Name: capturing the cardiac injury response of targeted cell populations via cleared heart three-dimensional imaging
Uploaded: 2020-03-17T13:00:00
Description: Watch this Scientific Journal Video about Capturing the Cardiac Injury Response of Targeted Cell Populations via Cleared Heart Three-Dimensional Imaging at JoVE.com

Funding for this project was provided by the UW School of Medicine and Public Health from the Wisconsin Partnership Program (A.I.M.), and an American Heart Association Career Development Award 19CDA34660169 (A.I.M.).

All experiments were conducted in accordance with the Guide for the Use and Care of Laboratory Animals and in compliance with the Institutional Animal Care and Use Committee in the School of Medicine and Public Health at the University of Wisconsin&#8211;Madison. All methods were performed on wild type C57BL/6J (B6) and transgenic mouse lines obtained from Jackson Laboratories.
1. Coronary Artery Occlusion (Myocardial Infarction) Induced via Ligation of the Left Anterior Descending Artery (LAD) ...

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Cell-cell interactions between cardiomyocytes and non-myocyte populations are a determining factor of whether the heart will undergo fibrosis or repair following injury. Discoveries have been made demonstrating that a variety of cell types, including nerves14, epicardial cells24, peritoneal macrophages25, arterioles12,13, and lymphatic endothelial cells26, all play an ...

The heart has long been considered to be a post-mitotic organ, yet recent evidence demonstrates that cardiomyocyte renewal occurs in the adult human heart at about 1% per year1. However, these low rates of cardiomyocyte turnover are insufficient to replenish the massive loss of tissue that occurs following injury. A heart that has suffered a myocardial infarction will lose around one billion cardiomyocytes, often serving as a prelude to heart failure and sudden cardiac death2,3. With over 26 million people affected by heart failure worldwide, there is an unmet need for therapeutics that can reverse the damages inflicted by heart disease4.
In order to bridge this gap in therapeutics, scientists have begun investigating evolutionarily conserved mechanisms that underlie endogenous regeneration following injury. One model for studying mammalian cardiac regeneration is the neonatal mouse. Within the week following birth, neonatal mice have a robust regenerative response following cardiac damage5. We have previously demonstrated that neonatal mice can regenerate their heart via cardiomyocyte proliferation following an apical resection5. Although this technique can evoke cardiac regeneration in the neonates, the surgery lacks clinical relevance to human heart injuries. In order to mimic a human injury in the neonatal mouse model, we have developed a technique to induce a myocardial infarction through a coronary artery occlusion6. This technique requires surgical ligation of the left anterior descending artery (LAD), which is responsible for delivering 40%&#8211;50% of the blood to the left ventricular myocardium6,7. Thus, the surgery results in an infarct that impacts a significant portion of the left ventricular wall. This damage to the myocardium will stimulate cardiomyocyte proliferation and heart regeneration in neonates5.
The coronary artery occlusion surgery provides a highly reproducible and directly translational method to uncover the inner workings of cardiac regeneration. The neonatal surgery parallels coronary artery atherosclerosis in the human heart, where accumulation of plaque within the inner walls of the arteries can cause an occlusion and subsequent myocardial infarction8. Due to a void in therapeutic treatments for heart failure patients, an occlusion in the LAD is associated with mortality rates reaching up to 26% within a year following injury9, and consequently has been termed the &#34;widow maker.&#34; Advancements in therapeutics require a model that accurately reflects the complex physiological and pathological effects of cardiac injury. Our surgical protocol for neonatal mouse cardiac injury provides a platform that allows researchers to investigate the molecular and cellular cues that signal mammalian heart regeneration after injury.
Recent research highlights the dynamic relationship between the extracellular environment and proliferating cardiomyocytes. For example, the postnatal regenerative window can be extended by decreasing the stiffness of the extracellular matrix surrounding the heart10. Biomaterials from the neonatal extracellular matrix can also promote heart regeneration in adult mammalian hearts following cardiac injury11. Also accompanying cardiomyocyte proliferation is an angiogenic response12,13; collateral artery formation unique to the regenerating heart of the neonatal mouse was shown to be essential for stimulating cardiac regeneration12. Moreover, our lab has demonstrated that nerve signaling regulates cardiomyocyte proliferation and heart regeneration via modulation of growth factor levels, as well as the inflammatory response following injury14. These findings emphasize the need to trace non-myocyte cell populations in response to cardiac injury. In order to accomplish this goal, we have taken advantage of the Cre-lox recombination system in transgenic mice lines to incorporate constitutive or conditional expression of fluorescent reporter proteins for lineage tracing. Furthermore, we can use advanced methods to determine clonal expansion patterning with the Rainbow mouse line, which relies on stochastic expression of the Cre-dependent, multi-color fluorescent reporters to determine the clonal expansion of targeted cell populations15. Employing lineage tracing with the neonatal coronary artery occlusion surgery is a powerful tool for dissecting the intricate cellular mechanisms of cardiac regeneration.
Tracking the lineage of fluorescently labeled cells with three-dimensional (3D) whole organ imaging is difficult to achieve using traditional sectioning and reconstruction technique &#8211; especially when cell populations are fragile, such as nerve fibers or blood vessels. While direct whole-mount imaging of the organ by optical sectioning can capture superficial cell populations, structures that reside deep within the tissue remain inaccessible. To circumvent these barriers, tissue clearing techniques have been developed to reduce the opacity of whole organ tissues. Recently, significant advances have been made to Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging compatible Tissue hYdrogel (CLARITY)&#8211;based methods, which clear fixed tissue via lipid extraction16. Steps are also taken to homogenize the refractive index and subsequently reduce light scattering while imaging17. One such method is active CLARITY, which expedites lipid decomposition by using electrophoresis to penetrate the detergent throughout the tissue18. Although effective, this tissue clearing method requires expensive equipment and can cause tissue damage, making the approach incompatible with fragile cell populations such as the cardiac nerves19. Thus, we employ the passive CLARITY approach, which relies on heat to gently facilitate detergent penetration, therefore aiding in the retention of intricate cell structures20,21.
Passive CLARITY is typically thought to be less efficient than active CLARITY18, as the technique is often accompanied by two major obstacles: the inability to clear the entire organ depth and the extensive amount of time required to clear adult tissues. Our passive CLARITY approach overcomes both of these barriers with an expeditated clearing process that is capable of fully clearing neonatal and adult heart tissue. Our passive CLARITY tissue clearing technique has reached an efficiency that permits the visualization of a variety of cardiac cell populations, including rare populations distributed throughout the adult heart. When the cleared heart is imaged with confocal microscopy, the architecture of cell-specific patterning during development, disease, and regeneration can be illuminated.

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			<td>1-thioglycerol</td>
			<td></td>
			<td></td>
			<td></td>
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			<td>6-0 Prolene Sutures</td>
			<td>Ethicon</td>
			<td>8889H</td>
			<td>Polypropylene Sutures</td>
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			<td>Acrylamide</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
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			<td>Boric acid</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Forceps</td>
			<td>Excelta</td>
			<td>16-050-146</td>
			<td>Half Curved, Serrated, 4 in</td>
		</tr>
		<tr>
			<td>Dressing Forceps</td>
			<td>Fisherbrand</td>
			<td>13-812-39</td>
			<td>Dissecting, 4.5 in</td>
		</tr>
		<tr>
			<td>Glass Vial</td>
			<td>Fisherbrand</td>
			<td>03-339-26A</td>
			<td>12 x 35 mm Vial with Cap</td>
		</tr>
		<tr>
			<td>Histodenz</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>Density gradient medium</td>
		</tr>
		<tr>
			<td>Iridectomy Scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-03</td>
			<td>2 mm Cutting Edge</td>
		</tr>
		<tr>
			<td>Large Dissecting Scissors</td>
			<td>Fisherbrand</td>
			<td>08-951-20</td>
			<td>Straight, 6 in</td>
		</tr>
		<tr>
			<td>Needle Holder</td>
			<td>Fisherbrand</td>
			<td>08-966</td>
			<td>Mayo-Hegar, 6 in</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sharp Forceps</td>
			<td>Sigma-Adrich</td>
			<td>Z168777</td>
			<td>Fine Tip, Straight, 4.25 in</td>
		</tr>
		<tr>
			<td>Small Dissecting Scissor</td>
			<td>Walter Stern Inc</td>
			<td>25870-002</td>
			<td>30 mm Cutting Edge</td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate (SDS)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Forceps</td>
			<td>Excelta</td>
			<td>16050133</td>
			<td>Medium Tissue, 1X2 Teeth</td>
		</tr>
		<tr>
			<td>VA-044</td>
			<td>Wako Chemicals</td>
			<td></td>
			<td>Water-soluble azo initiator</td>
		</tr>
	</tbody>
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capturing the cardiac injury response of targeted cell populations via cleared heart three-dimensional imaging

Cardiovascular disease outranks all other causes of death and is responsible for a staggering 31% of mortalities worldwide. This disease manifests in cardiac injury, primarily in the form of an acute myocardial infarction. With little resilience following injury, the once healthy cardiac tissue will be replaced by fibrous, non-contractile scar tissue and often be a prelude to heart failure. To identify novel treatment options in regenerative medicine, research has focused on vertebrates with innate regenerative capabilities. One such model organism is the neonatal mouse, which responds to cardiac injury with robust myocardial regeneration. In order to induce an injury in the neonatal mouse that is clinically relevant, we have developed a surgery to occlude the left anterior descending artery (LAD), mirroring a myocardial infarction triggered by atherosclerosis in the human heart. When matched with the technology to track changes both within cardiomyocytes and non-myocyte populations, this model provides us with a platform to identify the mechanisms that guide heart regeneration. Gaining insight into changes in cardiac cell populations following injury once relied heavily on methods such as tissue sectioning and histological examination, which are limited to two-dimensional analysis and often damage the tissue in the process. Moreover, these methods lack the ability to trace changes in cell lineages, instead providing merely a snapshot of the injury response. Here, we describe how technologically advanced methods in lineage tracing models, whole organ clearing, and three-dimensional (3D) whole-mount microscopy can be used to elucidate mechanisms of cardiac repair. With our protocol for neonatal mouse myocardial infarction surgery, tissue clearing, and 3D whole organ imaging, the complex pathways that induce cardiomyocyte proliferation can be unraveled, revealing novel therapeutic targets for cardiac regeneration.

Often the two most challenging steps are guiding the heart out of the chest cavity and ligating the LAD. To troubleshoot these steps, adjustments may be made in the placement of the initial puncture between the fourth intercostal muscles; if the puncture and blunt dissection are too close in proximity to the sternum, the heart may not be able to exit the chest cavity (Figure 1A).
Additionally, increased pressure on the left abdomen may be needed t...

Watch this Scientific Journal Video about Capturing the Cardiac Injury Response of Targeted Cell Populations via Cleared Heart Three-Dimensional Imaging at JoVE.com

Capturing the Cardiac Injury Response of Targeted Cell Populations via Cleared Heart Three-Dimensional Imaging

Cardiomyocyte proliferation following injury is a dynamic process that requires a symphony of extracellular cues from non-myocyte cell populations. Utilizing lineage tracing, passive CLARITY, and three-dimensional whole-mount confocal microscopy techniques, we can analyze the influence of a variety of cell types on cardiac repair and regeneration.

Cardiovascular disease outranks all other causes of death and is responsible for a staggering 31% of mortalities worldwide. This disease manifests in ...

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