We thank the Magdi Yacoub Institute (MYI) for supporting microscopy analysis and the projects involving cardiac repair, the Technicians and the Manager of our animal facility. This work has been supported by the British Heart Foundation (BHF), Project grant PG/10/019. MPS is supported by the MYI and BHF. TP is a BHF-Research Excellence Fellow. NR is an NH&amp;MRC Australia Fellow.

All animal studies were performed in compliance with international (Directive 2010/63/EU of the European Parliament) and national (UK Home Office, Act 1986) regulations. The procedures herein described are part of our plan of work under UK license authorities and have not been undertaken for the purpose of recording.
1. Preparation of Cells
This protocol describes the preparation of a specific cell line (Human Embryonic Kidney, HEK293 cells) for demonstration purposes. Cell-specific protocols must be employed for growing and eventually differentiating pluripotent or adult stem cells into specific cell lineages.
<ol>
	<li>Grow cells in DMEM media (high glucose, 4,500 mg/L) containing 1% sodium pyruvate, 2 mM glutamine, 1% antibiotic Pen/Strep and 10% Fetal Bovine Serum (FBS). One 10 cm plate should be normally split at 1:3 and media supplemented fresh every two days.</li>
	<li>Treat cells mildly with trypsin 1x&#160;and resuspend in DMEM medium as described in 1:1.</li>
	<li>Count cells in a hemocytometer chamber and collect 3 x 106 cells into a separate tube.</li>
	<li>Centrifuge in a swing bucket rotor at 100 g for 5 min.</li>
	<li>Discard the supernatant and wash the cell pellet with sterile phosphate buffer 2x.</li>
	<li>Resuspend in PBS 1x&#160;at a concentration of 106/50 ul for injection into the left ventricle of immunodepleted mice.</li>
</ol>
2. Presurgical Conditions
<ol>
	<li>Maintain immunodepleted mice (NOD.CB17-Prkdscid/JHliHSD) with sterile food pellets and water in a temperature-controlled isolator (22 &#176;C; Table 1) before each surgery.</li>
	<li>Perform surgery of immunodepleted mice under a laminar flow hood. Clean the working area with 70% Ethanol and autoclave surgical tools (forceps and scissors).</li>
	<li>Turn on heating pads for the surgery and for the recovery.&#160;Heating pads are important to block the decrease of body temperature occurring during and post-surgery.</li>
	<li>Place a clean and autoclaved cage on the recovery-heating pad.</li>
	<li>Transport the mice in their original cages from the isolator to the surgery room into a sterile autoclaved bag.</li>
	<li>Weigh the mouse and according to the body weight inject subcutaneously (s.c.) a solution containing medetomidine&#160;and ketamine hydrochloride&#160;diluted in 0.9%&#160; sterile saline solution to anesthetize the mouse, as well as to relax the musculature (medetomidine&#160;1 mg/kg; ketamine hydrochloride&#160;75 mg/kg).</li>
	<li>Remove the mouse from the cage when fully anesthetized (within 1-2 min, no toe-pinch reflex after stimulation)</li>
	<li>Apply hair removal cream on the left side of the chest and the throat area. After an appropriate amount of time (approximately 2-5 min), wipe away the loose hairs with a water-wet tissue.</li>
	<li>Place the mouse on the surgery panel in a supine position. From the surgeon&#39;s viewpoint the position is vertical, tail down-head up.</li>
	<li>Inject analgesic solution at 0.1-0.2 &#956;g/g diluted in sterile saline solution (buprenorphine) into the hind limb subcutaneously and cover the shaved area with the aseptic solution povidone iodine&#160;using a cotton tip applicator.</li>
	<li>Focus the microscope (2.5X objective) on the throat area.</li>
</ol>
3. Surgical Conditions
<ol>
	<li>With blunt scissors make a midline ventral skin incision of about 0.5-1 cm length slightly below the cricoid cartilage. Separate the skin from connective tissue to visualize the salivary glands.</li>
	<li>Split the salivary glands on their natural midline division by simultaneously pulling each part sideward with forcep and magnetic chest retractor and expose the trachea.</li>
	<li>Pull the tongue gently to the side to expose the larynx. Slide the intubation tube carefully into the trachea. The tube tip is visible through the displayed larynx and trachea. Importantly, if the tube is in, but not clearly visible, it is in the esophagus. Remove it and change the position of the tube tip.</li>
	<li>Connect the tube to the ventilation machine. Set the stroke volume at 200 &#956;l and&#160;150&#160;stroke/min. Secure the ventilation tubing on the surgery panel with a tape.</li>
	<li>With blunt scissors and forceps, make a vertical tail to head 1- to 1.5-cm-long skin incision over the left thorax area.</li>
	<li>Loosen the skin from the connective tissue/muscle layers and the major and minor pectoral muscles without making incisions.</li>
	<li>Perform thoracotomy between the third and fourth ribs as follows:
	<ol>
		<li>Perforate the intercostal muscle layer by pinching and scratching with small, rounded forceps.</li>
		<li>Once the intercostal muscle layer is perforated, place the chest retractor to maximally open the chest cavity. The upper and middle parts of the left ventricle (LV) with its overlying auricle (atrium) are now visible.</li>
	</ol>
	</li>
	<li>Prepare a precision syringe with cells. The syringe should be able to deliver an amount of volume of 10 ul for each injection. Cast a 30 G needle at the tip of the syringe.</li>
	<li>Fix with tape a small plastic cannula (from an introcan-W 20 G) on the needle, leaving exposed only 1 mm including the open tip of the needle. This will prevent the needle from perforating the entire left ventricular wall and injecting the cells into the left ventricle cavity.</li>
	<li>With the help of a 5X objective, visualize at this magnification the left ventricle and inject the cells into the left ventricular wall in 5 different locations (each injection will deliver 10 ul for a total of 106 cells injected). If the injection is successful, a white area and the absence of major backwash of the cells will be visible (if the injection was not successful, the animal would be excluded from functional analysis).</li>
	<li>Remove the retractor. Close the thoracic wall by enclosing the two separated ribs with two stitches of 6-0 silk suture.</li>
	<li>Moisturize the pectoral muscles and skin with a cotton tip drenched in PBS 1x&#160;and gently put the muscles back into the original position with the forceps.</li>
	<li>Close the skin with 3-4 stitches of 6-0 silk suture. Suture the throat incision with 2-3 stitches to close the skin.</li>
	<li>Inject atipamezole&#160;(1 mg/kg final concentration) to revert the anesthetic effects.</li>
	<li>As soon as the mouse starts breathing autonomously, take out the tube from the trachea and put the mouse on its right side. The mouse is expected to recover within minutes.</li>
	<li>When the mouse starts to turn and walk, place it in the warm recovery cage (small IVC cage with closed filtered top) for an hour.</li>
	<li>Leave the animals overnight in a controlled heated box (37 &#176;C).</li>
	<li>Fill a dish with water and pellet food&#160;to support recovery during the first 2 days after surgery.</li>
</ol>
4. Post-surgery Analyses
Ten hearts injected with cells and 10 control not injected hearts are analyzed histologically as follows:
<ol>
	<li>Perform analysis of the grafted cells 1 week after injection (Figures 1 and 3).
	<ol>
		<li>After euthanasia by overdose of isoflurane, perfuse the hearts with 20 ml of 4% paraformaldehyde (PFA) to fix the tissue. Dehydrate the fixed tissue in increasing percentages of ethanol (from 70-100%) and embed in paraffin blocks.</li>
		<li>Section the hearts in paraffin with a microtome at 5 &#956;m and stain with hematoxylin and eosin for morphological analysis.</li>
		<li>Visualize connective tissue by using Masson&#39;s Trichrome stain.</li>
		<li>Analyze the images under a slide scanner microscope (Figures 1A, 2A,&#160;3A, 3B, and 3C) and visualize the entire heart in short axis view using a digital slide scanner microscope (Figures 1B and 1C).</li>
	</ol>
	</li>
	<li>After the above analyses, deparaffinize the heart tissues in xylene, rehydrate by submerging specimens&#160;in decreasing percentages of ethanol (from 100% to water) and process for Scanning Electron Microscopy (SEM)&#160;as follows:
	<ol>
		<li>Incubate deparaffinized blocks with 1% tannic acid in 0.1 M phosphate buffer for 1 hr.</li>
		<li>Dehydrate specimens in increasing percentages of ethanol (from 25-100%).</li>
		<li>Dry samples with HMDS (hexamethyldisilazane).</li>
		<li>Mount the specimens on Stubbs with Acheson Silver Dag and coat them with gold-palladium.</li>
		<li>View samples in an analytical scanning electron microscope (Figure 2B).</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

In this manuscript, we have shown how to perform intramyocardial injection of cells in murine hearts. As a proof of this methodology, we have used HEK293 cells. It is important to emphasize that HEK293 cells are not used in any cell therapy study and therefore the findings of this manuscript are not appropriate for direct translation to a therapeutic approach. However, the fact that HEK293 cells are not contractile cells and do not transdifferentiate in other cell types focuses attention on technical aspects such as the ...

Various cell delivery protocols have been tested in murine and rat models of cardiovascular diseases with the aim of translating the efficiency, efficacy and safety of this experimental procedure in human patients. In small rodent hearts, intramyocardial cell delivery is the most feasible method of cell delivery1,2, whereas in the rat heart antegrade3 and retrograde4 intracoronary cell infusion can also be used. Both methods have limits and advantages. Cell delivery via the intracoronary route has theoretical advantages over direct intramuscular injection in promoting global cell dissemination3, but it also has the risk of causing coronary embolism3,5. Limitations on intramyocardial delivery are associated with mechanical injury, acute inflammation, and myocardial damage6,7. In humans, cells for cardiac repair are delivered by intramyocardial injection through an endocardial or surgical epicardial approach or by intracoronary arterial route8. Injection by transvascular routes is suited in patients with acute infarct and reperfused myocardium, but may not be possible in case of total occlusions or poor flow within the vessels of the effected territory9. Direct injection into the ventricular wall by transendocardial or transepicardial injection is technically feasible depending on the patient health status. Indeed, it has been shown that this technique is safe10,11, although for transepicardial injection an open chest surgery is necessary and for transendocardial approaches an electrophysiological mapping for each patient is required to differentiate sites of viable ischemic or scarred myocardium9.
Importantly, in cell therapy studies the choice of the best cell to be transplanted is still under investigation. Short-term analyses (4 weeks) showed that injection of cardiac stem cells defined as cardiospheres12 or side population cells from bone marrow13 induced cardiac functional recovery in murine14 and rat15 models of myocardial infarct by decreasing scar size and cell death. Allogeneic transplantation of cardiospheres in a rat myocardial infarct model without immunosuppression was found to be safe, promoted cardiac regeneration, and improved heart function through stimulation of endogenous repair mechanisms15. Lin-/c-kit+ adult progenitor cells in the heart were shown to be self-renewing, clonogenic, and multipotent in vitro and in vivo, and when injected into an ischemic rat heart reconstituted large portions of the injured myocardial wall16 and had the ability to form conductive and intermediate-sized coronary arteries17. These promising data fuelled phase I and II clinical trials in humans: injection of autologous and allogeneic mesenchymal stem cells (MSC)18, cardiospheres19, or c-Kit positive cardiac stem cells (CSC)20 in ischemic human hearts each showed beneficial effects in cardiac function in long term studies. Nevertheless, extensive long-term follow-up and retrospective meta-analysis demonstrated that stem cell therapy provides significant benefit to some patients, but not in others with a range of unpredictable outcomes21. It is possible that these limitations will necessitate the design of specific protocols of cell delivery for each individual and each disease.
In mouse and rat models, long-term studies revealed that cell injection did not further improve cardiac function (12 months). Indeed, grafts of human embryonic stem cell-derived cardiomyocytes (hESC-CM) were largely isolated from the host myocardium by a layer of fibrotic tissue22,23. Similar results have been observed after intramyocardial transplantation of skeletal myoblasts into the heart of infarcted mice24. Furthermore, the long-term ability of allogeneic MSCs to preserve function in the infarcted heart has been limited by transition from an immunoprivileged to an immunogenic state after differentiation25.
Taking into consideration the challenges and prospects outlined above, we show here how to deliver cells by intramyocardial injection in mice. We observe that cells without cardiomyocyte contractile properties are not connected with the host myocardium and form a cohesive mass with a thin fibrotic barrier. Although in some cases this outcome may be advantageous, the following analysis may be useful to understand how cell engraftment may be modulated to generate functionally connected myocardial structures as well.

<table border="0" cellpadding="0" cellspacing="0" width="560">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:103px;">Isolator</td>
			<td style="width:115px;">Pfi systems</td>
			<td style="width:88px;">-</td>
			<td style="width:255px;">Quotation needed</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:103px;">Heating Pad</td>
			<td style="width:115px;">Vet Tech Solutions</td>
			<td style="width:88px;">HE006</td>
			<td style="width:255px;">For small animals</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:103px;">medetomidine</td>
			<td style="width:115px;">National veterinary Service</td>
			<td style="width:88px;">-</td>
			<td style="width:255px;">Veterinary prescription is necessary</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:103px;">ketamine hydrochloride</td>
			<td style="width:115px;">National veterinary Service</td>
			<td style="width:88px;">-</td>
			<td style="width:255px;">Veterinary prescription is necessary</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:103px;">atipamezole</td>
			<td style="width:115px;">National veterinary Service</td>
			<td style="width:88px;">-</td>
			<td style="width:255px;">Veterinary prescription is necessary</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:103px;">Hair removal cream</td>
			<td style="width:115px;">Commercial shops</td>
			<td style="width:88px;">-</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:103px;">buprenorphine</td>
			<td style="width:115px;">NVS</td>
			<td style="width:88px;">-</td>
			<td style="width:255px;">Veterinary prescription is necessary</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:103px;">Leica MZFLIII microscope</td>
			<td style="width:115px;">Leica</td>
			<td style="width:88px;">Model S6E</td>
			<td style="width:255px;">With swing arm stand TS0</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:103px;">Hamamatsu Nanozoomer digital slide scanner</td>
			<td style="width:115px;">Hamamatsu</td>
			<td style="width:88px;">RS series</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:103px;">Scanning Electron Microscope</td>
			<td style="width:115px;">Jeol</td>
			<td style="width:88px;">JSM-6610</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:103px;">Blunt scissors</td>
			<td style="width:115px;">FST</td>
			<td style="width:88px;">14084-09</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:103px;">Minivent</td>
			<td style="width:115px;">Harvard apparatus</td>
			<td style="width:88px;">73-0043</td>
			<td style="width:255px;">Including small Y adapter (73-0027) and intubation cannula (73-2844)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:103px;">Forceps</td>
			<td style="width:115px;">FST</td>
			<td style="width:88px;">11052-10</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:103px;">Retraction system</td>
			<td style="width:115px;">FST</td>
			<td style="width:88px;">18200-20</td>
			<td style="width:255px;">Kit for animals up to 200grams</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:103px;">30G 12mm; &#189; inch</td>
			<td style="width:115px;">BBraun</td>
			<td style="width:88px;">A210</td>
			<td style="width:255px;">Fine yellow</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:103px;">microliter syringe</td>
			<td style="width:115px;">ESSLAB</td>
			<td style="width:88px;">81201</td>
			<td style="width:255px;">Also include a Hamilton repeating dispenser PB 600-1 Catalogue number 83700</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:103px;">6-0 silk suture</td>
			<td style="width:115px;">Ethicon</td>
			<td style="width:88px;">W1614T</td>
			<td style="width:255px;"></td>
		</tr>
	</tbody>
</table>

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.

We injected HEK293 cells, which are distinguishable from the heart cells by their different morphology (Figure 1), with a cobblestone shape compared to the elongated cardiomyocytes (Figure 1A). HEK293 cells were more reactive to hematoxylin dye (blue color) compared to cardiomyocytes (pink color), likely because of their increased nuclear content (Figure 1A). To further distinguish the injected cells from the host tissue, HEK293 cells were labeled with DAPI 2 hours prior...

Watch this Scientific Journal Video about Intramyocardial Cell Delivery: Observations in Murine Hearts at JoVE.com

Intramyocardial Cell Delivery: Observations in Murine Hearts

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 ...

intramyocardial-cell-delivery-observations-in-murine-hearts

Research

JoVE Journal

Medicine

13.4K Views.  Imperial College London. 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.

Video: Intramyocardial Cell Delivery: Observations in Murine Hearts

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. 
Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. 
Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. 
Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.

Numerous genetic manipulations and/or intramyocardial injections of genes, proteins, cells, and/or biomaterials are superimposed upon the dimension of time in studies of acute ischemia/ reperfusion injury and chronic remodeling in mice. This video illustrates the microsurgical procedures for ischemia/reperfusion, permanent coronary artery ligation, and intramyocardial injection studies.

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo.  With the advent of genetic modifications to ...

Reduction in Left Ventricular Wall Stress and Improvement in Function in Failing Hearts using Algisyl-LVR

Injection of Algisyl-LVR, a treatment under clinical development, is intended to treat patients with dilated cardiomyopathy. This treatment was recently used for the first time in patients who had symptomatic heart failure. In all patients, cardiac function of the left ventricle (LV) improved significantly, as manifested by consistent reduction of the LV volume and wall stress. Here we describe this novel treatment procedure and the methods used to quantify its effects on LV wall stress and function.
Algisyl-LVR is a biopolymer gel consisting of Na+-Alginate and Ca2+-Alginate. The treatment procedure was carried out by mixing these two components and then combining them into one syringe for intramyocardial injections. This mixture was injected at 10 to 19 locations mid-way between the base and apex of the LV free wall in patients.
Magnetic resonance imaging (MRI), together with mathematical modeling, was used to quantify the effects of this treatment in patients before treatment and at various time points during recovery. The epicardial and endocardial surfaces were first digitized from the MR images to reconstruct the LV geometry at end-systole and at end-diastole. Left ventricular cavity volumes were then measured from these reconstructed surfaces.
Mathematical models of the LV were created from these MRI-reconstructed surfaces to calculate regional myofiber stress. Each LV model was constructed so that 1) it deforms according to a previously validated stress-strain relationship of the myocardium, and 2) the predicted LV cavity volume from these models matches the corresponding MRI-measured volume at end-diastole and end-systole. Diastolic filling was simulated by loading the LV endocardial surface with a prescribed end-diastolic pressure. Systolic contraction was simulated by concurrently loading the endocardial surface with a prescribed end-systolic pressure and adding active contraction in the myofiber direction. Regional myofiber stress at end-diastole and end-systole was computed from the deformed LV based on the stress-strain relationship.

This article describes procedures for implanting a novel hydrogel in failing hearts and quantifying its effect on left ventricular wall stress and function. These procedures have been successfully applied in dogs and humans.

Injection of  Algisyl-LVR, a treatment under clinical development, is intended to treat  patients with dilated cardiomyopathy. This treatment was recently ...

Ultrasound-guided Transthoracic Intramyocardial Injection in Mice

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. Experiments involving myocardial injection are currently performed by direct surgical access through a thoracotomy. While convenient when performed at the time of another experimental manipulation such as coronary artery ligation, the need for an invasive procedure for intramyocardial delivery limits potential experimental designs. With ever improving ultrasound resolution and advanced noninvasive imaging modalities, it is now feasible to routinely perform ultrasound-guided, percutaneous intramyocardial injection. This modality efficiently and reliably delivers agents to a targeted region of myocardium. Advantages of this technique include the avoidance of surgical morbidity, the facility to target regions of myocardium selectively under ultrasound guidance, and the opportunity to deliver injectate to the myocardium at multiple, predetermined time intervals. With practiced technique, complications from intramyocardial injection are rare, and mice quickly return to normal activity on recovery from anesthetic. Following the steps outlined in this protocol, the operator with basic echocardiography experience can quickly become competent in this versatile, minimally invasive technique.

Echocardiography-guided percutaneous intramyocardial injection represents an efficient, reliable, and targetable modality for the delivery of gene transfer agents or cells into the murine heart. Following the steps outlined in this protocol, the operator can quickly become competent in this versatile, minimally invasive technique.

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. ...

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 &#181;m, C8 100 &#197;; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and lumacaftor.

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening ...

Percutaneous Contrast Echocardiography-guided Intramyocardial Injection and Cell Delivery in a Large Preclinical Model

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection fraction (HFrEF). Before they can be considered for use, and implemented in humans, extensive preclinical studies are required in large animal models to evaluate the safety, efficacy, and fate of the injectate (e.g., stem cells) once delivered into the myocardium. Small rodent models offer advantages (e.g., cost effectiveness, amenability for genetic manipulation); however, given inherent limitations of these models, the findings in these rarely translate into the clinic. Conversely, large animal models such as rabbits, have advantages (e.g., similar cardiac electrophysiology compared to humans and other large animals), whilst retaining a good cost-effective balance. Here, we demonstrate how to perform a percutaneous contrast echocardiography-guided intramyocardial injection (IMI) technique, which is minimally invasive, safe, well tolerated, and very effective in the targeted delivery of injectates, including cells, into several locations within the myocardium of a rabbit model. For the implementation of this technique, we also have taken advantage of a widely available clinical echocardiography system. After putting in practice the protocol described here, a researcher with basic ultrasound knowledge will become competent in the performance of this versatile and minimally invasive technique for routine use in experiments, aimed at hypothesis testing of the capabilities of cardiac regenerative therapeutics in the rabbit model. Once competency is achieved, the whole procedure can be performed within 25 min after anaesthetizing the rabbit.

Novel therapeutic strategies in cardiac regenerative medicine require extensive and detailed studies in large preclinical animal models before they can be considered for use in humans. Here, we demonstrate a percutaneous contrast echocardiography-guided intramyocardial injection technique in rabbits, which is valuable for hypothesis testing the efficacy of such novel therapies.

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection ...

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the luminal surface area in arteries thereby reducing adequate blood flow to organs and distal tissues. Clinically, revascularization procedures such as balloon angioplasty with or without stent placement aim to restore blood flow. Although these procedures reestablish blood flow by reducing plaque burden, they damage the vessel wall, which initiates the arterial healing response. The prolonged healing response causes arterial restenosis, or re-narrowing, ultimately limiting the long-term success of these revascularization procedures. Therefore, preclinical animal models are integral for analyzing the pathophysiological mechanisms driving restenosis, and provide the opportunity to test novel therapeutic strategies. Murine models are cheaper and easier to operate on than large animal models. Balloon or wire injury are the two commonly accepted injury modalities used in murine models. Balloon injury models in particular mimic the clinical angioplasty procedure and cause adequate damage to the artery for the development of restenosis. Herein we describe the surgical details for performing and histologically analyzing the modified, pressure-controlled rat carotid artery balloon injury model. Additionally, this protocol highlights how local periadventitial application of therapeutics can be used to inhibit neointimal hyperplasia. Lastly, we present light sheet fluorescence microscopy as a novel approach for imaging and visualizing the arterial injury in three-dimensions.

The rat carotid artery balloon injury mimics the clinical angioplasty procedure performed to restore blood flow in atherosclerotic vessels. This model induces the arterial injury response by distending the arterial wall, and denuding the intimal layer of endothelial cells, ultimately causing remodeling and an intimal hyperplastic response.

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the ...

Slicing and Culturing Pig Hearts under Physiological Conditions

Many novel drugs fail in clinical studies due to cardiotoxic side effects as the currently available in vitro assays and in vivo animal models poorly predict human cardiac liabilities, posing a multi-billion-dollar burden on the pharmaceutical industry. Hence, there is a worldwide unmet medical need for better approaches to identify drug cardiotoxicity before undertaking costly and time consuming &#39;first in man&#39; trials. Currently, only immature cardiac cells (human induced pluripotent stem cell-derived cardiomyocytes [hiPSC-CMs]) are used to test therapeutic efficiency and drug toxicity as they are the only human cardiac cells that can be cultured for prolonged periods required to test drug efficacy and toxicity. However, a single cell type cannot replicate the phenotype of the complex 3D heart tissue which is formed of multiple cell types. Importantly, the effect of drugs needs to be tested on adult cardiomyocytes, which have different characteristics and toxicity responses compared to immature hiPSC-CMs. Culturing human heart slices is a promising model of intact human myocardium. This technology provides access to a complete multicellular system that mimics the human heart tissue and reflects the physiological or pathological conditions of the human myocardium. Recently, through optimization of the culture media components and the culture conditions to include continuous electrical stimulation at 1.2 Hz and intermittent oxygenation of the culture medium, we developed a new culture system setup that preserves viability and functionality of human and pig heart slices for 6 days in culture. In the current protocol, we are detailing the method for slicing and culturing pig heart as an example. The same protocol is used to culture slices from human, dog, sheep, or cat hearts. This culture system has the potential to become a powerful predictive human in situ model for acute cardiotoxicity testing that closes the gap between preclinical and clinical testing results.

This protocol describes how to slice and culture heart tissue under physiological conditions for 6 days. This culture system could be used as a platform for testing the efficacy of novel heart failure therapeutics as well as reliable testing of acute cardiotoxicity in a 3D heart model.

Many novel drugs fail in clinical studies due to cardiotoxic side effects as the currently available in vitro assays and in vivo animal models poorly ...

Intratracheal Administration of Dry Powder Formulation in Mice

In the development of inhalable dry powder formulations, it is essential to evaluate their biological activities in preclinical animal models. This paper introduces a noninvasive method of intratracheal delivery of dry powder formulation in mice. A dry powder loading device that consists of a 200 &#181;L gel loading pipette tip connected to an 1 mL syringe via a three-way stopcock is presented. A small amount of dry powder (1-2 mg) is loaded into the pipette tip and dispersed by 0.6 mL of air in the syringe. Because pipette tips are disposable and inexpensive, different dry powder formulations can be loaded into different tips in advance. Various formulations can be evaluated in the same animal experiment without device cleaning and dose refilling, thereby saving time and eliminating the risk of cross-contamination from residual powder. The extent of powder dispersion can be inspected by the amount of powder remaining in the pipette tip. A protocol of intubation in mouse with a custom-made light source and a guiding cannula is included. Proper intubation is one of the key factors that influences the intratracheal delivery of dry powder formulation to the deep lung region of the mouse.

Dry powder formulations for inhalation have great potential in treating respiratory diseases. Before entering human studies, it is necessary to evaluate the efficacy of the dry powder formulation in preclinical studies. A simple and noninvasive method of the administration of dry powder in mice through the intratracheal route is presented.

In the development of inhalable dry powder formulations, it is essential to evaluate their biological activities in preclinical animal models. This paper ...

Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, cardiac stem cell therapy is studied by delivering SCs concurrently with the induction of myocardial injury. However, this approach presents two significant limitations: the early hostile pro-inflammatory ischemic environment may affect the survival of transplanted SCs, and it does not represent the subacute infarction scenario where SCs will likely be used. Here we describe a two-part series of surgical procedures for the induction of ischemia-reperfusion injury and delivery of mesenchymal stem cells (MSCs). This method of stem cell administration may allow for the longer viability and retention around damaged tissue by circumventing the initial immune response. A model of ischemia reperfusion injury was induced in mice accompanied by the delivery of mesenchymal stem cells (3.0 x 105), stably expressing the reporter gene firefly luciferase under the constitutively expressed CMV promoter, intramyocardially 7 days later. The animals were imaged via ultrasound and bioluminescent imaging for confirmation of injury and injection of cells, respectively. Importantly, there was no added complication rate when performing this two-procedure approach for SC delivery. This method of stem cell administration, collectively with the utilization of state-of-the-art reporter genes, may allow for the in vivo study of viability and retention of transplanted SCs in a situation of chronic ischemia commonly seen clinically, while also circumventing the initial pro-inflammatory response. In summary, we established a protocol for the delayed delivery of stem cells into the myocardium, which can be used as a potential new approach in promoting regeneration of the damaged tissue.

Stems cells are continuously investigated as potential treatments for individuals with myocardial damage, however, their decreased viability and retention within injured tissue can impact their long-term efficacy. In this manuscript we describe an alternative method for stem cell delivery in a murine model of ischemia reperfusion injury.

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, ...

Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of transplanted cells within the injured myocardium, limiting their therapeutic efficacy. Recently, the use of scaffolding biomaterials has gained attention for improving and maximizing stem cell therapy. The objective of this protocol is to introduce a simple and straightforward technique to transplant bone marrow-derived mesenchymal stem cells (MSCs) using injectable hydroxyphenyl propionic acid (GH) hydrogels; the hydrogels are favorable as a cell delivery platform for cardiac tissue engineering applications due to their ability to be cross-linked in situ and high biocompatibility. We present a simple method to fabricate MSC-loading GH hydrogels (MSC/hydrogels) and evaluate their survival and proliferation in three-dimensional (3D) in vitro culture. In addition, we demonstrate a technique for intramyocardial transplantation of MSC/hydrogels in mice, describing a surgical procedure to induce myocardial infarction (MI) via left anterior descending (LAD) coronary artery ligation and subsequent MSC/hydrogels transplantation.

Stem cell-based therapy has emerged as an efficient strategy to repair injured cardiac tissues after myocardial infarction. We provide an optimal in vivo application for stem cell transplantation using gelatin hydrogels that are able to be enzymatically cross-linked.

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of ...