The Wellcome Trust provided financial support (VIP Award to J.S.Y. and 074128 Research Fellowship to K.L.B.). Medical Research Council Studentship to R.J.A.
Leica_SP2_Stacker, an algorithm used to import TIFFs for &#x2018;reconstruction&#x2019; into a series, is based on Leica_tiff_sequence, a plugin for reading Leica SP2 TIFFs in ImageJ, developed by Tony Collins and used with his permission.
(<a href="http://www.uhnres.utoronto.ca/facilities/wcif/software/Plugins/LeicaTIFF.html">http://www.uhnres.utoronto.ca/facilities/wcif/software/Plugins/LeicaTIFF.html</a>)StackReg and TurboReg, algorithms used to correct for movement of tissue, are based on Th venaz and colleagues (1998)9.
Excel_writer.jar was written by Kurt De Vos (http://rsb.info.nih.gov/ij/plugins/excel-writer.html).
JNCT0.08JoVE.ijm, the particle detection algorithm, is based on an algorithm written by K.L. Brain and detailed previously5.

Part 1: Preparation of the Ca2+ indicator (Oregon Green 488 BAPTA-1 AM)
<ol>
<li>Oregon Green 488 BAPTA-1 AM comes in 500 &#x03BC;l vials as a small amount (50 &#x03BC;g) of powder. Add 40 &#x03BC;l Pluronic F-127 solution to the powder, shaking gently for 30 s, then add 460 &#x03BC;l PSS, and shake gently for 30 s. The final solution, 10 &#x03BC;M Oregon Green 488 BAPTA-1 AM, provides 4 x 125 &#x03BC;l aliquots. Avoid exposure to light and store at -20 C.</li>
</ol>
Part 2: Ca2+ loading
<ol>
<li>Remove the Ca2+ indicator (Oregon Green 488 BAPTA-1 AM) from the (-20 C) freezer and allow to thaw at room temperature.</li>
<li>Take 1 ml of PSS in a test-tube and place in water-bath. Bubble with 95% O2 / 5% CO2 at a rate of around 1 bubble appearing per second. The bubbler needs to be secured into the test-tube, e.g. with a bung or with Parafilm.</li>
<li>Dissect the tissue from the animal and, in a Sylgard-lined Petri dish, carefully remove connective tissue. Irrespective of smooth muscle organ type, PSS should be replaced every 10 mins and tissue should be washed well (e.g. by replacing the PSS three times) following dissection. For the vas deferens: consider cutting the organ length-ways to produce a sheet of tissue. For the urinary bladder: open longitudinally from the bladder neck (posterior) to the top of the dome (anterior). Prepare four strips, 6 &#x2013; 8 mm long and 1 &#x2013; 2 mm wide, along the dorsal surface of the detrusor, cutting in the direction of the dominant muscle bundles.</li>
<li>Place dissected tissue in the &#x2018;pre-warmed and bubbled&#x2019; PSS.</li>
<li>Add 125 &#x03BC;l of 10 &#x03BC;M Oregon Green 488 BAPTA-1 AM to the test-tube and give a little shake, by hand, to mix.</li>
<li>Resume bubbling and leave, covered with foil. The time is takes for sufficient tissue to take up the Ca2+ indicator varies with the size of tissue and thickness of the smooth muscle layer. For example: 70 mins (mouse detrusor, rat anococcygeus) to 120 mins (rat detrusor, mouse vas deferens).</li>
</ol>
Part 3: Preparing the &#x2018;Ca2+ loaded&#x2019; tissue for microscopy
Careful pinning of the tissue is necessary in order to minimize spontaneous movement of the tissue (a property of imaging of smooth muscle cells from relatively intact tissue) and to facilitate the location of a suitable site for imaging (as a flat piece of tissue may be surveyed in two dimensions, rather than three).
<ol>
<li>Rinse the tissue in bubbled PSS for at least 5 mins.</li>
<li>Mount in Sylgard-lined preparation dish, stretching the tissue (slightly) to form a flat sheet. Avoid using long &#x2018;pins&#x2019; that may scratch the objective; use instead short lengths of 25-50 &#x03BC;m diameter silver wire as &#x2018;pins&#x2019; and insert at an angle.</li>
<li>Position the preparation dish on the microscope stage, being careful to ensure that the PSS does not escape from the Sylgard-lined area of the dish (as this may lead to a large area being covered in PSS, when the superfusion begins).</li>
<li>Switch on the pump in order to superfuse the preparation with PSS. A rate of 100 ml per hour is often employed.</li>
<li>Switch on the heater unit.</li>
<li>Place the inflow and outflow tubes and temperature probe on the preparation dish (affixing each to the metal strip by the magnet on its base). The height of the tip of the outflow tube will determine the depth of the PSS. Be careful to ensure that the outflow is actually removing PSS (as it sometimes does not initially).</li>
<li>Set the temperature on the heater unit to reach the desired temperature, e.g. 33-35 C.</li>
<li>Allow the tissue to equilibrate for at least 10 mins.</li>
</ol>
Part 4: Selection of site to image
Exposure to excitation illumination will photobleach the indicator, so avoid using for more than a few seconds at a time.
<ol>
<li>With a low power objective (10x or 20x) use epifluorescence, exciting with a blue light, to view the smooth muscle and identify a suitable region to monitor. Try to pick a site based on: movement (which should be minimal) and the number of smooth muscle cells in the field. Bright &#x2018;structures&#x2019; may cause a high number of &#x2018;false positives&#x2019; in the image detection algorithm, so avoid sites with large numbers of (e.g.) interspersed epithelial cells or fibroblasts.</li>
<li>Switch to a higher power objective (40x).</li>
<li>Rotate the stage or the optical axis so the smooth muscle cell (s) lies horizontal (i.e. parallel to the rapid scan axis).</li>
</ol>
Part 5: Confocal microscopy
The details of how the confocal microscope is &#x2018;set up&#x2019; are specific to each microscope type, but the key considerations are: speed (minimum of 5 Hz is required for most Ca2+ transients); resolution and size of field (both of which are traded-off with speed). Some of the following protocol is specific to a Leica SP2 upright confocal microscope. A typical protocol is: 100 frame series, acquired at 5 Hz (256 x 256 pixels; approx. 100 x 100 &#x03BC;m) or 13.5 Hz (256 x 64 pixels; approx. 100 x 25 &#x03BC;m). The scanning head set to move bi-directionally (to maximize acquisition speed) and acquire at 1000 Hz per line.
<ol>
<li>Close the pinhole to one &#x2018;airy disk&#x2019;.</li>
<li>Set the laser (488 nm) power between 25 and 35% on the AOTF; this value is a trade-off between brightness and photobleaching. (The latter being a particular problem during long recordings.)</li>
<li>Acquire six series per site, and four - six sites per &#x2018;treatment&#x2019;. It&#x2019;s common to aim to monitor the same six sites pre-drug (i.e. &#x2018;control&#x2019;) and post-drug, although significant photobleaching may cause the experimenter to deviate from this.</li>
<li>It is essential with Ca2+ imaging that &#x2018;time controls&#x2019; are performed.</li>
</ol>
Part 6: Preparation for analysis
<ol>
<li>Download and install Image J from: http://rsb.info.nih.gov/ij/download.html. Download the platform-specific version, and then update to the latest version by downloading the latest imageJ.jar file from http://rsb.info.nih.gov/ij/upgrade/ij.jar, replacing the old file. Apple Mac users: if it runs slowly, make sure you&#x2019;re using the latest version of the java virtual machine (JVM) available from Apple (http://www.apple.com/macosx/features/java/).</li>
<li>Download Excel_writer.jar from: http://rsb.info.nih.gov/ij/plugins/excel-writer.html and install into the Plugins [directory] > jar directory.</li>
<li>Copy the following files into the Plugins directory: Leica_SP2_Stacker.class; JNCT0.08Release.ijm. Install each of these scripts using the Plugins [Menu] > Macros > Install&#x2026; function of ImageJ.</li>
<li>We use a Leica SP2 confocal microscope, the software (Leica LCS) for which generates each series as a single viewable &#x2018;movie&#x2019; within the software, but saves as individual frames. To reconstruct frames into series: (i) Make sure that all the &#x2018;frames&#x2019; to be reconstructed are in a single folder (e.g. &#x2018;DataFolder > Tiffs&#x2019;). (ii) Select Plugins [Menu] > Macros > Leica_SP2_Stacker. Select the root directory (e.g. &#x2018;DataFolder&#x2019;). Select the desired folder from the drop-down list (e.g. &#x2018;Tiffs/&#x2019;). Select an output directory or generate a new one (e.g. &#x2018;DataFolder > Stacks&#x2019;). The script will then reconstruct series from individual frames.</li>
</ol>
Part 7: Correcting for movement
Although movement of the imaged site may be minimized with good pinning and evenly-stretched tissue, some smooth muscle organs exhibit spontaneous contractions that cannot be abolished by careful pinning alone. Here we describe the use of a freely available algorithm that aligns or matches frames within a series.
<ol>
<li>Download &#x2018;StackReg&#x2019; (http://bigwww.epfl.ch/thevenaz/stackreg/) and &#x2018;TurboReg&#x2019; (http://bigwww.epfl.ch/thevenaz/turboreg/).</li>
<li>Install each into the Plugins directory using the Plugins > Macros > Install&#x2026;</li>
<li>Load a stack to be processed (File > Open&#x2026;) and then move to a frame that clearly shows the field of interest. Other frames will be aligned to this reference frame.</li>
<li>Plugins > Stacks > StackReg. Try each of the different &#x2018;Transformations&#x2019; (i.e. Translation, Rigid Body, Scaled Rotation, Affine) to determine what is most effective. (Further details: http://bigwww.epfl.ch/thevenaz/stackreg/)</li>
</ol>
Part 8: Particle detection algorithm
For each &#x2018;site&#x2019; imaged, factors will vary that affect the detection of Ca2+ transients. By measuring certain properties of each site (&#x2018;particle parameters&#x2019; - detailed below), the process of detection is facilitated. Thus for each site, one calculates &#x2018;threshold for subtracted&#x2019;, &#x2018;threshold for ratio&#x2019; and &#x2018;cell edge threshold&#x2019;.
<ol>
<li>Plugins [Menu] > Macros > Install&#x2026; and select &#x2018;JNCT0.08Release.ijm&#x2019;.</li>
<li>Plugins [Menu] > Macros > load stack (shortcut: l). Select a series and calculate particle parameters:<ol style="list-style:none;">
<li>a. Threshold for subtracted (= background)</li>
</ol></li>
<li>Select a rectangular ROI over what you believe to be &#x2018;background&#x2019;.</li>
<li>Measure (shortcut: m). Note down MEAN.<ol style="list-style:none;">
<li>b. Calculating threshold for ratio</li>
</ol></li>
<li>Select a rectangular ROI around an optically flat area of the cell(s).</li>
<li>Measure (shortcut: m). Note down MEAN and STANDARD DEVIATION.</li>
<li>Repeat a further two times OR you can draw three boxes at once by pressing &#x2018;shift&#x2019;.</li>
<li>Calculate and note down threshold for ratio = (1+SD/mean) x 32, averaging values from the three replicates / sites.<ol style="list-style:none">
<li>c. Cell edge threshold</li>
</ol></li>
<li>Image > Stacks > Z project... Choose projection type: &#x2018;Average intensity&#x2019;</li>
<li>Image > Adjust > Threshold.</li>
<li>Select black and white.</li>
<li>Define &#x2018;under&#x2019; (upper slider) limit and note down the corresponding value (to the right of the slider). Only events occurring in the black regions will be counted.</li>
<li>Click &#x2018;reset&#x2019;.</li>
<li>Analyse stacks (shortcut: 2)</li>
<li>Select just one series at this stage, i.e. the &#x2018;first file&#x2019; and &#x2018;last file&#x2019; should be the same.</li>
<li>Enter values of &#x2018;threshold for subtracted&#x2019;, &#x2018;threshold for ratio&#x2019; and &#x2018;cell edge threshold&#x2019;. Leave other settings at their default values.</li>
<li>The algorithm will produce a dual-pane window in which the processed series is shown on the left and the original on the right. Carefully go through this to assess the number of false positives and the occasions on which events, visible by eye, have not been detected. To get a more accurate detection of Ca2+ transients it may be necessary to adjust slightly some of the &#x2018;particle parameters&#x2019;. While the process may seem a little &#x2018;hit and miss&#x2019; on the first attempt, one quickly gets a feel as to what parameters are key.</li>
<li>For each series, the algorithm produces an Excel file that details characteristics of the detected event (such as amplitude, area and position). &#x2018;False positives&#x2019; - identified by reviewing the image files that the algorithm outputs (e.g. step 8.17) - may be removed, by hand, from this Excel file.</li>
</ol>

<ol>
	<li>Haugland, R. P., Gregory, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indicators+for+Ca2+,+Mg2+,+Zn2++and+other+metal+ions.">Indicators for Ca2+, Mg2+, Zn2+ and other metal ions.</a> Handbook of fluorescent probes and research products. , 771-826 (2002).</li><li>Boland, B., Himpens, B., Gillis, J. M., Casteels, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+force+and+Ca2++in+anococcygeal+and+vas+deferens+smooth+muscle+cells+of+the+mouse.">Relationship between force and Ca2+ in anococcygeal and vas deferens smooth muscle cells of the mouse.</a> Pflugers Arch. 421, 43-51 (1992).</li><li>Hashitani, H., Fukuta, H., Takano, H., Klemm, M. F., Suzuki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+and+propagation+of+spontaneous+excitation+in+smooth+muscle+of+the+guinea-pig+urinary+bladder.">Origin and propagation of spontaneous excitation in smooth muscle of the guinea-pig urinary bladder.</a> J Physiol. 150, 273-286 (2001).</li><li>Heppner, T. J., Bonev, A. D., Nelson, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elementary+purinergic+Ca2++transients+evoked+by+nerve+stimulation+in+rat+urinary+bladder+smooth+muscle.">Elementary purinergic Ca2+ transients evoked by nerve stimulation in rat urinary bladder smooth muscle.</a> J. Physiol. 564, 201-212 (2005).</li><li>Brain, K. L., Jackson, V. M., Trout, S. J., Cunnane, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intermittent+ATP+release+from+nerve+terminals+elicits+focal+smooth+muscle+Ca2++transients+in+mouse+vas+deferens.">Intermittent ATP release from nerve terminals elicits focal smooth muscle Ca2+ transients in mouse vas deferens.</a> J. Physiol. 541, 849-862 (2002).</li><li>Fleichman, M., Schneider, T., Fetscher, C., Michel, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+transduction+underlying+carbachol-induced+contraction+of+rat+urinary+bladder+II.+Protein+kinases.">Signal transduction underlying carbachol-induced contraction of rat urinary bladder II. Protein kinases.</a> J. Pharmacol. Exp. Ther. 308, 54-58 (2004).</li><li>Young, J. S., Meng, E., Cunnane, T. C., Brain, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+purinergic+neurotransmission+in+the+mouse+urinary+bladder.">Spontaneous purinergic neurotransmission in the mouse urinary bladder.</a> J. Physiol. 586, 5743-5755 (2008).</li><li>Nelson, M. T., Cheng, H., Rubart, M., Santana, L. F., Bonev, A. D., Knot, H. J., Lederer, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relaxation+of+arterial+smooth+muscle+by+calcium+sparks.">Relaxation of arterial smooth muscle by calcium sparks.</a> Science. 270, 633-637 (1995).</li><li>Th&#233;venaz, P., Ruttimann, U. E., Unser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pyramid+approach+to+subpixel+registration+based+on+intensity.">A pyramid approach to subpixel registration based on intensity.</a> IEEE Trans. Image Process. 7, 27-41 (1998).</li></ol>

Efforts were made to minimize the number of animals used and their suffering; all experiments were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986 and European Communities Council Directive 86/09/EEC.

Choice of fluorescent Ca2+ indicator
Oregon Green 488 BAPTA-1 AM was chosen as the Ca2+ indicator because it (i) is bright compared to other indicators (e.g. Fluo-4 and Calcium Green)1; (ii) is sensitive to physiological changes in Ca2+ concentration with a Kd of similar magnitude1 to the intracellular Ca2+ concentration [Ca2+]i (e.g. of vas deferens2); (iii) loads many smooth muscle cells, enabling smooth muscle cell coupling to be monitored. However, Oregon Green 488 BAPTA-1 AM is a non-ratioable Ca2+ indicator, and as such cannot easily be used to determine the absolute [Ca2+]i. Furthermore, it may also buffer Ca2+ if present at high concentrations and becomes saturated with high amplitude changes in [Ca2+]i.
Inhibition of spontaneous contractility
The spontaneous contractility of smooth muscle organs may be reduced pharmacologically, such as by blocking the movement of Ca2+ through L-type Ca2+ channels (e.g. by: nifedipine3; diltiazem4). Note that these drugs will greatly reduce the amplitude of whole-cell Ca2+ flashes / transients.
Contraction of the vas deferens results from a combination of primarily purinergic and noradrenergic neurotransmission. Focal Ca2+ transients in this tissue are, however, insensitive to noradrenergic receptor blockade (e.g. &#x03B1;1 adrenoceptor, by prazosin5) so movement may be reduced without affecting focal Ca2+ transients.
Alternatively, contraction may be prevented &#x2018;downstream&#x2019; through the inhibition of myosin light chain kinase e.g. by wortmannin6.
Conclusions
Monitoring Ca2+ fluorescence at submicron resolution is a powerful technique to study the post-junctional effects of neurotransmitter release5,7 and release of Ca2+ from internal stores (e.g. &#x2018;sparks&#x2019;8). The analysis of Ca2+ transients using the methodology outlined here is cost-effective compared to commercially-available image analysis packages and allows tailor-made analysis through custom-written Java plugins for ImageJ.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Oregon Green 488 BAPTA-1 AM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>O6807</td>
<td>10 x 50 &mu;g</td>
</tr>
<tr>
<td>Pluronic F-127 solution</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>P3000MP</td>
<td>20% solution in DMSO</td>
</tr>
<tr>
<td>Leica SP2 upright confocal microscope</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Air table (&#x2018;vibration isolated workstation&#x2019;)</td>
<td>&nbsp;</td>
<td>Newport</td>
<td>M-VW-3046-OPT-012140</td>
<td>&nbsp;</td>
<tr>
<td>Sylgard 184 elastomer</td>
<td>&nbsp;</td>
<td>Dow Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

focal ca2+ transient detection in smooth muscle

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ from internal stores, and allows multiple cells to be monitored simultaneously to assess, for example, coupling in syncytial tissue. Subcellular Ca2+ transients are common in smooth muscle, yet are difficult to measure accurately because of the problems caused by their stochastic occurrence, over an often wide field of view, in an organ that it prone to contract. To overcome this problem, we've developed a series of imaging protocols and analysis routines to acquire and then analyse, in an automated fashion, the frequency, location and amplitude of such events. While this approach may be applied in other contexts, our own work involves the detection of local purinergic Ca2+ transients for locating transmitter release with submicron resolution. 
ATP is released as a cotransmitter from autonomic nerves, where it binds to P2X1 receptors on the smooth muscle of the detrusor and vas deferens. Ca2+ enters the smooth muscle, resulting in purinergic neuroeffector Ca2+ transients (NCTs). The focal Ca2+ transients allow the optical monitoring of neurotransmitter release in a manner that has many advantages over electrophysiology. Apart from the greatly improved spatial resolution, optical recording has the additional advantage of allowing the recording of transmitter release from many distinguishable sites simultaneously. Furthermore, the optical plane of focus is easier to maintain or correct during long recording series than is the repositioning of an intracellular sharp microelectrode. 
In summary, a method for imaging of Ca2+ fluorescence is outlined which details the preparation of tissue, and the acquisition and analysis of data. We outline the use of several scripts for the analysis of such Ca2+ transients.

Watch this Scientific Journal Video about Focal Ca2+ Transient Detection in Smooth Muscle at JoVE.com

Focal Ca2+ Transient Detection in Smooth Muscle

Details methods for high-resolution Ca2+ imaging of smooth muscle within isolated organs, including: preparation of the tissue, image acquisition and data analysis.

Focal Ca2+ Transient Detection in Smooth Muscle

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ ...

focal-ca2-transient-detection-in-smooth-muscle

Research

JoVE Journal

Biology

11.2K visualizações.  University of Oxford. Detalhes métodos de alta resolução Ca 2 + Imagem do músculo liso dentro órgãos isolados, incluindo: preparação do tecido de aquisição de imagem e análise de dados.

Vídeo: Focal Ca2+ Transient Detection in Smooth Muscle

Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded and their collection does not imply any added risk to the newborn or mother s health. Moreover no ethical concerns are raised. The UC is composed by one vein and two arteries from which both endothelial cells (ECs) 1 and smooth muscle cells (SMCs) 2, two of the main cellular components of blood vessels, can be isolated. In this project the SMCs were obtained after enzymatic treatment of the UC arteries accordingly the experimental procedure previously described by Jaffe et al 3. After cell isolation they were kept in t-flash with DMEM-F12 supplemented with 5% of fetal bovine serum and were cultured for several passages. Cells maintained their morphological and other phenotypic characteristics in the different generations. The aim of this study was to isolate smooth muscle cells in order to use them as models for future assays with constrictor drugs, isolate and structurally characterize L-type calcium channels, to study cellular and molecular aspects of the vascular function 4 and to use them in tissue engineering.

Isolation of Human Umbilical Arterial Smooth Muscle Cells HUASMC

The umbilical cords are used to isolate smooth muscle cells by different ways. In this work we used the enzymatic treatment to isolated smooth muscle cells.

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded ...

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography

Contraction or relaxation of smooth muscle cells within the walls of resistance arteries determines the artery diameter and thereby controls flow of blood through the vessel and contributes to systemic blood pressure. The contraction process is regulated primarily by cytosolic calcium concentration ([Ca2+]cyt), which is in turn controlled by a variety of ion transporters and channels. Ion channels are common intermediates in signal transduction pathways activated by vasoactive hormones to effect vasoconstriction or vasodilation. And ion channels are often targeted by therapeutic agents either intentionally (e.g. calcium channel blockers used to induce vasodilation and lower blood pressure) or unintentionally (e.g. to induce unwanted cardiovascular side effects).
Kv7 (KCNQ) voltage-activated potassium channels have recently been implicated as important physiological and therapeutic targets for regulation of smooth muscle contraction. To elucidate the specific roles of Kv7 channels in both physiological signal transduction and in the actions of therapeutic agents, we need to study how their activity is modulated at the cellular level as well as evaluate their contribution in the context of the intact artery.
The rat mesenteric arteries provide a useful model system. The arteries can be easily dissected, cleaned of connective tissue, and used to prepare isolated arterial myocytes for patch clamp electrophysiology, or cannulated and pressurized for measurements of vasoconstrictor/vasodilator responses under relatively physiological conditions. Here we describe the methods used for both types of measurements and provide some examples of how the experimental design can be integrated to provide a clearer understanding of the roles of these ion channels in the regulation of vascular tone.

Measurements of Kv7 (KCNQ) potassium channel activity in isolated arterial myocytes (using patch clamp electrophysiological techniques) in parallel with measurements of constrictor/dilator responses (using pressure myography) can reveal important information about the roles of Kv7 channels in vascular smooth muscle physiology and pharmacology.

Contraction  or relaxation of smooth muscle cells within the walls of resistance arteries  determines the artery diameter and thereby controls flow of ...

Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al.26 that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 &#956;M in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

We have developed a novel and reproducible technique to isolate primary cultures of pulmonary artery smooth muscle cells (PASMC) from mice as young as P7, thereby allowing better study of the signaling pathways involved in neonatal smooth muscle cell contraction and relaxation.

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling ...

Measurement of Smooth Muscle Function in the Isolated Tissue Bath-applications to Pharmacology Research

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues. While this technique has been implemented for over 100 years, the versatility, simplicity and reproducibility of this assay helps it to remain an indispensable tool for pharmacologists and physiologists alike. Tissue bath systems are available in a wide array of shapes and sizes, allowing a scientist to evaluate samples as small as murine mesenteric arteries and as large as porcine ileum &#8211; if not larger. Central to the isolated tissue bath assay is the ability to measure concentration-dependent changes to isometric contraction, and how the efficacy and potency of contractile agonists can be manipulated by increasing concentrations of antagonists or inhibitors. Even though the general principles remain relatively similar, recent technological advances allow even more versatility to the tissue bath assay by incorporating computer-based data recording and analysis software. This video will demonstrate the function of the isolated tissue bath to measure the isometric contraction of an isolated smooth muscle (in this case rat thoracic aorta rings), and share the types of knowledge that can be created with this technique. Included are detailed descriptions of aortic tissue dissection and preparation, placement of aortic rings in the tissue bath and proper tissue equilibration prior to experimentation, tests of tissue viability, experimental design and implementation, and data quantitation. Aorta will be connected to isometric force transducers, the data from which will be captured using a commercially available analog-to-digital converter and bridge amplifier specifically designed for use in these experiments. The accompanying software to this system will be used to visualize the experiment and analyze captured data.

This protocol describes the measurement of isometric contraction in an isolated smooth muscle preparation, using an isolated tissue bath system and computer-based data acquisition.

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues.  ...

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

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 from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

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

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell (VSMC) proliferation. Common murine models study small vessels such as the carotid and femoral arteries. Herein we describe an in vivo system in which both VSMC proliferation and endothelial barrier function can be simultaneously assessed in a large vessel. We studied the infrarenal aortic response to injury in C57BL/6 mice. The aorta was injured from the left renal vein to the aortic bifurcation by 30 transmural crushes of 5-seconds duration with a cotton-tipped applicator. Morphological changes were assessed with conventional histology. Aorta wall thickness was measured from the luminal surface to the adventitia. EdU integration and counter staining with DAPI and alpha-actin was used to demonstrate VSMC proliferation. Activation of ERK1/2, a known moderator of intimal hyperplasia formation, was determined by Western Blot analysis. The effect of inflammation was determined by immunohistochemistry for B-cells, T-cells, and macrophages. En face sections of endothelium were visualized with scanning electron microscopy (SEM). Endothelial barrier function was determined with Evans Blue staining. Transmural injury resulted in aortic wall thickening. This injury induced VSMC proliferation, most prominently at 3 days after injury, and early activation of ERK1/2 and decreased p27kip1 expression. Injury did not result in increased B-cells, T-cells, or macrophages infiltration in the vessel wall. Injury caused partial endothelial cell denudation and loss of cell-cell contact. Injury resulted in a significant loss of endothelial barrier function, which returned to baseline after seven days. The murine transmural blunt aortic injury model provides an efficient system to simultaneously study both VSMC proliferation and endothelial barrier function in a large vessel.

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Restenosis following cardiovascular procedures (bypass surgery, angioplasty, or stenting) is a significant problem reducing the durability of these procedures. An ideal therapy would inhibit smooth muscle cell (VSMC) proliferation while promoting regeneration of the endothelium. We describe a model for simultaneous assessment of VSMC proliferation and endothelial function in vivo.

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell ...

Measuring Proliferation of Vascular Smooth Muscle Cells Using Click Chemistry

The ability of a cell to proliferate is integral to the normal function of most cells, and dysregulation of proliferation is at the heart of many disease processes. For these reasons, measuring proliferation is a common tool used to assess cell function. Cell proliferation can be measured simply by counting; however, this is an indirect means of measuring proliferation. One common means of directly detecting cells preparing to divide is by incorporation of labeled nucleoside analogs. These include the radioactive nucleoside analog 3H-thymidine plus non-radioactive nucleoside analogs such as 5-bromo-2&#8217; deoxyuridine (BrdU) and 5-ethynyl-2&#8242;-deoxyuridine (EdU). Incorporation of EdU is detected by click chemistry, which has several advantages when compared to BrdU. In this report, we provide a protocol for measuring proliferation by the incorporation of EdU. This protocol includes options for various readouts, along with the advantages and disadvantages of each. We also discuss places where the protocol can be optimized or altered to meet the specific needs of the experiment planned. Finally, we touch on the ways that this basic protocol can be modified for measuring other cell metabolites.

Proliferation is a critical part of cellular function, and a common readout used to assess potential toxicity of new drugs. Measuring proliferation is, therefore, a frequently used assay in cell biology. Here we present a simple, versatile method of measuring proliferation that can be used in adherent and non-adherent cells.

The ability of a cell to proliferate is integral to the normal function of most cells, and dysregulation of proliferation is at the heart of many disease ...

Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro

Smooth muscle cells (SMCs) are the predominant cell type in the aortic media. Their contractile machinery is important for the transmission of force in the aorta and regulates vasoconstriction and vasodilation. Mutations in genes encoding for the SMC contractile apparatus proteins are associated with aortic diseases, such as thoracic aortic aneurysms. Measuring SMC contraction in vitro is challenging, especially in a high-throughput manner, which is essential for screening patient material. Currently available methods are not suitable for this purpose. This paper presents a novel method based on electric cell-substrate impedance sensing (ECIS). First, an explant protocol is described to isolate patient-specific human primary SMCs from aortic biopsies and patient-specific human primary dermal fibroblasts for the study of aortic aneurysms. Next, a detailed description of a new contraction method is given to measure the contractile response of these cells, including the subsequent analysis and suggestion for comparing different groups. This method can be used to study the contraction of adherent cells in the context of translational (cardiovascular) studies and patient and drug screening studies.

Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro

This paper describes an explant culture-based method for the isolation and culturing of primary, patient-specific human aortic smooth muscle cells and dermal fibroblasts. Furthermore, a novel method is presented for measuring cell contraction and subsequent analysis, which can be used to study patient-specific differences in these cells.

Smooth muscle cells (SMCs) are the predominant cell type in the aortic media. Their contractile machinery is important for the transmission of force in ...