This work was supported by the National Natural Science Foundation of China (Grant No. 82270352), Beijing Research Ward Construction Clinical Research Project (2022-YJXBF-04-03), National High Level Hospital Clinical Research Funding (2022-NHLHCRF-YSPY-01), Capital&#39;s Founds for Health Improvement and Research (No. 2022-1-4062), National Key Clinical Specialty Discipline Construction Program (Grant No. 2020-QTL-009), and Chinese Society of Cardiology&#39;s Foundation (No. CSCF2021B02).

All experiments were approved by the Animal Care and Use Committee of Peking University. 8-12 week-old male C57 mice were used for the present study. The animals were obtained from a commercial source (see Table of Materials).
1. Animal preparation
<ol>
	<li>Remove the hair from the precordial area using hair removal cream (see Table of Materials). Then, proceed with acquiring the ultrasound images following step 2.</li>
</ol>
2. Ultrasound imaging before IR surgery
<ol>
	<li>Anesthetize the mouse by placing it into the isoflurane-induction chamber (see Table of Materials) and initiate isoflurane delivery at a concentration of 1.5% with 1.5-2.0 L/min O2 flow.</li>
	<li>Position the anesthetized mouse in a supine position on the physiologic monitoring platform (ultrasound system&#39;s imaging platform, see Table of Materials). Apply a small amount of conductive gel to the copper sheet of the physiological monitoring table and secure the mouse paws with low-adhesive tape&#160;on it to obtain physiological information on ECG and respiration. Maintain the body temperature at 37-38 &#176;C using the built-in warming platform. 
	NOTE: Switch the control switch of the gas anesthesia machine from the chamber to the mask, and keep the anesthetic dose constant at 1.5% during this time. The heart rate of the mice at this point should be approximately 350-450 beats/min.</li>
	<li>Apply ophthalmic vet ointment to the eyes before imaging to prevent dryness.</li>
	<li>Perform transthoracic echocardiography using a 40 MHz linear probe9 (e.g., MS550 probe for mice) (Figure 1A). 
	NOTE: The ultrasound machine is equipped with the MS550 probe.</li>
	<li>Liberally apply ultrasound transmission gel (see Table of Materials) to ensure optimal image quality.</li>
	<li>Adjust the position of the probe and platform to form a modified parasternal long-axis (PLAX) view9 (Figure 1A) to examine the left anterior descending artery (LAD) in B-mode. Briefly, image the heart using a parasternal long-axis view with the platform laterally aligned and positioned at a small angle between the probe and platform.</li>
	<li>Locate the suitable heart section in B-mode and activate Color Doppler on the touchscreen (Figure 1B). The LAD (white arrow) can be identified within the left ventricular wall (Figure 1B). The real-time red color indicates the direction of blood flow (i.e., blood flow is towards the probe). 
	NOTE: By adjusting the x-axis, Color Doppler allows for the visualization of the complete LAD (from the aortic sinus to the distal branch site) within this image window.</li>
	<li>Slightly move the probe to find the right position and reduce the influence of pulmonary vein blood flow. 
	NOTE: To reduce the influence of pulmonary vein blood flow, slightly adjust the probe and differentiate the LAD from the pulmonary vein. The LAD runs in the left ventricular wall, while the pulmonary vein drains into the left atrium. Locate the LAD using Color Doppler, and use B-Mode to select the suitable section.</li>
	<li>After visualizing the LAD in Color Doppler mode, press the Pulse Wave (PW) button and switch to the PW-mode. Position the yellow-indicator line on the coronary artery, ensuring it is parallel to the direction of the flow. Then press the Save Clip button to record the baseline data.</li>
	<li>Increase the isoflurane concentration to 3% and wait for 30 s. Ensure that the flow velocity gradually increases with time. Press the Save Clip button frequently to capture the highest blood flow velocity. 
	NOTE: The isoflurane stimulus causes the heart to work harder, leading to possible movement of the heart and the LAD. Once the image in PW-mode is captured or cine-stored, click on the PW-mode to ensure the yellow-indicator line is on the LAD. If displacement occurs, adjust the yellow-indicator line slightly to the point of blood flow and continue recording. This process requires quick mastery and switching.</li>
	<li>After collecting coronary blood flow images, turn off the anesthetic and adjust the probe to the normal PLAX view. Wait for the mouse heart rate to increase slowly to about 500 bpm, then measure the mouse heart function by switching the probe to the parasternal short-axis section (PSAX).</li>
	<li>Upon completing the echocardiographic imaging, remove the animal from the platform and allow it to recover in its home cage.&#160;Remove the gel and allow the animal to dry to prevent hypothermia.</li>
	<li>Use the Peak Vel and LV function tools to obtain the peak diastolic velocities and heart systolic function from the images, respectively.</li>
	<li>Calculate CFR as the ratio of diastolic peak coronary flow velocity (CFV) at maximal flow to diastolic peak CFV at baseline.</li>
</ol>
3. Myocardial ischemia-reperfusion procedure
NOTE: The initial&#160;measurement was baseline, and then surgery was performed on the same animal.

<ol>
	<li>Anesthetize the mice with sodium pentobarbital (60 mg/kg) and administer the analgesic carprofen (5 mg/kg, subcutaneous injection). Use a prewarmed heating pad (37 &#176;C) throughout the surgical procedure. Ensure appropriate depth of anesthesia by the absence of a withdrawal reflex to toe pinching and blink reflexes. Place the mouse supine on the heating pad and apply&#160;ophthalmic ointment to the eyes to prevent them from drying out. Perform endotracheal intubation for mechanical ventilation: disinfect the area with 3 alternating scrubs of betadine or chlorhexidine and 70% alcohol before and after removing the hair of the neck.&#160;Locate the trachea and gently insert the catheter. Then, connect the mice to the ventilator (inspiratory tidal volume of 250 &#181;L at 120 breaths/min) (see Table of Materials).</li>
	<li>Disinfect the skin of the precordial area with 3 alternating scrubs of betadine or chlorhexidine and 70% alcohol. Expose the heart by performing a left thoracotomy10 at the fourth intercostal space.</li>
	<li>Ligate the left anterior descending artery (LAD) with a slipknot using a 5-0 silk suture (see Table of Materials), inserting 1-2 mm below the root of the left atrial appendage. Ensure that the stitching direction is parallel to the lower edge of the left atrial appendage. Visually confirm ischemia in all mice via local color darkening of the ischemic myocardium.</li>
	<li>Occlude the LAD artery for 30 min11. After 30 min, release the ligature and verify reperfusion by observing the reddening of the previously discolored area of heart muscle for 1-2 min.</li>
	<li>After the coronary occlusion/reperfusion, close the chest in layers and allow the mice to recover for about half an hour11.</li>
</ol>
4. Ultrasound imaging after IR surgery
<ol>
	<li>Measure the CFR again at 1 h, 3 h, 5 h, 8 h, 24 h, and 48 h after reperfusion, respectively, following the steps outlined in section 2. Compare these measurements to the CFR values obtained before the ischemia-reperfusion (IR) procedure.</li>
</ol>
5. Statistical analysis
<ol>
	<li>Perform statistical analyses using appropriate statistical analysis software (see Table of Materials). 
	NOTE: For comparison of continuous variables between paired samples, utilize paired t-tests if the data is normally distributed. For non-normally distributed data or when assumptions of normality are not met, use Friedman&#39;s ANOVA. Consider p &#60; 0.05 as statistically significant.</li>
</ol>

CFR Changes Post-Ischemia: Implications for Microvascular Dysfunction

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transthoracic+Doppler+echocardiography-noninvasive+diagnostic+window+for+coronary+flow+reserve+assessment.">Transthoracic Doppler echocardiography-noninvasive diagnostic window for coronary flow reserve assessment.</a> Cardiovascular ultrasound. 1, 4 (2003).</li><li>Picano, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+echocardiography:+a+historical+perspective.">Stress echocardiography: a historical perspective.</a> The American Journal of Medicine. 114 (2), 126-130 (2003).</li><li>Lim, D. S., Kim, Y. H., Lee, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+flow+reserve+is+reflective+of+myocardial+perfusion+status+in+acute+anterior+myocardial+infarction.">Coronary flow reserve is reflective of myocardial perfusion status in acute anterior myocardial infarction.</a> Catheterization and Cardiovascular Interventions: Official Journal of The Society For Cardiac Angiography &amp; Interventions. 51 (3), 281-286 (2000).</li><li>Feldman, L. J., Himbert, D., Juliard, J. 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A., Seligman, H., Howard, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+flow+reserve+and+cardiovascular+outcomes:+a+systematic+review+and+meta-analysis.">Coronary flow reserve and cardiovascular outcomes: a systematic review and meta-analysis.</a> European Heart Journal. 43 (16), 1582-1593 (2022).</li><li>Taqueti, V. R., Di Carli, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+microvascular+disease+pathogenic+mechanisms+and+therapeutic+options:+JACC+state-of-the-art+review.">Coronary microvascular disease pathogenic mechanisms and therapeutic options: JACC state-of-the-art review.</a> Journal of the American College of Cardiology. 72 (21), 2625-2641 (2018).</li><li>Wikstr&#246;m, J., Gr&#246;nros, J., Gan, L. 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The authors declare that they have no known competing commercial or financial relationships that could have appeared to influence the work reported in this paper.

This study presents a protocol that uses a modified parasternal long-axis view to dynamically assess CFR after ischemia-reperfusion. The major findings indicate a significant decrease in CFR in IR mice, with the most pronounced reduction observed at 1 h after reperfusion. However, cardiac function was not affected within 48 h.
CFR serves as an indicator of myocardial blood supply, offering a noninvasive approach to evaluate both coronary artery stenosis and coronary microvascular circulation. Clinical studies have demonstrated that lower CFR values are associated with worse prognoses14,15,16, and a CFR cut-off value of 1.75 has been established as optimal for risk stratification14. A recent meta-analysis further showed that the risk of death increases by 16% for every 0.1 unit decrease in CFR, indicating that CFR represents a continuum of risk, with lower levels predisposing patients to poorer clinical outcomes17. In this study, CFR showed a trend of increasing with the extension of reperfusion time but remained lower than before the procedure, emphasizing the importance of monitoring patients not only immediately after opening the culprit vessel through PCI but also at 48 h. Moreover, CFR serves as a measure of coronary microvascular dysfunction, integrating the hemodynamic effects of focal, diffuse, and small-vessel disease on myocardial tissue perfusion18. Therefore, CFR is a crucial noninvasive technique for diagnosing coronary microvascular diseases. As CFR on LAD is a strong and independent indicator of mortality6,7, this study aims to provide reference values for clinical decisions. Furthermore, the use of ultrasound machines can potentially reduce the need for coronary angiography in a healthcare cost-containment environment. By appropriately training and upgrading technology, risk stratification can be tailored to individual patient needs.
The modified PLAX view offers greater convenience and time savings for scientific researchers. Continued improvement of this technology will facilitate its broader application in other coronary microvascular diseases. Key steps in this protocol include visualizing the coronary artery and obtaining high-quality PW velocity images. Blood flow velocity gradually increases with an increasing anesthetic concentration, so continuous capture is recommended to avoid missing the maximum blood flow velocity. As increasing anesthetic concentration can alter the heart rate, briefly returning to Color Doppler mode during the measurement is advised to ensure consistent positioning before and after the measurement.
It is essential to acknowledge limitations, including limitations inherent to ultrasound measurement of CFR. Due to the curvature of the coronary artery, it is not possible to display the entire artery fully, leading to measurement in only one segment. Operators must aim to measure the beginning of the coronary artery to identify the point of maximum coronary blood flow velocity as accurately as possible. Additionally, CFR should ideally be determined based on changes in coronary blood flow volume, but this study uses blood flow velocity instead of blood flow volume, overlooking the effect of vessel diameter. However, previous studies have demonstrated a strong correlation between CFR and CFVR (coronary flow velocity reserve)19. Further research on coronary microvascular function may aid in understanding the complex alterations in ischemia and enhance our comprehension of coronary microvascular dysfunction.

Coronary heart disease stands as one of the leading causes of mortality worldwide1. Even when the culprit coronary artery is re-opened through primary percutaneous coronary intervention (PCI) after cardiac ischemia, coronary microvascular perfusion often remains diminished. Additionally, there is no guarantee of reperfusion in the downstream capillaries supplying the myocardium1. This phenomenon, known as the "no-reflow phenomenon," is linked to clinical progression and a poor prognosis. Consequently, achieving adequate microvascular reflow after successful reperfusion therapy becomes critical for myocardial salvage. Hence, early assessment of microvascular function following revascularization is crucial for clinical practices.
Various techniques, such as invasive intracoronary temperature/pressure guide wire to index of microcirculatory resistance (IMR) and hyperemic microvascular resistance (HMR), non-invasive cardiovascular magnetic resonance (CMR), single-photon emission computed tomography (SPECT), and Positron emission tomography (PET), can be employed to assess microvascular function2. However, these methods are either invasive or semi-invasive, expensive, and often not readily available, limiting their clinical utility. On the other hand, assessing coronary flow reserve (CFR) by transthoracic Doppler echocardiography offers a noninvasive, relatively simple, and cost-effective approach without exposing patients to ionizing radiation, as seen in other methods3.
Though previous studies have employed transthoracic Doppler echocardiography to measure CFR in mice and rats, challenges remain for operators to locate the complex angles between the platform, mice, and probe. This protocol overcomes this issue by providing an easier method to locate the left anterior descending artery (LAD) coronary artery and measure CFR quickly using the modified parasternal long-axis (PLAX) view.
Furthermore, CFR obtained in the infarct-related artery (IRA) distal to the culprit lesion has shown a strong correlation with perfusion status assessed by myocardial contrast echocardiography (MCE)4. It has also been identified as a predictive marker for viability and left ventricular (LV) function recovery after an acute myocardial infarction (AMI)5. Additionally, CFR has been established as a reliable marker for all-cause mortality and adverse cardiovascular outcomes6,7. Previous reports have described the use of echocardiography to assess CFR in rat models of myocardial infarction8. Nevertheless, the CFR in the early stage of ischemia-reperfusion has not been thoroughly studied. Therefore, this study provides a reference value for diagnosing microvascular dysfunction and assessing the therapeutic effect of ischemia-reperfusion through dynamic tests in IR mice in the early stage of reperfusion.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5-0 silk suture</td>
			<td>Ningbo MEDICAL Needle Co., Ltd.</td>
			<td>210322</td>
			<td></td>
		</tr>
		<tr>
			<td>C57 mice</td>
			<td>Peking University Health Science Center Department of Laboratory Animal Science</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Depilating agent</td>
			<td>Nair</td>
			<td>NAR-255-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrode gel</td>
			<td>Cofoe</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>High Frequency Ultrasound</td>
			<td>FUJIFILM VisualSonics, Inc.</td>
			<td>Vevo3100</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>REWARD</td>
			<td>R510-22-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Linear array high frequency transducer</td>
			<td>FUJIFILM VisualSonics, Inc.</td>
			<td>MS550</td>
			<td></td>
		</tr>
		<tr>
			<td>Rodent Ventilator</td>
			<td>Shanghai Alcott Biotech</td>
			<td>ALC-V9</td>
			<td></td>
		</tr>
		<tr>
			<td>Small Animal Anesthesia Machine</td>
			<td>REWARD</td>
			<td>R530</td>
			<td></td>
		</tr>
		<tr>
			<td>SPSS</td>
			<td>IBM Corp, Armonk, NY, USA</td>
			<td>version 23.0&#160;</td>
			<td>statistical analysis software</td>
		</tr>
		<tr>
			<td>Ultrasound Gel</td>
			<td>Cofoe</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vevo Lab Software</td>
			<td>FUJIFILM VisualSonics, Inc.</td>
			<td></td>
			<td>Verison 5.7.0</td>
		</tr>
	</tbody>
</table>

dynamic assessments of coronary flow reserve after myocardial ischemia reperfusion in mice

After cardiac ischemia, there is often insufficient myocardial perfusion, even if flow has been successfully and completely restored in an upstream artery. This phenomenon, known as the "no-reflow phenomenon," is attributed to coronary microvascular dysfunction and has been associated with poor clinical outcomes. In clinical practice, a reduction in coronary flow reserve (CFR) is frequently used as an indicator of coronary artery disease. CFR is defined as the ratio of the peak flow velocity induced by pharmacologic or metabolic factors to the resting flow velocity.
This protocol focused on assessing the dynamic changes in CFR before and after ischemia-reperfusion (IR) using pulse wave Doppler measurements. In this study, normal mice exhibited the ability to increase the peak velocity of coronary blood flow up to two times higher than the resting values under isoflurane stimulation. However, after ischemia-reperfusion, the CFR at 1 h significantly decreased compared to the pre-operation baseline. Over time, the CFR showed gradual recovery, but it remained below the normal level. Despite the preservation of systolic function, early detection of microvascular dysfunction is crucial, and establishing a practical guide could aid doctors in this task, while also facilitating the study of cardiovascular disease progression over time.

This study utilized male C57 mice (BW ~18-20 g) to characterize the dynamic change of CFR. The modified PLAX image was employed to assess the coronary artery flow characteristics of LAD (Figure 1A,B). CFR was calculated as the ratio of the maximal flow velocity during maximal vasodilation induced by 3% isoflurane to the maximal flow velocity at a baseline of 1.5% isoflurane concentration12,13. All measurements and calculations were repeated over three consecutive cardiac cycles and averaged, with representative results shown in Figure 2.
Before the IR surgery, baseline CFR was measured, and the mice had a normal CFR value near 2.14 &#177; 0.43. However, after the IR surgery, CFR significantly decreased at reperfusion 1 h compared to before IR surgery (1.18 &#177; 0.14 vs. 2.14 &#177; 0.43) (Figure 3A). This reduction indicated that microcirculation was not immediately restored even after opening the culprit vessel. As the reperfusion time was prolonged, the CFR values remained at a consistently low level, with values of 1.21 &#177; 0.20 at 3 h, 1.39 &#177; 0.33 at 5 h, 1.44 &#177; 0.38 at 8 h, 1.34 &#177; 0.36 at 24 h, and 1.48 &#177; 0.47 at 48 h after reperfusion, suggesting that hypoperfusion could persist for at least two days (Figure 3A). Furthermore, there was no statistical significance between CFR values at 1 h, 3 h, 5 h, 8 h, 24 h, and 48 h.
The mice were also monitored for cardiac function, and it was observed that there were no significant changes in left ventricular cardiac function when the CFR was significantly reduced in the mice (Figure 3B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65391/65391fig01.jpg" /> 
Figure 1: Modified parasternal long-axis view. (A) illustrates the positioning of the probe and platform while obtaining the coronary artery velocity of LAD. (B) shows the placement of the Pulse Wave velocity sensor on the LAD coronary artery. The blue color indicates movement away from the ultrasound probe, while the orange color indicates movement toward the ultrasound probe. <a href="https://www.jove.com/files/ftp_upload/65391/65391fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65391/65391fig02.jpg" /> 
Figure 2: Visualization and recording of the Pulse Wave Velocity imaging of LAD. (A) Pulse Wave image during the rest condition of the LAD coronary artery. (B) Maximum hyperemic coronary Pulse Wave image of the LAD coronary artery. The blue color illustrates movement away from the ultrasound probe, while the orange color illustrates movement toward the ultrasound probe. <a href="https://www.jove.com/files/ftp_upload/65391/65391fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65391/65391fig03.jpg" /> 
Figure 3: Measurement of coronary flow reserve and ejection fraction. (A) Statistical analysis of CFR in animals before ischemia and at 1 h, 3 h, 5 h, 8 h, 24 h, and 48 h after reperfusion, respectively (n = 9). (B) Assessment of ejection fraction from each group at each time point (n = 9). *p &#60; 0.05; data presented as mean &#177; SD, analyzed with paired t-tests and Friedman&#39;s ANOVA. <a href="https://www.jove.com/files/ftp_upload/65391/65391fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Dynamic Assessments of Coronary Flow Reserve after Myocardial Ischemia Reperfusion in Mice at JoVE.com

Author Spotlight: Improved Localization and Monitoring of Coronary Flow Reserve Using Modified PLAX View in Mice

The present protocol describes a modified parasternal long-axis view for swift and precise localization of the left anterior descending artery. This approach is designed to be simpler and more user-friendly while facilitating the examination of dynamic changes in coronary flow reserve following myocardial ischemia-reperfusion in mice.

Dynamic Assessments of Coronary Flow Reserve after Myocardial Ischemia Reperfusion in Mice

After cardiac ischemia, there is often insufficient myocardial perfusion, even if flow has been successfully and completely restored in an upstream ...

Our protocol describes a modified parasternal long axis view for swift and precise localization of the left anterior descending artery. Using this approach, we studied dynamic changes in coronary flow reserve following myocardial ischemia-reperfusion in mice. This study presents a modified PLAX view that saves time and offers convenience for scientific researchers.Additionally, the study found that CFR increased the base reperfusion time extension, but remained lower than pre-procedure, highlighting the importance of early monitoring after PCI. Our protocol simplifies locating complex angles between the platform, mice, probe, and murine CFR.

dynamic-assessments-coronary-flow-reserve-after-myocardial-ischemia

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Biology

861 Views.  Peking University China-Japan Friendship School of Clinical Medicine. Our protocol describes a modified parasternal long axis view for swift and precise localization of the left anterior descending artery. Using this approach, we studied dynamic changes in coronary flow reserve following myocardial ischemia-reperfusion in mice. This study presents a modified PLAX view that saves time and offers convenience for scientific researchers.Additionally, the study found that CFR increased the base reperfusion time extension, but remained lower than pre-procedure...

Video: Author Spotlight: Improved Localization and Monitoring of Coronary Flow Reserve Using Modified PLAX View in Mice

Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these acrylics do not bond to the bone, screws have been used as anchors. However, in small experimental animals like finches or mouse, their craniums are very fragile and can not successfully hold the anchors. In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium. Therefore, anchor screws are not required and surgical field can be reduced considerably. Past experiences show that the bonding effect maintains more than 2 months. Conventional resin's window of time when the materials are pliable and workable is a few minutes. However, the window of working time for these dental adhesives is significantly wider and adjustable.

In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium.

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

Environmentally Induced Heritable Changes in Flax

Some flax varieties respond to nutrient stress by modifying their genome and these modifications can be inherited through many generations. Also associated with these genomic changes are heritable phenotypic variations 1,2. The flax variety Stormont Cirrus (Pl) when grown under three different nutrient conditions can either remain inducible (under the control conditions), or become stably modified to either the large or small genotroph by growth under high or low nutrient conditions respectively. The lines resulting from the initial growth under each of these conditions appear to grow better when grown under the same conditions in subsequent generations, notably the Pl line grows best under the control treatment indicating that the plants growing under both the high and low nutrients are under stress. One of the genomic changes that are associated with the induction of heritable changes is the appearance of an insertion element (LIS-1) 3, 4 while the plants are growing under the nutrient stress. With respect to this insertion event, the flax variety Stormont Cirrus (Pl) when grown under three different nutrient conditions can either remain unchanged (under the control conditions), have the insertion appear in all the plants (under low nutrients) and have this transmitted to the next generation, or have the insertion (or parts of it) appear but not be transmitted through generations (under high nutrients) 4. The frequency of the appearance of this insertion indicates that it is under positive selection, which is also consistent with the growth response in subsequent generations. Leaves or meristems harvested at various stages of growth are used for DNA and RNA isolation. The RNA is used to identify variation in expression associated with the various growth environments and/or t he presence/absence of LIS-1. The isolated DNA is used to identify those plants in which the insertion has occurred.

Growing some flax varieties under nutrient stress results in genomic variation within a subset of the genome and phenotypic variation. A complex insertion at a specific site is associated with growth under various nutrient regimes and with changes in gene expression around this site.

Some flax varieties respond to nutrient stress by modifying their genome and these modifications can be inherited through many generations. Also ...

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae zygotes result from the fusion of haploid cells of distinct mating type (MATa, MATalpha) and give rise to corresponding stable diploids that successively generate as many as 20 diploid progeny as a result of their strikingly asymmetric mitotic divisions 2. Zygote formation is orchestrated by a complex sequence of events: In this process, soluble mating factors bind to cognate receptors, triggering receptor-mediated signaling cascades that facilitate interruption of the cell cycle and culminate in cell-cell fusion. Zygotes may be considered a model for progenitor or stem cell function.
Although much has been learned about the formation of zygotes and although zygotes have been used to investigate cell-molecular questions of general significance, almost all studies have made use of mating mixtures in which zygotes are intermixed with a majority population of haploid cells 3-8. Many aspects of the biochemistry of zygote formation and the continuing life of the zygote therefore remain uninvestigated.
Reports of purification of yeast zygotes describe protocols based on their sedimentation properties 9; however, this sedimentation-based procedure did not yield nearly 90% purity in our hands. Moreover, it has the disadvantage that cells are exposed to hypertonic sorbitol. We therefore have developed a versatile purification procedure. For this purpose, pairs of haploid cells expressing red or green fluorescent proteins were co-incubated to allow zygote formation, harvested at various times, and the resulting zygotes were purified using a flow cytometry-based sorting protocol. This technique provides a convenient visual assessment of purity and maturation. The average purity of the fraction is approximately 90%. According to the timing of harvest, zygotes of varying degrees of maturity can be recovered. The purified samples provide a convenient point of departure for "-omic" studies, for recovery of initial progeny, and for systematic investigation of this progenitor cell.

Flow Cytometry-based Purification of S. cerevisiae Zygotes

To purify zygotes of S. cerevisiae, haploid cells of opposite mating type were engineered to express red or green fluorescent proteins, co-incubated to allow zygote formation, and fractionated using a flow cytometry-based protocol. The highly-enriched fraction enables subsequent "-omic" studies, recovery of initial progeny, and systematic investigation of zygote morphogenesis.

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed under anoxia for 20 hr&#160;(simulated ischemia), and subsequently moved to a normal atmosphere with food (simulated reperfusion). This experimental paradigm results in increased death and neuronal damage, and techniques are presented to assess organism viability, alterations to the morphology of touch neuron processes, as well as touch sensitivity, which represents the behavioral output of neuronal function. Finally, a method for constructing hypoxic incubators using common kitchen storage containers is described. The addition of a mass flow control unit allows for alterations to be made to the gas mixture in the custom incubators, and a circulating water bath allows for both temperature control and makes it easy to identify leaks. This method provides a low cost alternative to commercially available units.

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

A protocol is described that uses anoxia/starvation in C. elegans to model ischemia/reperfusion. Functional outcomes include increased mortality, visible abnormalities in GFP-labeled neuronal processes, and impaired behavioral responses that require neuronal function.

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed ...

Floral-Dip Transformation of Flax (Linum usitatissimum) to Generate Transgenic Progenies with a High Transformation Rate

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be successful for many plant species. However, flax (Linum usitatissimum) transformation by floral-dip has not been reported. The goal of this protocol is to establish that Agrobacterium and the floral-dip method can be used to generate transgenic flax. We show that this technique is simple, inexpensive, efficient, and more importantly, gives a higher transformation rate than the current available methods of flax transformation.
In summary, inflorescences of flax were dipped in a solution of Agrobacterium carrying a binary vector plasmid (T-DNA fragment plus the Linum Insertion Sequence, LIS-1) for 1 - 2 min. The plants were laid flat on their side for 24 hr. Then, plants were maintained under normal growth conditions until the next treatment. The process of dipping was repeated 2 - 3 times, with approximately 10 - 14 day intervals between dipping. The T1 seeds were collected and germinated on soil. After approximately two weeks, treated progenies were tested by direct PCR; 2 - 3 leaves were used per plant plus the appropriate T-DNA primers. Positive transformants were selected and grown to maturity. The transformation rate was unexpectedly high, with 50 - 60% of the seeds from treated plants being positive transformants. This is a higher transformation rate than those reported for Arabidopsis thaliana and other plant species, using floral-dip transformation. It is also the highest, which has been reported so far, for flax transformation using other methods for transformation.

Floral-Dip Transformation of Flax Linum usitatissimum to Generate Transgenic Progenies with a High Transformation Rate

Here, we present a protocol to transform flax using Agrobacterium-mediated plant transformation via floral-dip. This protocol is simple to perform and inexpensive, yet yields a higher transformation rate than the current available methods for flax transformation.

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be ...

Isolation of Mouse Coronary Endothelial Cells

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new vascular formation. Endothelial cell dysfunction is implicated in the development and progression of many cardiovascular diseases including ischemic heart disease. To examine the function and characterization of coronary endothelial cells, cell isolation is the first step and it requires high purity and quantity to conduct subsequent experiments. This protocol describes an efficient method to isolate adult mouse coronary endothelial cells. The mouse heart is dissected and minced into small pieces. After the digestion of the heart using dispase and collagenase II, cells are washed&#160;and incubated with magnetic beads which are conjugated with anti-CD31 antibody. The beads with endothelial cells are washed several times and are ready to use in various applications, including imaging and molecular biological experiments. Efficient isolation yields approximately 104 cells per one heart with over 90% purity.

This protocol is prepared to share our method of isolating mouse coronary endothelial cells for the purpose of imaging or to conduct molecular biological experiments.

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...