The authors&#160;thank Drs. James Hawkins, Zu-Xi Yu and Xuan Qu from the National Heart, Lung, and Blood Institute (NHLBI) for their assistance with mouse surgery and preparation and staining of paraffin sections. The authors are&#160;grateful to the NIH Fellows Editorial Board for editorial assistance.

All mouse experiments were approved by the Animal Care and Use Program at the National Institutes of Health (NIH) with protocol number H0083R3.&#160;The NHLBI IACUC approved the protocol without analgesics.
1. Hypothermia Anesthesia in Neonatal Mice
<ol>
	<li>Sterilize sponges and surgical equipment in an autoclave before surgery. Prepare all the surgical materials and switch on a hot bead sterilizer 15-20 min&#160;in advance to reach 240&#176;C to 270&#176;C.</li>
	<li>Transfer all C57BL/6 pups (age P1) from their nursing mother to a clean mouse cage with fresh bedding and nesting materials. Once the pups are taken into a surgery room, perform the apical surgery promptly to minimize their time spent separated from the mother and to reduce the risk of maternal cannibalization.</li>
	<li>Put sponges on an ice bed and then place one pup on the sponge for ~3 min&#160;to achieve hypothermia anesthesia. Confirm anesthesia by observing apnea and akinesia and pinching a rear foot. Check the status of neonates frequently because too little time will not make the pup akinetic and apneic, and an excessive duration of anesthesia can lower the survival rate12.</li>
</ol>
2. Thoracotomy
<ol>
	<li>Transfer the pup from the ice bed onto a surgical benchtop area and use tape to immobilize its arms, legs, and tail in a supine position.</li>
	<li>Disinfect the chest using betadine and gently clean it using a 70% alcohol prep pad.</li>
	<li>Make a transverse skin incision along the forth intercostal area of the chest cavity using a Vannas Spring Scissor, and then blunt dissect the forth intercostal muscles to facilitate access to the heart. 
	NOTE: Survival rates improve when blood loss is minimized during the surgical process.</li>
</ol>
3. Surgical Amputation of the Heart Ventricular Apex
<ol>
	<li>By hand, gently apply pressure on the abdomen to exteriorize the apex of the heart. Absorb blood around the surgical area with sterile cotton-tipped applicators for clear visualization. For sham-operated control neonates, proceed directly to Step 4 (Suture and Recovery of Mice).</li>
	<li>Under a magnifying lamp and using iridectomy scissors, gently perform piecemeal resection of the left ventricle (LV) until the LV chamber is exposed. Be careful to minimize the resected portions of the LV. Approximately 15% resection is necessary to achieve optimum exposure of the LV chamber.</li>
	<li>Ensure that the heart returns to the chest cavity once the LV chamber is exposed.</li>
</ol>
4. Suture and Recovery of Mice
<ol>
	<li>Suture the ribs and muscles together to seal the chest cavity using sterile Proline 6-0 sutures, and then carefully close the skin incision site using skin glue.</li>
	<li>Warm the neonate under a heat lamp for ~3 min&#160;for recovery, and then clean the blood and glue traces using a 70% alcohol prep pad before reintroducing it to its littermates. Try to complete the whole surgical procedure within 10 min&#160;because minimizing the time spent separated from the mother improves pup survival after apical resection.</li>
	<li>Following each surgery, place the surgical tools in contact with the beads of the hot bead sterilizer for approximately 20 sec&#160;for complete sterilization. Allow the surgical tools to cool to RT before each surgery.</li>
	<li>After completing surgeries for all pups in a litter, mix the pups with the mother&#39;s bedding and excrement before returning them to the mother&#39;s nest. 
	NOTE: Generally, ICR/CD-1 mice are better foster mothers than C57BL/6, but even among nursing mothers of the same genetic background, fostering instincts vary. If changing a nursing mother is necessary to reduce mortality by maternal cannibalization, remove the P0 pups to a nursing mother and then perform apical resection at P1. Only perform either the sham operation or the apical resection in one litter of pups, because mixing sham and surgical pups could decrease the survival rates of the surgical group of pups6.</li>
</ol>
5. Post-Surgical Analysis
<ol>
	<li>One day after surgery, monitor the pups, ensure that there is no difference between sham and surgical groups, and count the number of pups to measure the survival rate. If the surgeries are performed properly, the survival rates should be similar and greater than 60% in both sham and surgical groups.</li>
	<li>Heart isolation and fixation.
	<ol>
		<li>Euthanize the pups at days 1 and 2 post-surgery via decapitation and day 21 post-surgery by CO2 with an assurance of death by cervical dislocation, then clean the chest using a 70% alcohol prep.</li>
		<li>Incise the midline skin and muscle of the chest and then open the chest.</li>
		<li>Excise the entire hearts from the thoracic cavity and fix the entire heart of each sample in 5 ml of 4% paraformaldehyde O/N at RT.</li>
	</ol>
	</li>
	<li>On the next day, transfer the samples to 70% ethanol. Samples can be stored up to one week before paraffin embedding.</li>
	<li>Slice 5-&#181;m-thick paraffin sections through the entire ventricle and perform standard hematoxylin and eosin (H&#38;E) and Masson&#39;s Trichrome staining to examine the regenerative response5, 7. Specifically, H&#38;E stain is used to examine muscle replacement and Masson&#39;s Trichrome stain is used to examine fibrotic responses5.</li>
	<li>Select the area of interest and set up at least three foci per sample manually using a slide scanner. Image and analyze the staining slides at 40&#215; magnification according to the manufacturer&#39;s instructions with default parameters.</li>
</ol>

<ol>
	<li>Lin, Z., Pu, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+cardiac+regeneration+and+repair.">Strategies for cardiac regeneration and repair.</a> Sci Transl Med. 6, 231-239 (2014).</li><li>Xin, M., Olson, E. N., Bassel-Duby, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mending+broken+hearts:+cardiac+development+as+a+basis+for+adult+heart+regeneration+and+repair.">Mending broken hearts: cardiac development as a basis for adult heart regeneration and repair.</a> Nat Rev Mol Cell Biol. 14, 529-541 (2013).</li><li>Mahmoud, A. I., Porrello, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turning+back+the+cardiac+regenerative+clock:+lessons+from+the+neonate.">Turning back the cardiac regenerative clock: lessons from the neonate.</a> Trends Cardiovasc Med. 22, 128-133 (2012).</li><li>Porrello, E. R., Olson, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+neonatal+blueprint+for+cardiac+regeneration.">A neonatal blueprint for cardiac regeneration.</a> Stem Cell Res. 13, 556-570 (2014).</li><li>Porrello, E. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+regenerative+potential+of+the+neonatal+mouse+heart.">Transient regenerative potential of the neonatal mouse heart.</a> Science. 331, 1078-1080 (2011).</li><li>Mahmoud, A. I., Porrello, E. R., Kimura, W., Olson, E. N., Sadek, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+models+for+cardiac+regeneration+in+neonatal+mice.">Surgical models for cardiac regeneration in neonatal mice.</a> Nat Protoc. 9, 305-311 (2014).</li><li>Allen, T. C., Prophet, E. B., Mills, B., Arrington, J. B., Sobin, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematoxylin+and+eosin.">Hematoxylin and eosin.</a> Laboratory methods in Histotechnology. , 53-58 (1992).</li><li>Andersen, D. C., Ganesalingam, S., Jensen, C. H., Sheikh, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do+neonatal+mouse+hearts+regenerate+following+heart+apex+resection?.">Do neonatal mouse hearts regenerate following heart apex resection?.</a> Stem cell reports. 2, 406-413 (2014).</li><li>Kotlikoff, M. I., Hesse, M., Fleischmann, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comment+on+'Do+neonatal+mouse+hearts+regenerate+following+heart+apex+resection?'.">Comment on 'Do neonatal mouse hearts regenerate following heart apex resection?'.</a> Stem cell reports. 3, 2 (2014).</li><li>Sadek, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-investigator+letter+on+reproducibility+of+neonatal+heart+regeneration+following+apical+resection.">Multi-investigator letter on reproducibility of neonatal heart regeneration following apical resection.</a> Stem cell reports. 3, 1 (2014).</li><li>Andersen, D. C., Jensen, C. H., Sheikh, S. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+to+Sadek+et+al.+and+Kotlikoff+et+al.">Response to Sadek et al. and Kotlikoff et al.</a> Stem cell reports. 3, 3-4 (2014).</li><li>Phifer, C. B., Terry, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+hypothermia+for+general+anesthesia+in+preweanling+rodents.">Use of hypothermia for general anesthesia in preweanling rodents.</a> Physiol Behav. 38, 887-890 (1986).</li><li>Laflamme, M. A., Murry, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerating+the+heart.">Regenerating the heart.</a> Nat Biotechnol. 23, 845-856 (2005).</li><li>Laflamme, M. A., Murry, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+regeneration.">Heart regeneration.</a> Nature. , 326-335 (2011).</li><li>Jesty, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=c-kit++precursors+support+postinfarction+myogenesis+in+the+neonatal,+but+not+adult,+heart.">c-kit+ precursors support postinfarction myogenesis in the neonatal, but not adult, heart.</a> Proc Natl Acad Sci U S A. 109, 13380-13385 (2012).</li><li>Gonzalez-Rosa, J. M., Martin, V., Peralta, M., Torres, M., Mercader, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+scar+formation+and+regression+during+heart+regeneration+after+cryoinjury+in+zebrafish.">Extensive scar formation and regression during heart regeneration after cryoinjury in zebrafish.</a> Development. 138, 1663-1674 (2011).</li></ol>

Publication fees for this article were sponsored by a gift from Fine Science Tools to Dr. Jian Hou.

Cardiac regeneration shows potential for the treatment and prevention of heart failure1, 2. Animal models are indispensable and play a critical role in understanding how cardiac regeneration occurs3-6. Many amphibians and fish regenerate heart tissue in response to injury, providing insight into our understanding of human cardiovascular disease13, 14. In terms of evolution, however, the pathophysiology should be more conserved between a mouse model and humans. Heart regeneration in mammalian animal models is a relatively new but burgeoning research field for the therapeutics of heart failure3-6. 
Recently, two surgical approaches, apical resection and myocardial infarction, have been established in the study of heart regeneration in neonatal mice6. Although apical resection provides a relatively easy and direct model for loss and/or injury of cardiomyocytes, the main limitation of apical resection compared to myocardial infarction is that this surgical method cannot be performed beyond P7 due to high mortality6. There are, however, a number of ways to reduce mortality with this method. For apical surgery on P7 pups, the pups are cooled to 16&#176;C for optimum hypothermia anesthesia. Generally, sterile conditions and surgical proficiency are critical for successful apical resection surgery. To improve survival, the surgery should be performed as quickly and gently as possible and a minimally invasive operation will reduce bleeding. Additionally, ICR/CD-1 mice are thought as good foster mothers and can effectively avoid of severe maternal cannibalization. For example, placing the P0 C57BL/6 pups with an ICR/CD-1 mother increases the survival rates of P1 pups with apical resection approximately from 60% to 80%ref.6. In addition, cryoinjury is an alternative procedure for study of cardiac regeneration. Compared to apical resection, cryoinjury model represents a different regenerative response and may be a more reproducible model15, 16. Thus far, a comparison of apical resection with other models warrants further investigations. 
Using phospho-histone H3 and 5-bromo-2-deoxyuridine staining, Porrello and colleagues showed that cardiomyocyte proliferation contributes to the heart regeneration of neonatal P1 mice after apical resection response. Moreover, no significant cardiac hypertrophy and increase of cell size have been observed in mice with apical resection relative to sham-operated controls. After apical resection of 7-day-old mice, there is no regenerative response for myocardium5. A more detailed time course analysis of morphology and cardiac fibrosis after apical resection has been demonstrated5. Of note, the usefulness of apical resection in neonatal mouse models for heart regeneration research has been questioned by Anderson and colleagues8. Although many independent groups have demonstrated the reproducibility of apical resection, variations in the details of surgical procedures and different amounts of resected myocardium might be responsible for confusing results10. Quantification of the genomic DNA in resected heart is helpful for control of minimal chamber exposition and reproducibility of this technique. Additional research will be needed to fully understand neomyogenesis and complete vs. incomplete heart regeneration in the apical resection model8-11.
The main aim of this study is to present the procedures of apical resection in a visualized way. This video of apical resection in the neonatal mouse will be helpful to increase the use of this method and teach it to heart regeneration researchers. In addition, it may promote the pursuit of better approaches for mammalian heart regeneration in animal models and facilitate the discovery and translation of the molecular and cellular mechanisms responsible for heart regeneration in humans.

Prolonged human life span leads to various aging- and lifestyle-related diseases, including heart failure, a leading cause of mortality. However, without replacement of lost or dysfunctional cardiac muscle cells, current therapeutics can only transiently improve cardiac function1, 2. Thus, it is necessary to discover and develop innovative strategies for cardiac regeneration and repair. The adult mammalian heart has limited regenerative potential. Studies from lower vertebrates, such as urodele amphibians and teleost fish, have provided unprecedented insights into the molecular and cellular mechanisms underlying heart regeneration3, 4. Recently, a neonatal mouse model of heart regeneration has emerged that might enable identification and characterization of more evolutionarily conserved pathophysiological events required for human heart regeneration5. 
Apical resection refers to the surgical removal of left ventricular apex. This procedure is restricted to 1- to 7-day-old (P1 to P7) mice due to its high lethality in older mice6. The cardiac regeneration process in neonatal mice after apical resection is expected to be as follows: (I) rapid and effective formation of a hematoma to seal the apex and prevent exsanguination; (II) cardiomyocyte regeneration and restoration of systolic function5, 7. Recent work has stimulated debate on the significance and efficiency of this model8-11. Thus, it is important to present the apical resection clearly and in detail. To this end, this protocol vividly and specifically describes a video of how I did apical resection based on a previous protocol6. 
Understanding the molecular mechanisms underlying cardiac regeneration is of importance for treatment of heart disease characterized by loss and/or injured cardiomyocytes, such as heart failure1, 2. Given the current and promising progress of apical resection in the research of cardiac regeneration, this study could promote the use of this technique and its uses in cardiac regeneration research.

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apical resection mouse model to study early mammalian heart regeneration

Cardiovascular disease plagues the whole world due to intensive lifestyle changes. Heart regeneration holds great promise for repairing and restoring cardiomyocytes lost due to injury and disease. In contrast to the robust cardiac regeneration of certain lower vertebrates, adult mammalian hearts typically show minimal capacity for heart regeneration and repair. However, recent studies have sparked considerable scientific interest with the finding that, between postnatal day 1 to 7 (P1 to P7), the neonatal mouse heart retains significant regenerative potential after apical resection (i.e., surgical amputation and exposure of left ventricular apex). One major controversy over this finding might be due to the diverse surgery-related procedures used in efforts to replicate or expand upon this important finding. These instructions dynamically present the materials and methodology for apical resection in a mouse model. The salient steps of this rodent survival surgery involve hypothermia anesthesia, thoracotomy, surgical amputation of heart ventricular apex, and suture and recovery of mice. The approach described could expand the application of the apical resection mouse model for cardiovascular research.

Mouse pups were euthanized 1, 2, and 21 days post-apical resection, and their hearts were collected for H&#38;E and Masson&#39;s Trichrome stain. Blue color in Masson&#39;s Trichrome stain indicates the deposition of epicardial extracellular matrix5. With successful apical resection, a blood clot is effectively formed to seal the LV one day post-apical resection, as shown in Figure 1A. A gradual resorption of the blood clot and early cardiac fibrosis is observed, and replacement by myocardial tissue occurs until complete regeneration of myocardium takes place 21 days after apical resection (Figure 1B and C). Morphological analysis shows no difference between resected and sham-operated hearts at 21 days after surgical operation (Figure 1C and D).
<img alt="Figure 1" src="/files/ftp_upload/53488/53488fig1.jpg" /> 
Figure 1. Representative H&#38;E and Masson&#39;s Trichrome stain. Ventricular myocardium of neonatal mouse hearts 1 (A), 2 (B), or 21 (C) days after apical resection and 21 days after sham operation (D). For each subfigure, the scale bar of the left panel is 1 mm and the scale bar of the right panel is 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53488/53488fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Apical Resection Mouse Model to Study Early Mammalian Heart Regeneration at JoVE.com

Apical Resection Mouse Model to Study Early Mammalian Heart Regeneration

A step-by-step video protocol of apical resection is demonstrated in this study. Apical resection is a recently highlighted surgical approach in mammalian heart regeneration research. This study may promote the application of apical resection as a standard methodology in research into the mechanism underlying heart regeneration.

Cardiovascular disease plagues the whole world due to intensive lifestyle changes. Heart regeneration holds great promise for repairing and restoring ...

apical-resection-mouse-model-to-study-early-mammalian-heart

Nanyang Technological University

Research

JoVE Journal

Medicine

11.2K Views.  National Institutes of Health. A step-by-step video protocol of apical resection is demonstrated in this study. Apical resection is a recently highlighted surgical approach in mammalian heart regeneration research. This study may promote the application of apical resection as a standard methodology in research into the mechanism underlying heart regeneration.

Video: Apical Resection Mouse Model to Study Early Mammalian Heart Regeneration

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

Renal Ischaemia Reperfusion Injury: A Mouse Model of Injury and Regeneration

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable during renal transplantation. Experimental models that accurately and reproducibly recapitulate renal IRI are crucial in dissecting the pathophysiology of AKI and the development of novel therapeutic agents. Presented here is a mouse model of renal IRI that results in reproducible AKI. This is achieved by a midline laparotomy approach for the surgery with one incision allowing both a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia of the left kidney. By careful monitoring of the clamp position and body temperature during the period of ischaemia this model achieves reproducible functional and structural injury. Mice sacrificed 24 hr&#160;following surgery demonstrate loss of renal function with elevation of the serum or plasma creatinine level as well as structural kidney damage with acute tubular necrosis evident. Renal function improves and the acute tissue injury resolves during the course of 7 days following renal IRI such that this model may be used to study renal regeneration. This model of renal IRI has been utilized to study the molecular and cellular pathophysiology of AKI as well as analysis of the subsequent renal regeneration.

The mouse model of renal ischaemia reperfusion injury described here comprises of a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia that results in acute kidney injury. This model uses a midline laparotomy approach with all steps performed via one incision.

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable ...

Ferric Chloride-induced Thrombosis Mouse Model on Carotid Artery and Mesentery Vessel

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric chloride injury is now a well-established technique to rapidly and accurately induce the formation of thrombi in exposed veins or artery of small and large diameter. This model has played a key role in the study of the pathophysiology of thrombosis, in the discovery and validation of novel antithrombotic drugs and in the understanding of the mechanism of action of these new agents. Here, the implementation of this technique on a mesenteric vessel and carotid artery in mice is presented. The method describes how to label circulating leukocytes and platelets with a fluorescent dye and to observe, by intravital microscopy on the exposed mesentery, their accumulation at the injured vessel wall which leads to the formation of a thrombus. On the carotid artery, the occlusion caused by the clot formation is measured by monitoring the blood flow with a Doppler probe.

The FeCl3 induced thrombosis model in mice is described herein. A method to monitor thrombus growth by intravital microscopy observation on a mesenteric vessel and by blood flow measurement in the carotid artery is presented.

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric ...

Vein Interposition Model: A Suitable Model to Study Bypass Graft Patency

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. Research models for bypass graft failure are essential for a better understanding of pathobiological and pathophysiological processes during graft patency loss. Large animal models, such as pigs or sheep, resemble human anatomical structures but require special facilities and equipment. This video describes a rat vein interposition model to investigate vein graft patency loss. Rats are inexpensive and easy to handle. Compared to mouse models, the convenient size of rats permits better operability and enables a sufficient amount of material to be obtained for further diverse analysis. In brief, the inferior epigastric vein of a donor rat is harvested and used to replace a segment of the femoral artery. Anastomosis is conducted via single stitches and sealed with fibrin glue. Graft patency can be monitored non-invasively using duplex sonography. Myointimal hyperplasia, which is the main cause for graft patency loss, develops progressively over time and can be calculated from histological cross sections.

This video demonstrates a model to study the development of myointimal hyperplasia after venous interposition surgery in rats.

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. ...

A Mouse Model of Orthopedic Surgery to Study Postoperative Cognitive Dysfunction and Tissue Regeneration

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and significantly diminish the outcome. Indeed, after routine orthopedic surgery to repair a fracture, as many as 50% of elderly patients suffer from neurologic complications like delirium. Also, the capacity to heal and regenerate tissue after surgery decreases with age, and can impact the quality of fracture repair and even osseous integration of implants. Thus, a better understanding of mechanisms that drive these age-dependent changes could provide strategic targets to minimize risk for such complications and optimize outcomes. Here, we introduce a clinically relevant mouse model of tibial fracture. The postoperative changes in these mice mimic some of the cognitive impairments commonly observed after routine orthopedic surgery in humans. Briefly, an incision is performed in the right hind limb under strictly aseptic conditions. Muscles are disassociated, and a 0.38-mm stainless steel pin is inserted into the upper crest of the tibia, inside the intramedullary canal. Osteotomy is then performed, and the wound is stapled. We have used this model to investigate the effects of surgical trauma on postoperative neuroinflammation and behavioral changes. By applying this fracture model in combination with parabiosis, a surgical model in which 2 mice are anastomosed, we have studied cells and secreted factors that systemically rejuvenate organ function and tissue regeneration after injury. By following our step-by-step protocol, these models can be reproduced with high fidelity, and can be adapted to interrogate many biologic pathways that are altered by surgical trauma.

This protocol describes a mouse model of orthopedic surgery that has been used to study mechanisms of postoperative neuroinflammation and behavioral changes, and when combined with parabiosis, to study tissue regeneration during aging.

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

A Cryoinjury Model to Study Myocardial Infarction in the Mouse

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we demonstrate a murine cryoinjury infarct model that generates precise infarct sizes with high reproducibility and replicability. In brief, after intubation and sternotomy of the animal, the heart is lifted from the thorax. The probe of a handheld liquid nitrogen delivery system is applied onto the myocardial wall to induce cryoinjury. Impaired ventricular function and electrical conduction can be monitored with echocardiography or optical mapping. Transmural myocardial remodeling of the infarcted area is characterized by collagen deposition and loss of cardiomyocytes. Compared to other models (e.g., LAD-ligation), this model utilizes a handheld liquid nitrogen delivery system to generate more uniform infarct sizes.

This article demonstrates a model to study cardiac remodeling after myocardial cryoinjury in mice.

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we ...

Blood Circuit Reconstruction in an Abdominal Mouse Heart Transplantation Model

The surgical technique of heterotopic abdominal heart transplantation in mice is a standard model for research in transplantation immunology. Here, the established technique for a modified blood circuit reconstruction in a heterotopic abdominal heart transplantation model is presented. This method uses the intrathoracic inferior vena cava (IIVC) instead of the pulmonary artery of the donor heart for the anastomosis to the inferior vena cava of the recipient. It is facilitating and improving success rates for abdominal heart transplantation in mice.

A novel technique for blood circuit reconstruction in a heterotopic abdominal mouse heart transplantation model is demonstrated.

The surgical technique of heterotopic abdominal heart transplantation in mice is a standard model for research in transplantation immunology. Here, the ...

An In Vivo Mouse Model of Total Intravenous Anesthesia During Cancer Resection Surgery

Anesthesia is a routine component of cancer care that is used for diagnostic and therapeutic procedures. The anesthetic technique has recently been implicated in impacting long-term cancer outcomes, possibly through modulation of adrenergic-inflammatory responses that impact cancer cell behavior and immune cell function. Emerging evidence suggests that propofol-based total intravenous anesthesia (TIVA) may be beneficial for long-term cancer outcomes when compared to inhaled volatile anesthesia. However, the available clinical findings are inconsistent. Preclinical studies that identify the underlying mechanisms involved are critically needed to guide the design of clinical studies that will expedite insight. Most preclinical models of anesthesia have been extrapolated from the use of anesthesia in in vivo research and are not optimally designed to study the impact of anesthesia itself as the primary endpoint. This paper describes a method for delivering propofol-TIVA anesthesia in a mouse model of breast cancer resection that replicates key aspects of clinical delivery in cancer patients. The model can be used to study mechanisms of action of anesthesia on cancer outcomes in diverse cancer types and can be extrapolated to other non-cancer areas of preclinical anesthesia research.

An In Vivo Mouse Model of Total Intravenous Anesthesia During Cancer Resection Surgery

This paper describes a method for modeling total intravenous anesthesia (TIVA) during cancer resection surgery in mice. The goal is to replicate key features of anesthesia delivery to patients with cancer. The method allows investigation of how anesthetic technique affects cancer recurrence after resection surgery.

Anesthesia is a routine component of cancer care that is used for diagnostic and therapeutic procedures. The anesthetic technique has recently been ...