The authors thank Alessandra Bergadano, DVM, PhD, for the dedicated supervision of long-term animal health. This work was supported by the research funds of the Research Council, Kantonsspital Aarau, Aarau, Switzerland, and the Swiss national science foundation SNF (310030_182450).

Veterinary support was performed according to institutional guidelines. Experiments were approved by the Local Ethics Committee, Switzerland (BE 60/19). The ARRIVE guidelines and 3R principles have been strictly followed6,7. Thirty-one male Lewis rats, 12 weeks old and weighing 492&#160;&#177; 8 g, were included. House all rats at a room temperature of 23 &#176;C and a 12 h light/dark cycle. Provide free access to water and pellets. Statistical analyses have been performed using the nonparametric Wilcoxon-Mann-Whitney U test. Probability values (p) of &#8804; 0.05 and/or &#8804; 0.01 were considered significant.
1. Preoperative phase-general preparation and anesthesiological aspects
<ol>
	<li>Randomize rats into either coil or stent treatment groups (Figure 2) via a web-based randomization system. Now, perform a preoperative clinical examination of all animals planned for surgery next to a quiet, aseptic operating room maintaining a room temperature of 23 &#177; 3 &#176;C. Analyze the animals&#39; behavior and inspect the mucous membranes and turgor as part of the preoperative clinical examination.</li>
	<li>Record the weight of each animal.</li>
	<li>Prior to surgery, incubate the arterial pouches from donor rats in 0.1% sodium dodecyl sulfate for 10 h at 37 &#176;C to obtain decellularized aneurysms8. Collect these pouches from donor animals a few days before the surgery.
	<ol>
		<li>Prepare the full length of the abdominal aorta with microscissors and forceps and apply 6-0 nonabsorbable ligatures at an interval of 3-4 mm.</li>
		<li>Directly generate vital aneurysms intraoperatively by a previously ligated arterial vessel pouch from the thoracic part of a donor animal9. Perform thoracotomy with scissors and surgical forceps at the indicated FU time point and ligate the vessel pouch at the desired length.</li>
	</ol>
	</li>
	<li>Directly implant the pouch into the recipient and harvest the aneurysm from the donor animal for further macroscopic analysis and histological processing.</li>
	<li>For anesthesia induction, place all rats in a clean box provided with oxygen (O2) until loss of consciousness after 5-10 min. Anesthetize rats with a subcutaneous (SC) injection of a mixture of fentanyl 0.005 mg/kg, medetomidine 0.15 mg/kg, and midazolam 2 mg/kg. 
	NOTE: This ensures a surgical plane of at least 45 min.</li>
	<li>Check the depth of anesthesia by the absence of the pedal withdrawal reflex.</li>
	<li>Place the rats in a supine position and shave the thoracoabdominal part with an electric shaver.</li>
	<li>Fixate the 4 paws of the rats with tape on a board, covered by a heating pad connected to an autoregulating rectal probe. Insert the rectal probe in the rat&#39;s anus to maintain the desired temperature of 37 &#176;C with the help of the heating pad.</li>
	<li>Now, install a sensor on the right hind leg connected to a computerized system for checking vital signs intraoperatively.</li>
	<li>Cover the rat&#39;s nose and mouth with a face mask. If requiring prolonged anesthesia, start isoflurane (1.0-2.0% titrated to effect in 100% O2).</li>
	<li>Disinfect the surgical field with povidone-iodine or alternating disinfectants and drape the surgical field in a sterile fashion.</li>
	<li>For perianesthetic care, apply a sterile ophthalmic lubricant to the eyes and cover them with an opaque foil mask to prevent drying and damage from the surgical lamp.</li>
	<li>Throughout surgery, supply oxygen continuously via the face mask, monitor the body temperature, and provide heat using a heating pad, maintaining normothermia.</li>
	<li>Monitor other vital signs continuously (pulse and breath distension, heart and breath rate, and oxygen saturation).</li>
</ol>
2. Operative phase - cell-tracer injection
NOTE: The detailed surgical approach in the Helsinki rat microsurgical sidewall aneurysm model9 and techniques for coil- and stent-implantation are described elsewhere8,10,11.
<ol>
	<li>Store the fluorescent lipophilic cell-tracer at &#8804; -20 &#176;C all the time, protected from light.</li>
	<li>Perform the surgery by preparing the rat aorta and caval vein, followed by the separation of both, as well as proximal and distal temporary clamping of the aorta. 
	NOTE: This technique has been described previously9.
	<ol>
		<li>Clamp the proximal and distal parts of the aorta with two temporary titan clips.</li>
	</ol>
	</li>
	<li>Put one microswab with purple padding each under the proximal and distal parts of the aorta for better visualization of the artery.</li>
	<li>Now, protect the abdomen with wet gauze.</li>
	<li>On the day of the operation, dissolve 2 &#181;L of the cell-tracer by pipetting in 1 mL of phosphate-buffered saline (PBS).</li>
	<li>Transfer the mixture to a 1 mL syringe fitted with a 27-1/2 G (0.4 x 13 mm) sterile cannula. 
	NOTE: Be careful to avoid light exposure while performing steps 2.5 and 2.6.</li>
	<li>Turn off the light in the operating room. While looking under a microscope, perform the one-point injection in the middle ventral part of the aorta using micro forceps and carefully inject 1 mL of heparinized 0.9% saline solution.</li>
	<li>Inject the cell-tracer carefully (Video 1) and immediately turn off the operating microscope as well. Again, protect the abdomen with wet gauze.</li>
	<li>Let the dye incubate for at least 15 min. After the incubation period, turn on the microscope and operating room lights.</li>
	<li>Perform the longitudinal arteriotomy and suturing of the aneurysm, as described elsewhere11.
	<ol>
		<li>Use microforceps and microscissors to perform the arteriotomy so that its length averages the diameter of the harvested aneurysm (step 1.3). To ensure the correct length, place the aneurysm beside the aorta before performing arteriotomy. Suture the aneurysm with 8-10 single-stitches using a nonabsorbable 10-0 suture, and carefully remove the temporary clamps-starting distally-under continuous irrigation with heparinized saline. Close the wound in a layered fashion. Of note, use a coil packing density of 1 cm. 
		NOTE: The technique of coil- or stent-implantation has been described elsewhere8,10.</li>
	</ol>
	</li>
</ol>
3. Postoperative phase-monitoring and analgetic care
<ol>
	<li>At the end of the surgery, reverse the anesthesia with an SC injection mixture of buprenorphine 0.05 mg/kg, atipamezol 0.75 mg/kg, and flumazenil 0.2 mg/kg. Let each operated animal recover in a clean cage until fully awake and warm, as needed, with a heating lamp.</li>
	<li>For 3 days, administer 1 mg/kg meloxicam (one injection or oral application per day) and buprenorphine (0.05 mg/kg four times each day) SC. Overnight, provide buprenorphine continuously in the drinking water with the same dosing: 6 mL&#160;buprenorphine 0.3 mg/mL, 360 mL&#160;drinking water, 10 mL&#160;of 5% glucose.</li>
	<li>In the immediate postoperative phase, house each animal in a single cage for protection. Regroup the animals after 24 h.</li>
	<li>If any rat shows distressed or aggressive behavior after SC injection, administer buprenorphine in the drinking water during the day.</li>
	<li>Provide soft feed on the cage floor to support feeding and recovery postoperatively.</li>
	<li>Observe and take care of all the animals according to the wellbeing and pain score sheet.</li>
	<li>Administer rescue analgesia SC (meloxicam 1 mg/kg and 0.05 mg/kg buprenorphine) when needed.</li>
</ol>

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<li>Li, Z. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endothelial+progenitor+cells+contribute+to+neointima+formation+in+rabbit+elastase-induced+aneurysm+after+flow+diverter+treatment%5BTitle%5D)+AND+%22CNS+Neuroscience+%26+Therapeutics%22%5BJournal%5D)">Endothelial progenitor cells contribute to neointima formation in rabbit elastase-induced aneurysm after flow diverter treatment.</a> CNS Neuroscience & Therapeutics. 19 (5), 352-357 (2013).</li>
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</ol>

The authors are solely responsible for the design and conduct of the presented study and declare no competing interests.

This study demonstrates that neointima formation is mediated via endothelial cells originating in the parent artery of the aneurysm complex but is supported by the recruitment of cells derived from the aneurysm wall in vital aneurysms. Nevertheless, the role of circulating progenitor cells in aneurysm healing remains controversial12,13. Overall, 31 male Lewis rats were included in this investigation; only 4 died prematurely (12.9% mortality).
<p class="jove_c...

Subarachnoid hemorrhage caused by the rupture of an intracranial aneurysm (IA) is a devastating neurosurgical condition associated with high morbidity and mortality1,2,3,4. In addition to microsurgical clipping, which provides direct endothelium-to-endothelium contact, endovascular devices have gained increasing importance over the past decades for treating ruptured and incidentally discovered IAs. The healing response in endovascularly treated IAs mainly depends on neointima formation and thrombus organization. Both are synergic processes, depending on cell migration from the adjacent vessel and the aneurysm wall.5 To date, the origin of endothelial cells in neointima formation of endovascular treated aneurysms remains unclear. There is an ongoing debate in the literature about the source from which neointima-forming cells are recruited.
By using a cell-tracer injection of CM-Dil dye (see the Table of Materials) in the abdominal aorta of rats, we aimed to analyze the role of endothelial cells, originating in the parent artery, in neointima formation at two different FU time points (day 7 and day 21) (Figure 1). An advantage of the model is the direct local cell-tracer incubation in vivo in a parent artery prior to aneurysm suture, allowing FU at later time points. In vivo injection techniques, such as cell-tracer incubation, have not been described in the literature. An advantage of this technique is the direct, one-point, intraoperative, in vivo injection, which makes the model robust and reproducible.

<table><tbody><tr><td>3-0 resorbable suture</td><td>Ethicon Inc., USA</td><td>VCP428G</td><td></td></tr><tr><td>4-0 non-absorbable suture</td><td>B. Braun, Germany</td><td>G0762563</td><td></td></tr><tr><td>6-0 non-absorbable suture</td><td>B. Braun, Germany</td><td>C0766070</td><td></td></tr><tr><td>9-0 non-absorbable suture</td><td>B. Braun, Germany</td><td>G1111140</td><td></td></tr><tr><td>Atipamezol</td><td>Arovet AG, Switzerland</td><td></td><td></td></tr><tr><td>Bandpass filter blue</td><td>Thorlabs</td><td>FD1B</td><td>any other</td></tr><tr><td>Bandpass filter green</td><td>Thorlabs</td><td>FGV9</td><td>any other</td></tr><tr><td>Bipolar forceps</td><td></td><td></td><td>any other</td></tr><tr><td>Bicycle spotlight</td><td></td><td></td><td>any other</td></tr><tr><td>Board (20 x 10 cm)</td><td></td><td></td><td>any other</td></tr><tr><td>Buprenorphine</td><td>Indivior, Switzerland</td><td>1014197</td><td></td></tr><tr><td>Camera</td><td>Sony NEX-5R, Sony, Tokyo, Japan</td><td></td><td></td></tr><tr><td>Cannula (27-1/2 G)</td><td></td><td></td><td>any other</td></tr><tr><td>Cell count software</td><td>Image-J version 1.52n, U.S. National Institutes of Health, Bethesda, Maryland, USA, https://imagej.nih.gov/ij/</td><td></td><td></td></tr><tr><td>CellTracker CM-Dil dye</td><td>ThermoFisher SCIENTIFIC, USA</td><td>C7000</td><td></td></tr><tr><td>Coil-Device</td><td>Styker, Kalamazoo, MI, USA</td><td></td><td>2 cm of Target 360 TM Ultra, 2-mm diameter</td></tr><tr><td>Desinfection</td><td></td><td></td><td>any other</td></tr><tr><td>Eye-lubricant</td><td></td><td></td><td>any other</td></tr><tr><td>Fentanyl</td><td>Sintetica, S.A., Switzerland</td><td>98683</td><td>any generic</td></tr><tr><td>Flumazenil</td><td>Labatec-Pharma, Switerzland</td><td></td><td></td></tr><tr><td>Fluoresceine</td><td>Curatis AG</td><td>5030376</td><td>any generic</td></tr><tr><td>Fluorescence microscope</td><td>Olympus BX51, Hamburg, Germany; Cell Sens Dimension Imaging software v1.8</td><td></td><td></td></tr><tr><td>Foil mask</td><td></td><td></td><td>any other</td></tr><tr><td>Glucose (5%)</td><td></td><td></td><td>any other</td></tr><tr><td>Heating pad</td><td>Homeothermic Control Unit, Harvard, Edenbridge, England</td><td></td><td>any other</td></tr><tr><td>Isotonic sodium chloride solution (0.9%)</td><td>Fresenius KABI</td><td>336769</td><td>any generic</td></tr><tr><td>Isoflurane</td><td></td><td></td><td>any generic</td></tr><tr><td>Longuettes</td><td></td><td></td><td>any other</td></tr><tr><td>Meloxicam</td><td>Boehringer Ingelheim</td><td>P7626406</td><td>any generic</td></tr><tr><td>Medetomidine</td><td>Virbac, Switzerland</td><td>QN05CM91</td><td></td></tr><tr><td>Micro needle holder</td><td></td><td></td><td>any other</td></tr><tr><td>Midazolam</td><td>Roche, Switzerland</td><td></td><td></td></tr><tr><td>Monitoring-system</td><td>Starr Life Sciences Corp., 333 Allegheny Ave, Oakmont, PA 15139, United States</td><td></td><td></td></tr><tr><td>Needle holder</td><td></td><td></td><td>any other</td></tr><tr><td>O2-Face mask</td><td></td><td></td><td>any other</td></tr><tr><td>Operation microscope</td><td>OPMI, Carl Zeiss AG, Oberkochen, Germany</td><td></td><td>any other</td></tr><tr><td>Oxygen</td><td></td><td></td><td>any other</td></tr><tr><td>Rectal temperature probe</td><td></td><td></td><td>any other</td></tr><tr><td>Scalpell</td><td>Swann-Morton</td><td>210</td><td>any other</td></tr><tr><td>Small animal shaver</td><td></td><td></td><td>any other</td></tr><tr><td>Smartphone</td><td></td><td></td><td>any other</td></tr><tr><td>Sodium dodecyl sulfate (0.1%)</td><td>Sigma-Aldrich</td><td>11667289001</td><td></td></tr><tr><td>Soft feed</td><td>Emeraid Omnivore</td><td></td><td>any generic</td></tr><tr><td>Soft tissue forceps</td><td></td><td></td><td>any other</td></tr><tr><td>Soft tissue spreader</td><td></td><td></td><td>any other</td></tr><tr><td>Stainless steel sponge bowls</td><td></td><td></td><td>any other</td></tr><tr><td>Stent-Device</td><td>Biotroni, B&amp;uuml;lach, Switzerland</td><td></td><td>modified magmaris device, AMS with polymer coating, 6-mm length, 2-mm diameter</td></tr><tr><td>Sterile micro swabs</td><td></td><td></td><td>any other</td></tr><tr><td>Straight and curved microforceps</td><td></td><td></td><td>any other</td></tr><tr><td>Straight and curved microscissors</td><td></td><td></td><td>any other</td></tr><tr><td>Straight and curved forceps</td><td></td><td></td><td>any other</td></tr><tr><td>Surgery drape</td><td></td><td></td><td>any other</td></tr><tr><td>Surgical scissors</td><td></td><td></td><td>any other</td></tr><tr><td>Syringes 1 mL, 2 mL, and 5 mL</td><td></td><td></td><td>any other</td></tr><tr><td>Tape</td><td></td><td></td><td>any other</td></tr><tr><td>Vascular clip applicator</td><td>B. Braun, Germany</td><td>FT495T</td><td></td></tr><tr><td>Yasargil titan standard clip (2x)</td><td>B. Braun Medical AG, Aesculap, Switzerland</td><td>FT242T</td><td>temporary</td></tr></tbody></table>

using a cell-tracer injection to investigate the origin of neointima-forming cells in a rat saccular side wall model

Microsurgical clipping creates a subsequent barrier of blood flow into intracranial aneurysms, whereas endovascular treatment relies on neointima and thrombus formation. The source of endothelial cells covering the endoluminal layer of the neointima remains unclear. Therefore, the aim of the present study was to investigate the origin of neointima-forming cells after cell-tracer injection in the already well-established Helsinki rat microsurgical sidewall aneurysm model.
Sidewall aneurysms were created by suturing decellularized or vital arterial pouches end-to-side to the aorta in male Lewis rats. Before arteriotomy with aneurysm suture, a cell-tracer injection containing CM-Dil dye was performed into the clamped aorta to label endothelial cells in the adjacent vessel and track their proliferation during follow-up (FU). Treatment followed by coiling (n = 16) or stenting (n = 15). At FU (7 days or 21 days), all rats underwent fluorescence angiography, followed by aneurysm harvesting and macroscopic and histological evaluation with immunohistological cell counts for specific regions of interest. 
None of the 31 aneurysms had ruptured upon follow-up. Four animals died prematurely. Macroscopically residual perfusion was observed in 75.0% coiled and 7.0% of stented rats. The amount of cell-tracer-positive cells was significantly elevated in decellularized stented compared to coiled aneurysms with respect to thrombus on day 7 (p = 0.01) and neointima on day 21 (p = 0.04). No significant differences were found in thrombus or neointima in vital aneurysms. 
These findings confirm worse healing patterns in coiled compared to stented aneurysms. Neointima formation seems particularly dependent on the parent artery in decellularized aneurysms, whereas it is supported by the recruitment from aneurysm wall cells in vital cell-rich walls. In terms of translation, stent treatment might be more appropriate for highly degenerated aneurysms, whereas coiling alone might be adequate for aneurysms with mostly healthy vessel walls.

A total of 31 animals were included in the laboratory setting: 27 rats were included in the final statistical analysis; 4 rats died prematurely (12.9% mortality rate). Intraoperatively, breath distension was significantly (p = 0.03) reduced in stent- (12.9 &#956;m &#177; 0.7) compared to coil-treated (13.5 &#956;m &#177; 0.6) rats. Fluorescence angiography was performed for every rat at the end of the final FU. Reperfusion was indicated in all 6 coil-treated animals, whereas reperfusion was observed in only 12.5...

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Using a Cell-Tracer Injection to Investigate the Origin of Neointima-Forming Cells in a Rat Saccular Side Wall Model

We performed a one-point, lipophilic cell-tracer injection to track endothelial cells, followed by an arteriotomy and suturing of sidewall aneurysms on the abdominal rat aorta. Neointima formation seemed dependent on the parent artery in decellularized aneurysms and was promoted by the recruitment from aneurysm wall cells in vital cell-rich walls.

Microsurgical clipping creates a subsequent barrier of blood flow into intracranial aneurysms, whereas endovascular treatment relies on neointima and ...

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Research

JoVE Journal

Neuroscience

2.2K Views.  Kantonsspital Aarau. Door gebruik te maken van een directe intra-aortale cel-tracer applicatie, kunnen we de rol van endotheelcellen afkomstig uit de bovenliggende slagader in neointimavorming op twee verschillende tijdstippen analyseren. Een groot voordeel van deze techniek is een directe, one-point, interpretatieve in-vivo injectie die dit model tot een robuuste en reproduceerbare techniek maakt. Incubeer vóór de operatie de arteriële zakjes van donorratten in 0,1% natriumdodecylodecylaat gedurende 10 uur bi...

Video: Using a Cell-Tracer Injection to Investigate the Origin of Neointima-Forming Cells in a Rat Saccular Side Wall Model

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic lamellae and smooth muscle cells (SMCs), and an outer layer of fibroblasts and extracellular matrix. In contrast to the widespread study of pathological models (e.g., atherosclerosis) in the adult aorta, much less is known about the embryonic and perinatal aorta. Here, we focus on SMCs and provide protocols for the analysis of the morphogenesis and pathogenesis of embryonic and perinatal aortic SMCs in normal development and disease. Specifically, the four protocols included are: i) in vivo embryonic fate mapping and clonal analysis; ii) explant embryonic aorta culture; iii) SMC isolation from the perinatal aorta; and iv) subcutaneous osmotic mini-pump placement in pregnant (or non-pregnant) mice. Thus, these approaches facilitate the investigation of the origin(s), fate, and clonal architecture of SMCs in the aorta in vivo. They allow for modulating embryonic aorta morphogenesis in utero by continuous exposure to pharmacological agents. In addition, isolated aortic tissue explants or aortic SMCs can be used to gain insights into the role of specific gene targets during fundamental processes such as muscularization, proliferation, and migration. These hypothesis-generating experiments on isolated SMCs and the explanted aorta can then be assessed in the in vivo context through pharmacological and genetic approaches.

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

Protocols for studying the embryonic and perinatal murine aorta using in vivo clonal analysis and fate mapping, aortic explants, and isolated smooth muscle cells are detailed here. These diverse approaches facilitate the investigation of the morphogenesis of the embryonic and perinatal aorta in normal development and the pathogenesis in disease.

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic ...

Developmental Biology

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

Medicine

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

Intramyocardial Cell Delivery: Observations in Murine Hearts

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue revascularization and cell survival. In addition, further observations revealed that specific stem cells, such as cardiac stem cells, mesenchymal stem cells and cardiospheres have the ability to integrate within the surrounding myocardium by differentiating into cardiomyocytes, smooth muscle cells and endothelial cells.
Here, we present the materials and methods to reliably deliver noncontractile cells into the left ventricular wall of immunodepleted mice. The salient steps of this microsurgical procedure involve anesthesia and analgesia injection, intratracheal intubation, incision to open the chest and expose the heart and delivery of cells by a sterile 30-gauge needle and a precision microliter syringe.
Tissue processing consisting of heart harvesting, embedding, sectioning and histological staining showed that intramyocardial cell injection produced a small damage in the epicardial area, as well as in the ventricular wall. Noncontractile cells were retained into the myocardial wall of immunocompromised mice and were surrounded by a layer of fibrotic tissue, likely to protect from cardiac pressure and mechanical load.

Intramyocardial cell delivery in murine models of cardiovascular diseases, such as hypertension or myocardial infarction, is widely used to test the therapeutic potential of different cell types in regenerative studies. Therefore, a detailed description and a clear visualization of this surgical procedure will help to define the limits and advantages of cardiovascular cell therapeutic analyses in small rodents.

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue ...

Vascular Balloon Injury and Intraluminal Administration in Rat Carotid Artery

The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and cellular mechanisms involved in vascular smooth muscle dedifferentiation, neointima formation and vascular remodeling. Male Sprague-Dawley rats are the most frequently employed animals for this model. Female rats are not preferred as female hormones are protective against vascular diseases and thus introduce a variation into this procedure. The left carotid is typically injured with the right carotid serving as a negative control. Left carotid injury is caused by the inflated balloon that denudes the endothelium and distends the vessel wall. Following injury, potential therapeutic strategies such as the use of pharmacological compounds and either gene or shRNA transfer can be evaluated. Typically for gene or shRNA transfer, the injured section of the vessel lumen is locally transduced for 30 min with viral particles encoding either a protein or shRNA for delivery and expression in the injured vessel wall. Neointimal thickening representing proliferative vascular smooth muscle cells usually peaks at 2 weeks after injury. Vessels are mostly harvested at this time point for cellular and molecular analysis of cell signaling pathways as well as gene and protein expression. Vessels can also be harvested at earlier time points to determine the onset of expression and/or activation of a specific protein or pathway, depending on the experimental aims intended. Vessels can be characterized and evaluated using histological staining, immunohistochemistry, protein/mRNA assays, and activity assays. The intact right carotid artery from the same animal is an ideal internal control. Injury-induced changes in molecular and cellular parameters can be evaluated by comparing the injured artery to the internal right control artery. Likewise, therapeutic modalities can be evaluated by comparing the injured and treated artery to the control injured only artery.

This protocol uses a balloon catheter to cause an intraluminal injury on the rat carotid artery and henceforth elicit neointimal hyperplasia. This is a well-established model for studying the mechanisms of vascular remodeling in response to injury. It is also widely used to determine the validity of potential therapeutic approaches.

The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and ...

Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease conditions1, 2. Network growth is commonly attributed to angiogenesis, defined as the growth of new vessels from pre-existing vessels. The angiogenic process is also directly linked to arteriogenesis, defined as the capillary acquisition of a perivascular cell coating and vessel enlargement. Needless to say, angiogenesis is complex and involves multiple players at the cellular and molecular level3. Understanding how a microvascular network grows requires identifying the spatial and temporal dynamics along the hierarchy of a network over the time course of angiogenesis. This information is critical for the development of therapies aimed at manipulating vessel growth.
The exteriorization model described in this article represents a simple, reproducible model for stimulating angiogenesis in the rat mesentery. It was adapted from wound-healing models in the rat mesentery4-7, and is an alternative to stimulate angiogenesis in the mesentery via i.p. injections of pro-angiogenic agents8, 9. The exteriorization model is attractive because it requires minimal surgical intervention and produces dramatic, reproducible increases in capillary sprouts, vascular area and vascular density over a relatively short time course in a tissue that allows for the two-dimensional visualization of entire microvascular networks down to single cell level. The stimulated growth reflects natural angiogenic responses in a physiological environment without interference of foreign angiogenic molecules. Using immunohistochemical labeling methods, this model has been proven extremely useful in identifying novel cellular events involved in angiogenesis. Investigators can readily correlate the angiogenic metrics during the time course of remodeling with time specific dynamics, such as cellular phenotypic changes or cellular interactions4, 5, 7, 10, 11.

This article describes a simple model for stimulating angiogenesis in the rat mesentery. The model produces dramatic increases in capillary sprouting, vascular area and vascular density over a relatively short time course in a tissue that allows en face visualization of entire microvascular networks down to the single cell level.

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease ...

Biology

A Novel Minimally Invasive Slow Injection Method for Reducing Tissue Injury

A Novel Cell Injection Method with Minimum Invasion

Directly injecting cells into tissues is a necessary process in cell administration and/or replacement therapy. The cell injection requires a sufficient amount of suspension solution to allow the cells to enter the tissue. The volume of the suspension solution affects the tissue, and this can cause major invasive injury as a result of the cell injection. This paper reports on a novel cell injection method, called slow injection, that aims to avoid this injury. However, pushing out the cells from the needle tip requires a sufficiently high injection speed according to Newton's law of shear force. To solve the above contradiction, a non-Newtonian fluid, such as gelatin solution, was used as the cell suspension solution in this work. Gelatins solution have temperature sensitivity, as their form changes from gel to sol at approximately 20 &#176;C. Therefore, to maintain the cell suspension solution in the gel form, the syringe was kept cooled in this protocol; however, once the solution was injected into the body, the body temperature converted it to a sol. The interstitial tissue fluid flow can absorb excess solution. In this work, the slow injection technique allowed cardiomyocyte balls to enter the host myocardium and engraft without surrounding fibrosis. This study employed a slow injection method to inject purified and ball-formed neonatal rat cardiomyocytes into a remote area of myocardial infarction in the adult rat heart. At 2 months following the injection, the hearts of the transplanted groups showed significantly improved contractile function. Furthermore, histological analyses of the slow-injected hearts revealed seamless connections between the host and graft cardiomyocytes via intercalated disks containing gap junction connections. This method could contribute to next-generation cell therapies, particularly in cardiac regenerative medicine.

Author Spotlight: A Novel Cell Injection Method with Minimum Invasion  

This method eliminates any major invasion during cell injections caused by the cell suspension solution.

Directly injecting cells into tissues is a necessary process in cell administration and/or replacement therapy. The cell injection requires a sufficient ...

Bioengineering

Quantitative Analysis of Cellular Composition in Advanced Atherosclerotic Lesions of Smooth Muscle Cell Lineage-Tracing Mice

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel interventions have remained elusive. This is likely due, in part, to a reliance on preclinical prevention models investigating the effects of genetic manipulations or pharmacological treatments on atherosclerosis development rather than the established disease. Also, results of these studies are often confounding because of the use of superficial lesion analyses and a lack of characterization of lesion cell populations. To help overcome these translational hurdles, we propose an increased reliance on intervention models that employ investigation of changes in cellular composition at a single cell level by immunofluorescent staining and confocal microscopy. To this end, we describe a protocol for testing a putative therapeutic agent in a murine intervention model including a systematic approach for animal dissection, embedding, sectioning, staining, and quantification of brachiocephalic artery lesions. In addition, due to the phenotypic diversity of cells within late-stage atherosclerotic lesions, we describe the importance of using cell-specific, inducible lineage tracing mouse systems and how this can be leveraged for unbiased characterization of atherosclerotic lesion cell populations. Together, these strategies may assist vascular biologists to more accurately model therapeutic interventions and analyze atherosclerotic disease and will hopefully translate into a higher rate of success in clinical trials.

We propose a standardized protocol to characterize the cellular composition of late-stage murine atherosclerotic lesions including systematic methods of animal dissection, tissue embedding, sectioning, staining, and analysis of brachiocephalic arteries from atheroprone smooth muscle cell lineage tracing mice.

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel ...

Murine Cervical Aortic Transplantation Model using a Modified Non-Suture Cuff Technique

With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. However, only minor improvement in the long-term results of transplanted solid organs could be observed over the past decades. In this context, chronic allograft vasculopathy (CAV) still represents the leading cause of late organ failure in cardiac, renal and pulmonary transplantation.
Thus far, the underlying pathogenesis of CAV development remains unclear, explaining why effective treatment strategies are presently missing and emphasizing a need for relevant experimental models in order to study the underlying pathophysiology leading to CAV formation. The following protocol describes a murine heterotopic cervical aortic transplantation model using a modified non-suture cuff technique. In this technique, a segment of the thoracic aorta is interpositioned in the right common carotid artery. With the use of the non-suture cuff technique, an easy to learn and reproducible model can be established, minimizing the possible heterogeneity of sutured vascular micro anastomoses.

Here, we present a protocol of heterotopic aortic transplantation in mice using the non-suture cuff technique in a cervical murine model. This model can be used to study the underlying pathology of chronic allograft vasculopathy (CAV) and can help evaluate new therapeutic agents in order to prevent its formation.

With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. ...

Cell Labeling and Injection in Developing Embryonic Mouse Hearts

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect their in vivo potential. Preferably, these cells should be placed in a proper embryonic environment in order to acquire their definite phenotype. Furthermore, cell lineage tracing studies in the mouse after labeling cells with dyes or retroviral vectors has remained mostly limited to early stage mouse embryos with still poorly developed organs. To overcome these limitations, we designed standard and ultrasound-mediated microinjection protocols to inject various agents in targeted regions of the heart in mouse embryos at E9.5 and later stages of development. &#160;Embryonic explant or embryos are then cultured or left to further develop in utero. These agents include fluorescent dyes, virus, shRNAs, or stem cell-derived progenitor cells. Our approaches allow for preservation of the function of the organ while monitoring migration and fate of labeled and/or injected cells. These technologies can be extended to other organs and will be very helpful to address key biological questions in biology of development.

We describe a series of methods to inject dyes, DNA vectors, virus, and cells in order to monitor both cell fate and phenotype of endogenous and grafted cells derived from embryonic or pluripotent cells within mouse embryos at embryonic day (E)9.5 and later stages of development.

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect ...

Using a Cell-Tracer Injection to Investigate the Origin of Neointima-Forming Cells in a Rat Saccular Side Wall Model

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