).</li><li>Samora, J. B., Frisbee, J. C., Boegehold, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth-dependent+changes+in+endothelial+factors+regulating+arteriolar+tone.">Growth-dependent changes in endothelial factors regulating arteriolar tone.</a> Am. J. Physiol. Heart Circ. Physiol. 292, H207-H214 (2007).</li><li>Samora, J. B., Frisbee, J. C., Boegehold, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+peroxide+emerges+as+a+regulator+of+tone+in+skeletal+muscle+arterioles+during+juvenile+growth.">Hydrogen peroxide emerges as a regulator of tone in skeletal muscle arterioles during juvenile growth.</a> Microcirculation. 15, 151-161 (2008).</li><li>Samora, J. B., Frisbee, J. C., Boegehold, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+myogenic+responsiveness+of+skeletal+muscle+arterioles+with+juvenile+growth.">Increased myogenic responsiveness of skeletal muscle arterioles with juvenile growth.</a> Am. J. Physiol. Heart Circ. Physiol. 294, 2344-2351 (2008).</li><li>Dacey, R. G., Duling, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+study+of+rat+intracerebral+arterioles:+methods,+morphology,+and+reactivity.">A study of rat intracerebral arterioles: methods, morphology, and reactivity.</a> Am. J. Physiol. Heart Circ. Physiol. 243, H598-H606 (1982).</li><li>Fredricks, K. T., Liu, Y., Lombard, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+extraparenchymal+resistance+arteries+of+rat+skeletal+muscle+to+reduce+PO2.">Response of extraparenchymal resistance arteries of rat skeletal muscle to reduce PO2.</a> Am. J. Physiol. 267, H706-H715 (1994).</li><li>Durand, M. J., Raffai, G., Weinberg, B. D., Lombard, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiotensin-(1-7)+and+low-dose+angiotensin+II+infusion+reverse+salt-induced+endothelial+dysfunction+via+different+mechanisms+in+rat+middle+cerebral+arteries.">Angiotensin-(1-7) and low-dose angiotensin II infusion reverse salt-induced endothelial dysfunction via different mechanisms in rat middle cerebral arteries.</a> Am. J. Physiol. Heart Circ. Physiol. 299, H1024-H1033 (2010).</li><li>LeBlanc, A. J., Cumpston, J. L., Chen, B. T., Frazer, D., Castranova, V., Nurkiewicz, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle+inhalation+impairs+endothelium-dependent+vasodilation+in+subepicardial+arterioles.">Nanoparticle inhalation impairs endothelium-dependent vasodilation in subepicardial arterioles.</a> J. Toxicol. Environ. Health A. 72, 1576-1584 (2009).</li><li>Jernigan, N. L., LaMarca, B., Speed, J., Galmiche, L., Granger, J. P., Drummond, 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=Dietary+salt+enhances+benzamil-sensitive+component+of+myogenic+constriction+in+mesenteric+arteries.">Dietary salt enhances benzamil-sensitive component of myogenic constriction in mesenteric arteries.</a> Am. J. Physiol. Heart Circ. Physiol. 294, H409-H420 (2008).</li><li>Stapleton, P. A., Goodwill, A. G., James, M. E., Frisbee, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+mechanisms+of+endothelium-dependent+dilation+in+skeletal+muscle+arterioles+with+genetic+hypercholesterolemia.">Altered mechanisms of endothelium-dependent dilation in skeletal muscle arterioles with genetic hypercholesterolemia.</a> Am. J. Physiol. Regul. Integr. Comp. Physiol. 293, R1110-R1119 (2007).</li><li>Goodwill, A. G., Stapleton, P. A., James, M. E., d'Audiffret, A. C., Frisbee, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+arachidonic+acid-induced+thromboxane+generation+impairs+skeletal+muscle+arteriolar+dilation+with+genetic+dyslipidemia.">Increased arachidonic acid-induced thromboxane generation impairs skeletal muscle arteriolar dilation with genetic dyslipidemia.</a> Microcirculation. 15, 621-631 (2008).</li><li>Baumbach, G. L., Hadju, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanics+and+composition+of+cerebral+arterioles+in+renal+and+spontaneously+hypertensive+rats.">Mechanics and composition of cerebral arterioles in renal and spontaneously hypertensive rats.</a> Hypertension. 21, 816-826 (1993).</li><li>Uchida, E., Bohr, D. F., Hoobler, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+studying+isolated+resistance+vessel+from+rabbit+mesentery+and+brain+and+their+responses+to+drugs.">A method for studying isolated resistance vessel from rabbit mesentery and brain and their responses to drugs.</a> Circ. Res. 4, 525-536 (1967).</li><li>Davis, M. J., Kuo, L., Chilian, W. M., Muller, J. M. I. s. o. l. a. t. e. d., Barker, J. H., Anderson, G. L., Menger, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+23.">Chapter 23.</a> Isolated, perfused microvessels. In: Clinically Applied Microcirculation Research. 32, 435-456 (1995).</li><li>Lombard, J. H., Liu, Y., Fredricks, K. T., Bizub, D. M., Roman, R. J., Rusch, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+and+mechanical+responses+of+rat+middle+cerebral+arterieal+to+reduced+PO2+and+prostacyclin.">Electrical and mechanical responses of rat middle cerebral arterieal to reduced PO2 and prostacyclin.</a> Am. J. Physiol. 276, H509-H516 (1994).</li></ol>

No conflicts of interest declared.

The protocol presented describes the isolation, removal and double cannulation of a skeletal muscle microvessel, although this general technique can be readily applied to most tissues. For the current manuscript, the term "arteriole" has been used by the authors to describe a resistance vessel ranging between 70-120 &mu;m in diameter under resting active tone, which is also a major contributor to the regulation of perfusion resistance to an organ or tissue.
With some modifications, this system can be fitted to multiple applications for the specific investigator and readily allows for the incorporation of additional techniques to provide more in depth experimentation (e.g., use of transmembrane electrodes for determination of membrane potential17). A critical step in the basic preparation is always insuring that the inflow and outflow pipettes that are used to cannulate the vessel are well matched, remain clear, and allow flow and pressure to be maintained in a facile manner. When the pipettes become clogged with minute pieces of debris, it may be necessary to break off very small pieces of the tips to restore flow. If the investigator is careful, pipettes can be reused multiple times until they reach a point where they either cannot be unclogged or the diameters sufficiently large that cannulation of the vessel becomes extremely difficult. However, with mastery of this surgical and experimental preparation, careful attention to detail, and the appropriate modifications to suit specific conditions and requirements, investigators can readily evaluate pathways determining vascular reactivity in response to a myriad of physiological and pharmacological challenges. This preparation will also allow the investigator to perform basic analyses on alterations to vascular wall mechanics under conditions of no active tone14. This is indeed a very powerful and adaptable technique and it is one that can readily be used for applications spanning the range of detailed mechanistic study to high-throughput screenings of more basic responses.

<table border="1">
	<tbody>
		<tr>
			<td>Reagents and Equipment</td>
			<td>Company</td>
			<td>Comments/Catalogue #</td>
		</tr>
		<tr>
			<td>Vessel Chamber</td>
			<td>Custom</td>
			<td>Dave Eick (MCW)</td>
		</tr>
		<tr>
			<td>Heated Circulating Water Bath</td>
			<td>PolyScience and Haake</td>
			<td>Haake DC 10</td>
		</tr>
		<tr>
			<td>Pipets</td>
			<td>Frederick Haer &amp; Co.</td>
			<td>Capillary Tubing 2.0 mm OD x 1.0 mm ID (27-33-1)</td>
		</tr>
		<tr>
			<td>Pressure Monitor</td>
			<td>World Precision Instruments</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Water Jacketed Reservoir</td>
			<td>Custom</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>External Light Source</td>
			<td>World Precision Instruments</td>
			<td>Novaflex</td>
		</tr>
		<tr>
			<td>Pipet Puller</td>
			<td>MicroData Instruments</td>
			<td>PMP102 Micropipet Puller</td>
		</tr>
		<tr>
			<td>Full complement of surgical tools</td>
			<td>Fine Science Tools</td>
			<td>Dumont</td>
		</tr>
		<tr>
			<td>Ultra Fine Forceps</td>
			<td>Fine Science Tools</td>
			<td>Inox #5</td>
		</tr>
		<tr>
			<td>Silk Suture Thread</td>
			<td>Ethilon</td>
			<td>#10-0 or 9-0</td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>Olympus</td>
			<td>Olympus SZ-11</td>
		</tr>
		<tr>
			<td>Analog Video Calipers</td>
			<td>Boeckeler</td>
			<td>Via Controller (Via-100)</td>
		</tr>
		<tr>
			<td>High Resolution Analog Camera</td>
			<td>Panasonic</td>
			<td>GP-MF 602</td>
		</tr>
		<tr>
			<td>Oxygen Tank</td>
			<td>Regional</td>
			<td>21% balance nitrogen and 5% CO2 balance nitrogen</td>
		</tr>
		<tr>
			<td>Tubing</td>
			<td>Tygon</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Drain Pump</td>
			<td>Cole Parmer Instrument Co.</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Modified Rat PSS</td>
			<td>See recipe below</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Van Breemen's Relaxant PSS</td>
			<td>See recipe below</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>
Table 1. A list of the major components of isolated microvessel station setup presented in the Figures.
<table border="1">
	<tbody>
		<tr>
			<td>Modified Rat PSS Recipe </td>
			<td>To make two liters of PSS </td>
			<td>20X Salt Stock (2L)</td>
			<td>20X Buffer Stock (2L) </td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>&nbsp;</td>
			<td>278.0 g</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>&nbsp;</td>
			<td>14.0 g</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MgSO4-7H2O</td>
			<td>&nbsp;</td>
			<td>11.5 g</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CaCl2-H2O</td>
			<td>&nbsp;</td>
			<td>9.4 g</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>80.8 g</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>0.4 g</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>0.28 g</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>1.98 g</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>20x Salt Stock</td>
			<td>100 mL</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>20x Buffer Stock</td>
			<td>100 mL</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Distilled Water</td>
			<td>1800 mL</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>
Table 2. Recipe for standard physiological salt solution (PSS) used in the isolated microvessel protocols.
Comments on Recipe: Make 2 L of Salt Stock and 2 L of Buffer Stock. These can be refrigerated when not being used, but shake them well and often before preparing PSS. The additional ingredients are added at the time of preparation of final PSS.
<table border="1">
	<tbody>
		<tr>
			<td>Van Breemen's Relaxant PSS </td>
			<td>To make 2 liters of PSS </td>
			<td>20X Salt Stock (1L)</td>
			<td>20X Buffer Stock (1L) </td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>&nbsp;</td>
			<td>107.4 g</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>&nbsp;</td>
			<td>7.0 g</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MgSO4-7H2O</td>
			<td>&nbsp;</td>
			<td>5.76 g</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MgCl2-6H2O</td>
			<td>&nbsp;</td>
			<td>81.32 g</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>40.4 g</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>0.2 g</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>15.22</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>0.28 g</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>1.98 g</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>20x Salt Stock</td>
			<td>100 mL</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>20x Buffer Stock</td>
			<td>100 mL</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Distilled Water</td>
			<td>1800 mL</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>
Table 3. Recipe for Van Breemen's relaxant physiological salt solution (PSS) used in the isolated microvessel protocols under conditions of zero active tone.
Comments on Recipe: Make 1 L of Salt Stock and 1 L of Buffer Stock. These can be refrigerated when not being used, but shake them well and often before preparing PSS. The additional ingredients are added at the time of preparation of final relaxant PSS.

the ex vivo isolated skeletal microvessel preparation for investigation of vascular reactivity

The isolated microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of factors that control vessel diameter, and thus, perfusion resistance1-5. This is a classic experimental preparation that was, in large measure, initially described by Uchida et al.15 several decades ago. This initial description provided the basis for the techniques that was extensively modified and enhanced, primarily in the laboratory of Dr. Brian Duling at the University of Virginia6-8, and we present a current approach in the following pages. This preparation will specifically refer to the gracilis arteriole in a rat as the microvessel of choice, but the basic preparation can readily be applied to vessels isolated from nearly any other tissue or organ across species9-13. Mechanical (i.e., dimensional) changes in the isolated microvessels can easily be evaluated in response to a broad array of physiological (e.g., hypoxia, intravascular pressure, or shear) or pharmacological challenges, and can provide insight into mechanistic elements comprising integrated responses in an intact, although ex vivo, tissue. The significance of this method is that it allows for facile manipulation of the influences on the integrated regulation of microvessel diameter, while also allowing for the control of many of the contributions from other sources, including intravascular pressure (myogenic), autonomic innervation, hemodynamic (e.g., shear stress), endothelial dependent or independent stimuli, hormonal, and parenchymal influences, to provide a partial list. Under appropriate experimental conditions and with appropriate goals, this can serve as an advantage over in vivo or in situ tissue/organ preparations, which do not readily allow for the facile control of broader systemic variables. 
The major limitation of this preparation is essentially the consequence of its strengths. By definition, the behavior of these vessels is being studied under conditions where many of the most significant contributors to the regulation of vascular resistance have been removed, including neural, humoral, metabolic, etc. As such, the investigator is cautioned to avoid over-interpretation and extrapolation of the data that are collected utilizing this preparation. The other significant area of concern with regard to this preparation is that it can be very easy to damage cellular components such as the endothelial lining or the vascular smooth muscle, such that variable source of error can be introduced. It is strongly recommended that the individual investigator utilize appropriate measurements to ensure the quality of the preparation, both at the initiation of the experiment and periodically throughout the course of a protocol.

Watch this Scientific Journal Video about The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity at JoVE.com

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

An ex vivo preparation is described for isolation of the largest gracilis muscle resistance arterioles for interrogation of both vascular responses to vasoactive stimuli and the assessment of basic structural properties via passive wall mechanics.

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

The isolated  microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of  factors that control vessel ...

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10.6K Views.  West Virginia University. An ex vivo preparation is described for isolation of the largest gracilis muscle resistance arterioles for interrogation of both vascular responses to vasoactive stimuli and the assessment of basic structural properties via passive wall mechanics.

Video: The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The contractile characteristics can be evaluated independently of external influences such as vascular tonus or neurohumoral status. It depicts a scientific approach between single cell measurements with isolated cardiac myocytes and in vivo studies like echocardiography. Thus, papillary muscle preparations serve as an excellent model to study cardiac physiology/pathophysiology and can be used for investigations like the modulation by pharmacological agents or the exploration of transgenic animal models. Here, we describe a method of isolating the murine left anterior papillary muscle to investigate cardiac contractility in an organ bath setup. In contrast to a muscle strip preparation isolated from the ventricular wall, the papillary muscle can be prepared in toto without damaging the muscle tissue severely. The organ bath setup consists of several temperature-controlled, gassed and electrode-equipped organ bath chambers. The isolated papillary muscle is fixed in the organ bath chamber and electrically stimulated. The evoked twitch force is recorded using a pressure transducer and parameters such as twitch force amplitude and twitch kinetics are analyzed. Different experimental protocols can be performed to investigate the calcium- and frequency-dependent contractility as well as dose-response curves of contractile agents such as catecholamines or other pharmaceuticals. Additionally, pathologic conditions like acute ischemia can be simulated.

Murine left ventricular papillary muscle can be used to investigate cardiac contractility in vitro. This article describes in detail the isolation and experimental protocols to study cardiac contractile characteristics.

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

Retinal Cryo-sections, Whole-Mounts, and Hypotonic Isolated Vasculature Preparations for Immunohistochemical Visualization of Microvascular Pericytes

Retinal pericytes play an important role in many diseases of the eye. Immunohistochemical staining techniques of retinal vessels and microvascular pericytes are central to ophthalmological research. It is vital to choose an appropriate method of visualizing the microvascular pericytes. We describe retinal microvascular pericyte immunohistochemical staining in cryo-sections, whole-mounts, and hypotonic isolated vasculature using antibodies for platelet-derived growth factor receptor &#946; (PDGFR&#946;) and nerve/glial antigen 2 (NG2). This allows us to highlight advantages and shortcomings of each of the three tissue preparations for the visualization of the retinal microvascular pericytes. Cryo-sections provide transsectional visualization of all retinal layers but contain only a few occasional transverse cuts of the microvasculature. Whole-mount provides an overview of the entire retinal vasculature, but visualization of the microvasculature can be troublesome. Hypotonic isolation provides a method to visualize the entire retinal vasculature by the removal of neuronal cells, but this makes the tissue very fragile.

We demonstrate three different tissue preparation techniques for immunohistochemical visualization of rat retinal microvascular pericytes, i.e., cryo-sections, whole-mounts, and hypotonic isolation of the vascular network.

Retinal pericytes play an important role in many diseases of the eye. Immunohistochemical staining techniques of retinal vessels and microvascular ...

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the choroidal vessels supply the photoreceptors. Alterations in retinal perfusion contribute to numerous sight-threatening disorders, including diabetic retinopathy, glaucoma and retinal branch vein occlusions. Understanding the molecular mechanisms involved in the control of blood flow through the retina and how these are altered during ocular disease could lead to the identification of new targets for the treatment of these conditions. Retinal arterioles are the main resistance vessels of the retina, and consequently, play a key role in regulating retinal hemodynamics through changes in luminal diameter. In recent years, we have developed methods for isolating arterioles from the rat retina which are suitable for a wide range of applications including cell physiology studies. This preparation has already begun to yield new insights into how blood flow is controlled in the retina and has allowed us to identify some of the key changes that occur during ocular disease. In this article, we describe methods for the isolation of rat retinal arterioles and include protocols for their use in patch-clamp electrophysiology, calcium imaging and pressure myography studies. These vessels are also amenable for use in PCR-, western blotting- and immunohistochemistry-based studies.

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

This manuscript describes a straightforward protocol for the isolation of arterioles from the rat retina that can be used in electrophysiological, calcium imaging and pressure myography studies.

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the ...

Assessment of Vascular Tone Responsiveness using Isolated Mesenteric Arteries with a Focus on Modulation by Perivascular Adipose Tissues

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic diseases. Endothelial dysfunction represents a major culprit for the reduced vasodilatation and enhanced vasoconstriction of arteries. Adipose (fat) tissues surrounding the arteries play important roles in the regulation of endothelium-dependent relaxation and/or contraction of the vascular smooth muscle cells. The cross-talks between the endothelium and perivascular adipose tissues can be assessed ex vivo using mounted blood vessels by a wire myography system. However, optimal settings should be established for arteries derived from animals of different species, ages, genetic backgrounds and/or pathophysiological conditions.

The protocol describes the use of wire myography to evaluate the transmural isometric tension of mesenteric arteries isolated from mice, with special consideration of the modulation by factors released from endothelial cells and perivascular adipose tissues.