The authors would like to thank Maria Hedman, Tiina Laitinen, Tomi Laitinen, Pekka Poutiainen, Annika Viren, and Severi Sormunen for assistance and permitting 15O-PET imaging at Kuopio University Hospital; and Heikki Karhunen, Minna T&#246;rr&#246;nen, and Riikka Ven&#228;l&#228;inen from National Laboratory Animal Center for their assistance in animal work.
This study is supported by grants from Finnish Academy, ERC, and CardioReGenix EU Horizon 2020 grant.

The experiments presented here are performed using about 10-week-old female domestic pigs and are approved by the Animal Experiment Board in Finland. Animals weigh 30-40 kg at the beginning of the protocol, allowing the same procedural equipment and imaging modalities as possible for humans. Chronic ischemia is induced 14 days before the gene transfer, and the follow-up time after the gene transfer depends on the viral vector used. The study protocol is shown in Figure 1. This protocol can be used to perform adenoviral or AAV-mediated gene therapy injections. The time of sample collection has to be adjusted to the transgene expression peak, which depends on the viral vector used. For example, when performing adenoviral gene transfers, the time of the sample collection is set to 6 days after the gene transfer.
1. Medication
<ol>
	<li>Administer a daily dose of 200 mg of amiodarone and 2.5 mg of bisoprolol to prevent fatal ventricular arrhythmias. The medication begins 1 week before the ischemia operation and is continued daily until the follow-up.</li>
	<li>In addition, administer peroral doses of clopidogrel (300 mg) and acetylsalicylic acid (300 mg) to the animals 1 day before the ischemia operation to prevent acute in-stent thrombosis after the stent placement.</li>
	<li>Administer 100 mg of lidocaine and 2.5 mL of (246 mg/mL) MgSO4 intravenously to the animals at the beginning of the ischemia operation to prevent ventricular arrhythmias.</li>
	<li>Administer intramuscular injection of cefuroxime (500 mg) at the beginning of each operation for infection prophylaxis.</li>
	<li>Administer 30 mg of enoxaparin intravenously at the beginning of the ischemia operations and subcutaneously after the operation procedure for thrombosis prevention.</li>
	<li>For anesthesia and analgesia, administer 1.5 mL of atropin, 6 mL of azaperone (40 mg/mL), propofol 20 mg/mL, at a rate of 15 mg/kg/h, and fentanyl 50 &#181;g/mL at a rate of 10 &#181;g/kg/h. Drug dosages were the same for each pig. Refer to local animal use guidelines for dose administration.</li>
	<li>Anesthetize the animals during all operations. All the operations should be performed in a sterile environment using a sterile technique.</li>
</ol>
2. Transthoracic echocardiography
<ol>
	<li>Perform transthoracic echocardiography before ischemia operation, gene transfer, and euthanasia to evaluate any detectable pericardial fluid and determine the myocardial strain.</li>
	<li>Place the transducer in the third or fourth intercostal space under the armpit of the pig to access parasternal short-axis views at the mitral valve level, papillary muscle, and apical levels (Video 1). The marker of the transducer should point to the sternum of the pig. To save a clip, press Acquire.</li>
</ol>
3. Endovascular operations under fluoroscopic guidance
<ol>
	<li>Perform left ventricle cine imaging after coronary angiograms before ischemia operation, gene transfer and tissue collection.</li>
	<li>Preparation of the operation
	<ol>
		<li>Prepare for the operations by sedating the pigs with an intramuscular injection of 1.5 mL of atropine and 6 mL of azaperone.</li>
		<li>After the sedation, induce general propofol and fentanyl anesthesia for the angiographic procedures to the pigs with doses of 15 mg/kg/h and 10 &#181;g/kg/h, respectively. 
		NOTE: The pigs are anesthetized for the entire procedure.</li>
		<li>Support the ventilation by intubation and ventilator and monitor the vital physiological parameters, such as ECG and respiratory parameters.</li>
	</ol>
	</li>
	<li>Introducer sheath placement
	<ol>
		<li>Place an introducer sheath into the right femoral artery for all the operations as standard practice in cardiology. Use ultrasound to track the femoral artery and pierce it with an entry needle (18 G). 
		NOTE: Use an 8F introducer sheath for intramyocardial gene transfers and a 6F sheath for all other operations. Introduce the guidewire of the sheath through the needle to thread the artery and hold the guidewire still while removing the needle.</li>
		<li>Insert the introducer sheath along the guidewire, and when placed, remove the guidewire and administer 1.25 mg of sublingual dinitrate to the pig to induce coronary vasodilation.</li>
	</ol>
	</li>
	<li>Coronary angiography
	<ol>
		<li>Perform coronary angiography directly before ischemia operation, gene transfer, and tissue collection. The machinery needed for the angiograms is shown in Figure 2.</li>
		<li>Use a 6F catheter under fluoroscopic guidance with an iodine contrast agent to image the right coronary artery and the left ascending coronary artery (Video 2).</li>
	</ol>
	</li>
	<li>Left ventricle cine imaging under rest and dobutamine stress
	<ol>
		<li>Administer a 21 mL bolus of iodine contrast agent into the left ventricle via a 5F pigtail catheter using an auto-injector. First, set the bolus duration at 3 s and the total volume for 21 mL. Then, press&#160;Single&#160;and&#160;Yes.</li>
		<li>Calculate the ejection fraction by the measurement software of the angiographic workstation. To perform the calculation, select&#160;Ventricular Analysis&#160;of the image in question. Scroll the image to select a time frame, one in diastole and one in systole. Select a tool to draw ventricular outlines of each time frame. 
		NOTE: The software now calculates the ejection fraction and stroke volume by the Simpson&#39;s method. The ejection fraction measurement is performed during rest and under dobutamine-induced stress.</li>
	</ol>
	</li>
	<li>Stress imaging
	<ol>
		<li>Dose dobutamine intravenously in escalating doses from 10 &#181;g/kg/min to 20 &#181;g/kg/min for the dobutamine-induced stress imaging until the target heart rate of 160 bpm is reached. Then, perform the cine imaging.</li>
	</ol>
	</li>
	<li>Ischemia operation
	<ol>
		<li>Place a bottleneck stent into the left coronary artery (LAD) 14 days before the gene transfer to induce chronic myocardial ischemia. After the bottleneck stent placement, check whether the bottleneck stent is placed correctly, restricting the coronary blood flow. 
		NOTE: The bottleneck stent is placed on a dilation catheter and consists of a bare-metal stent covered by a polytetrafluoroethylene tube formed in a bottleneck shape to reduce coronary blood flow9.</li>
	</ol>
	</li>
	<li>Defining the stent size
	<ol>
		<li>Choose the size of the stent, either 3.0/3.5/4.0 x 8 mm, according to the size of the left ascending coronary artery in the angiogram by using the automatic measurement software in the angiographic workstation (Video 3)12.</li>
	</ol>
	</li>
	<li>Stent placement
	<ol>
		<li>Place a coil to the left coronary artery and glide the bottleneck stent to the LAD, placing it distally to the first diagonal.</li>
		<li>Inflate the stent to nominal pressure in the artery using an in-deflator with a stent-to-lumen ratio of 1.3, anchoring the bottleneck in place. After an additional 15 s, deflate the stent and retract the equipment from the artery. 
		NOTE: Confirm the correct placement of the bottleneck stent by angiogram.</li>
	</ol>
	</li>
</ol>
4. PET imaging
NOTE: One day before the gene transfer, perform rest and stress 15O-labeled radiowater PET/CT scans (requires hospital environment and radiological technicians).
<ol>
	<li>Reference imaging
	<ol>
		<li>Perform computed tomography (CT) scans before rest and stress imaging. Use the CT information for attenuation correction.</li>
	</ol>
	</li>
	<li>15O-labeled radiowater imaging
	<ol>
		<li>Perform rest and stress imaging using an 800 MBq 15H2O bolus.</li>
	</ol>
	</li>
	<li>Stress imaging
	<ol>
		<li>Perform stress imaging with a further 800 MBq 15O-water bolus after a suitable radioactive decay of 12 min. 
		NOTE: Hyperemia is induced by adenosine (200 &#181;g/kg/min intravenous), as described previously12.</li>
	</ol>
	</li>
</ol>
5. Gene transfer
<ol>
	<li>Electroanatomical mapping
	<ol>
		<li>Proceed to electroanatomical mapping after a coronary angiogram and functional measurements (echocardiography, LV cine imaging).</li>
		<li>Introduce a mapping catheter to the left ventricle via femoral sheath in fluoroscopic guidance. 
		NOTE: Register about 100-150 points around the left ventricle with the mapping catheter to create the electroanatomical map.</li>
	</ol>
	</li>
	<li>Finishing the electroanatomical map
	<ol>
		<li>Delete the outlier points to ensure a more reliable electroanatomical map of the left ventricle.</li>
		<li>Do this by selecting Clip Planes of the map and delete the points that differ from the points forming the ventricular shape. Next, select Trajectories for the map view and delete the points that have traveled horizontally during point registering. 
		NOTE: Make sure the remaining points cover the left ventricle and register more points if needed.</li>
	</ol>
	</li>
	<li>Gene transfer injections
	<ol>
		<li>Introduce an intramyocardial injection catheter to the left ventricle via the femoral sheath under fluoroscopic guidance. Set the injection needle length to 3 mm.</li>
	</ol>
	</li>
	<li>Criteria for intramyocardial injections
	<ol>
		<li>Guide the gene transfers by electroanatomical mapping system and target the injections into viable but hypokinetic areas of the left ventricle. 
		NOTE: For viability, use a unipolar voltage over 5 mV as a criterion. For hypokinesia, select a local linear shortening (LLS) as low as available, at least below 12% but preferably below 6%13.</li>
	</ol>
	</li>
	<li>Intramyocardial injections
	<ol>
		<li>During 30 s, inject the vector material into the point of selection (step 5.4) and keep the injection needle inside the myocardium for an additional 5 s before retracting to prevent backflow to the left ventricle.</li>
	</ol>
	</li>
</ol>
6. Euthanasia and sample collection
NOTE: After the coronary angiogram and ejection fraction measurements described in steps 3.4.1 and 3.5.2, respectively, administer 50 mL of saturated potassium chloride intravenously to the anesthetized pig.
<ol>
	<li>Perfusion fixation of the heart
	<ol>
		<li>Harvest the heart from the thoracic cavity. Rinse with water. Place an 18 G needle above the aortic valve and attach the needle to a perfusion pump. Perfuse the heart with 750 mL of 1% paraformaldehyde (PFA).</li>
	</ol>
	</li>
	<li>Sample collection
	<ol>
		<li>Slice the heart into 1 cm thick slices using a sharp kitchen knife. Collect the samples from the gene transfer area into 4% PFA and liquid nitrogen. 
		NOTE: To harvest negative controls, collect a control sample from the posterior wall of the left ventricle.</li>
	</ol>
	</li>
	<li>Safety tissue collection
	<ol>
		<li>Harvest samples from remote tissues, such as the lung, liver, kidney, spleen, and ovaries. Take samples into 4% PFA and liquid nitrogen.</li>
	</ol>
	</li>
</ol>
7. Sample storing
<ol>
	<li>Store the samples for staining in 4% PFA for 48 h at 4 &#176;C. 
	NOTE: Replace the PFA daily with a fresh liquid.
	<ol>
		<li>After 48 h, replace the PFA with 15% sucrose in deionized water. Store for at least 24 h before embedding the samples into paraffin blocks. Snap frozen samples are stored at -70 &#176;C.</li>
	</ol>
	</li>
</ol>

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The authors declare no conflicts of interest.

The timepoints of this protocol may be modified according to the viral vector used. Also, the immunohistological analyses may be selected according to the therapeutic gene. It is also possible to add more timepoints and endpoints to the protocol if needed.
This protocol comprises stages, which are essential to succeed and impossible to correct afterward. First, if one fails to induce appropriate ischemia, the animal must be excluded from further procedures and analyses. Standardization of the ...

Coronary artery disease is accountable for the vast proportion of mortality and disease burden worldwide1. Current treatment strategies are percutaneous interventions, pharmacological treatment, and bypass surgery2. However, despite the progression of these current therapeutics, many patients suffer from so-called refractory angina, underlining the unmet need for novel treatment approaches3. Gene therapy-mediated therapeutic angiogenesis could target this patient group.
Myocardial gene therapy is most often delivered by using different viral vectors, most commonly replication-deficient adenovirus4. As therapeutic genes, various angiogenic growth factors are used. The most substantially studied angiogenic growth factors are the vascular endothelial growth factors (VEGFs) that mediate their angiogenic signaling through vascular endothelial growth factor receptors (VEGFRs) and their co-receptors5. Several clinical trials have proved the benefit and safety of cardiac gene therapy and made this novel treatment method a realistic option for treating ischemic heart diseases6,7. However, this concept still needs enhancement of the therapeutic genes and viral vectors put to the test in large animal models before entering the clinics. The pig has been frequently used as a laboratory animal since its heart is very similar to the human heart. The size of the cardiovascular system of a pig allows the usage of similar catheter inventions as used in humans. All imaging modalities available for humans can be used in pigs8.
There are several large animal models for chronic ischemia. The most commonly used is the ameroid constrictor model9,10,11. The downside of this method is the invasiveness since thoracotomy is needed to access the coronary vasculature. Previously in our group, a mini-invasive bottleneck stent model for chronic myocardial ischemia has been developed12. This method is also used in this manuscript to induce myocardial ischemia.
The usability of ultrasound imaging has evolved substantially despite the age of the imaging modality. For example, myocardial strain is still mainly in research usage due to its novelty. Myocardial strain reflects changes in the contractile function of the heart better than the traditional M-mode ejection fraction measurement13. Thus, here in the large animal model, myocardial strain measurement is utilized. To evaluate the function of the heart, cardiac output is also measured by cine imaging of the left ventricle during angiography. Cardiac output is measured both at rest and under dobutamine-induced stress to evaluate myocardial function under stress.
In addition to the measurements of the heart function, information on myocardial perfusion is essential in gene therapy studies aiming at therapeutic angiogenesis. In this animal model, animals are imaged with 15O-labeled radiowater positron emission tomography (15H2O-PET) as this is the golden standard for measuring myocardial perfusion. 15H2O-PET has been previously validated for measuring perfusion of ischemic porcine heart14.
Thus, the methods and modalities mentioned above constitute an excellent perspective for evaluating the efficacy of gene therapy in the ischemic heart.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1% PFA</td>
			<td>VWR</td>
			<td>VWRC28794.295</td>
			<td>Prepared from paraformaldehyde powder</td>
		</tr>
		<tr>
			<td>15 % sucrose</td>
			<td>VWR</td>
			<td>VWRC27480.294</td>
			<td>Prepared from solid sucrose</td>
		</tr>
		<tr>
			<td>4% PFA</td>
			<td>VWR</td>
			<td>VWRC28794.295</td>
			<td>Prepared from paraformaldehyde powder</td>
		</tr>
		<tr>
			<td>5 F pigtail catheter</td>
			<td>Cordis</td>
			<td>534-550S</td>
			<td></td>
		</tr>
		<tr>
			<td>6 F catheter AR2</td>
			<td>Cordis</td>
			<td>670-112-00</td>
			<td></td>
		</tr>
		<tr>
			<td>6 F introducer sheath</td>
			<td>Cordis</td>
			<td>504-606X</td>
			<td></td>
		</tr>
		<tr>
			<td>8 F introducer sheath</td>
			<td>Cordis</td>
			<td>504-608X</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetylsalicylic acid</td>
			<td></td>
			<td></td>
			<td>Varying producer</td>
		</tr>
		<tr>
			<td>Amiodarone</td>
			<td></td>
			<td></td>
			<td>Varying producer</td>
		</tr>
		<tr>
			<td>Angiographic station</td>
			<td>GE Healthcare</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Angiolaboratory set</td>
			<td>M&#246;lnlycke</td>
			<td></td>
			<td>designed for the needs of our angiolaboratory, contains sterile drapes, cups and swabs</td>
		</tr>
		<tr>
			<td>Bisoprolol</td>
			<td></td>
			<td></td>
			<td>Varying producer</td>
		</tr>
		<tr>
			<td>Cefuroxime</td>
			<td></td>
			<td></td>
			<td>Varying producer</td>
		</tr>
		<tr>
			<td>Clopidogrel</td>
			<td></td>
			<td></td>
			<td>Varying producer</td>
		</tr>
		<tr>
			<td>Coroflex Blue stent</td>
			<td>B.Braun Medical</td>
			<td>5029012</td>
			<td>Catalog number depends on stent size</td>
		</tr>
		<tr>
			<td>Crile forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cyclotron</td>
			<td>GE Healthcare</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dobutamine</td>
			<td></td>
			<td></td>
			<td>Varying producer</td>
		</tr>
		<tr>
			<td>Electroanatomical mapping system</td>
			<td colspan="3">Biologics Delivery Systems, Johnson &#38; Johnson company</td>
		</tr>
		<tr>
			<td>Enoxaparin</td>
			<td></td>
			<td></td>
			<td>Varying producer</td>
		</tr>
		<tr>
			<td>Fentanyl</td>
			<td></td>
			<td></td>
			<td>Varying producer</td>
		</tr>
		<tr>
			<td>Intramyocardial injection catheter</td>
			<td>Johnson &#38; Johnson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Iodine contrast agent</td>
			<td>Iomeron</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Kitchen knife</td>
			<td></td>
			<td></td>
			<td>Varying producer</td>
		</tr>
		<tr>
			<td>Lidocaine</td>
			<td></td>
			<td></td>
			<td>Varying producer</td>
		</tr>
		<tr>
			<td>Liquid nitrogen</td>
			<td></td>
			<td></td>
			<td>Varying producer</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td></td>
			<td></td>
			<td>Varying producer</td>
		</tr>
		<tr>
			<td>Needle 18 G</td>
			<td>Cordis</td>
			<td>12-004943</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusion pump</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PET-CT scanner</td>
			<td>Siemens Healthcare</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polytetrafluoroethylene tube</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Propofol</td>
			<td></td>
			<td></td>
			<td>Varying producer</td>
		</tr>
		<tr>
			<td>Scalpel no 11</td>
			<td>VWR</td>
			<td>SWAN0503</td>
			<td></td>
		</tr>
		<tr>
			<td>Sublingual dinitrate</td>
			<td>Takeda</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound machine</td>
			<td>Philips</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

large animal model for evaluating the efficacy of the gene therapy in ischemic heart

Coronary artery disease is one of the significant causes of mortality and morbidity worldwide. Despite the progression of current therapeutics, a considerable proportion of coronary artery disease patients remain symptomatic. Gene therapy-mediated therapeutic angiogenesis offers a novel therapeutic method for improving myocardial perfusion and relieving symptoms. Gene therapy with different angiogenic factors has been studied in few clinical trials. Due to the novelty of the method, the progress of myocardial gene therapy is a continuous path from bench to bedside. Therefore, large animal models are needed for evaluating the safety and efficacy. The more the large animal model identifies the original disease and the endpoints used in clinics, the more predictable outcomes are from clinical trials. Here, we introduce a large animal model for evaluating the efficacy of the gene therapy in the ischemic porcine heart. We use clinically relevant imaging methods such as ultrasound imaging and 15H2O-PET. For targeting the gene transfers into the desired area, electroanatomical mapping is used. The aim of this method is: (1) to mimic chronic coronary artery disease, (2) to induce therapeutic angiogenesis at hypoxic areas of the heart, and (3) to evaluate the safety and efficacy of the gene therapy by using relevant endpoints.

The success of the ischemia operation can be confirmed with this protocol by coronary angiogram and by determining the hypokinetic area by transthoracic ultrasound (Figure 1) before proceeding to the gene delivery. The state of the coronary occlusion can be evaluated by coronary angiogram, and the electroanatomical mapping ensures the ischemic and hibernating areas.
The efficacy of the gene therapy can be analyzed by measuring the circumferential strain, ejection ...

Watch this Scientific Journal Video about Large Animal Model for Evaluating the Efficacy of the Gene Therapy in Ischemic Heart at JoVE.com

Large Animal Model for Evaluating the Efficacy of the Gene Therapy in Ischemic Heart

Myocardial gene therapy for ischemic heart disease holds great promise for future therapeutics. Here, we introduce a large animal model for evaluating the efficacy of gene therapy in the ischemic heart.

Coronary artery disease is one of the significant causes of mortality and morbidity worldwide. Despite the progression of current therapeutics, a ...

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JoVE Journal

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1.7K Views.  University of Eastern Finland. Myocardial gene therapy for ischemic heart disease holds great promise for future therapeutics. Here, we introduce a large animal model for evaluating the efficacy of gene therapy in the ischemic heart.

Video: Large Animal Model for Evaluating the Efficacy of the Gene Therapy in Ischemic Heart

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

Gene Transfer for Ischemic Heart Failure in a Preclinical Model

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine their efficacy and safety.
Gene therapy using viral vectors is one of the most promising approaches for treating cardiac diseases. Viral delivery of various different genes by changing the carrier gene has immeasurable therapeutic potential.
In this video, the full process of an animal model of heart failure creation followed by gene transfer is presented using a swine model. First, myocardial infarction is created by occluding the proximal left anterior descending coronary artery. Heart remodeling results in chronic heart failure. Unique to our model is a fairly large scar which truly reflects patients with severe heart failure who require aggressive therapy for positive outcomes. After myocardial infarct creation and development of scar tissue, an intracoronary injection of virus is demonstrated with simultaneous nitroglycerine infusion. Our injection method provides simple and efficient gene transfer with enhanced gene expression. This combination of a myocardial infarct swine model with intracoronary virus delivery has proven to be a consistent and reproducible methodology, which helps not only to test the effect of individual gene, but also compare the efficacy of many genes as therapeutic candidates.

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine ...

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2- (oxide anion), peroxynitrite (ONOO-), hydrochlorous acid (HOCl), and hydroxyl radical (OH-).
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1, autoimmune diseases 2, etc&hellip;) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6, migration 7 etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12.
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.

Production and Detection of Reactive Oxygen Species ROS in Cancers

Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to stimulate microcirculation. The choice of delivery method for these agents is important because the route of administration profoundly affects the bioactivity and efficacy of these agents1,2. In this article, we demonstrate how to locally administer a substance in ischemic hindlimbs by using a catheterized osmotic pump. This pump can deliver a fixed volume of aqueous solution continuously for an allotted period of time. We also present our mouse model of unilateral hindlimb ischemia induced by ligation of the common femoral artery proximal to the origin of profunda femoris and epigastrica arteries in the left hindlimb. Lastly, we describe the in vivo cannulation and ligation of the infrarenal abdominal aorta and perfusion of the hindlimb vasculature with Microfil, a silicone radiopaque casting agent. Microfil can perfuse and fill the entire vascular bed (arterial and venous), and because we have ligated the major vascular conduit for exit, the agent can be retained in the vasculature for future ex vivo imaging with the use of small specimen micro-CT3.

We show here the in vivo insertion of an osmotic pump for constant local drug delivery and the creation of hindlimb ischemia in a mouse model. Moreover, the hindlimb vasculature is perfused with Microfil, a silicone radiopaque agent, to prepare for micro-computed tomography (micro-CT) imaging.

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

An Isolated Working Heart System for Large Animal Models

Since its introduction in the late 19th century, the Langendorff isolated heart perfusion apparatus, and the subsequent development of the working heart model, have been invaluable tools for studying cardiovascular function and disease1-15. Although the Langendorff heart preparation can be used for any mammalian heart, most studies involving this apparatus use small animal models (e.g., mouse, rat, and rabbit) due to the increased complexity of systems for larger mammals1,3,11. One major difficulty is ensuring a constant coronary perfusion pressure over a range of different heart sizes &#8211; a key component of any experiment utilizing this device1,11. By replacing the classic hydrostatic afterload column with a centrifugal pump, the Langendorff working heart apparatus described below allows for easy adjustment and tight regulation of perfusion pressures, meaning the same set-up can be used for various species or heart sizes. Furthermore, this configuration can also seamlessly switch between constant pressure or constant flow during reperfusion, depending on the user&#8217;s preferences. The open nature of this setup, despite making temperature regulation more difficult than other designs, allows for easy collection of effluent and ventricular pressure-volume data.

Most studies involving the Langendorff apparatus use small animal models due to the increased complexity of systems for larger mammals. We describe a Langendorff system for large animal models that allows for use across a range of species, including humans, and relatively easy data acquisition.

Since its introduction in the late 19th century, the Langendorff isolated heart perfusion apparatus, and the subsequent development of the working heart ...

A Quick Phenotypic Neurological Scoring System for Evaluating Disease Progression in the SOD1-G93A Mouse Model of ALS

The SOD1-G93A transgenic mouse is the most widely used animal model of amyotrophic lateral sclerosis (ALS). At ALS TDI we developed a phenotypic screening protocol, demonstrated in video herein, which reliably assesses the neuromuscular function of SOD1-G93A mice in a quick manner. This protocol encompasses a simple neurological scoring system (NeuroScore) designed to assess hindlimb function. NeuroScore is focused on hindlimb function because hindlimb deficits are the earliest reported neurological sign of disease in SOD1-G93A mice. The protocol developed by ALS TDI provides an unbiased assessment of onset of paresis (slight or partial paralysis), progression and severity of paralysis and it is sensitive enough to identify drug-induced changes in disease progression. In this report, the combination of a detailed manuscript with video minimizes scoring ambiguities and inter-experimenter variability thus allowing for the protocol to be adopted by other laboratories and enabling comparisons between studies taking place at different settings. We believe that this video protocol can serve as an excellent training tool for present and future ALS researchers.

This video protocol describes a sensitive, reliable, and quick method for evaluating the neuromuscular deficits in a transgenic mouse model of amyotrophic lateral sclerosis.

The SOD1-G93A transgenic mouse is the most widely used animal model of amyotrophic lateral sclerosis (ALS). At ALS TDI we developed a phenotypic screening ...

Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo

Plasma coagulation as a form of electrocautery is used in liver surgery for decades to seal the large liver cut surface after major hepatectomy to prevent hemorrhages at a later stage. The exact effects of plasma coagulation on liver tissue are only poorly examined. In our porcine model, the coagulation effects can be examined close to the clinical application. A combined laser Doppler flowmeter and spectrophotometer documents microcirculation changes during coagulation at 8 mm tissue depth noninvasively, providing quantifiable information about hemostasis beyond the subjective clinical impression. The temperature at coagulation site is assessed with an infrared thermometer prior and post coagulation and with a thermographic camera during coagulation, a measurement of the gas beam temperature is not possible due to the upper threshold of the devices. The depth of coagulation is measured microscopically on hematoxylin/eosin stained sections after calibration with an object micrometer and gives an exact information about the power setting-coagulation depth-relation. The sealing effect is examined on the bile ducts as it is not possible for a plasma coagulator to seal larger blood vessels. Burst pressure experiments are carried out on explanted organs to rule out blood pressure related effects.

Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo

Here we present a protocol to experimentally assess plasma coagulation in liver tissue in vivo. In a porcine model, microcirculation is examined by laser Doppler, coagulation depth is measured histologically, temperature at coagulation site by infrared thermometer and thermographic camera, and duct sealing effect is documented by burst pressure experiments.

Plasma coagulation as a form of electrocautery is used in liver surgery for decades to seal the large liver cut surface after major hepatectomy to prevent ...

Induction and Phenotyping of Acute Right Heart Failure in a Large Animal Model of Chronic Thromboembolic Pulmonary Hypertension

The development of acute right heart failure (ARHF) in the context of chronic pulmonary hypertension (PH) is associated with poor short-term outcomes. The morphological and functional phenotyping of the right ventricle is of particular importance in the context of hemodynamic compromise in patients with ARHF. Here, we describe a method to induce ARHF in a previously described large animal model of chronic PH, and to phenotype, dynamically, right ventricular function using the gold standard method (i.e., pressure-volume PV loops) and with a non-invasive clinically available method (i.e., echocardiography). Chronic PH is first induced in pigs by left pulmonary artery ligation and right lower lobe embolism with biological glue once a week for 5 weeks. After 16 weeks, ARHF is induced by successive volume loading using saline followed by iterative pulmonary embolism until the ratio of the systolic pulmonary pressure over systemic pressure reaches 0.9 or until the systolic systemic pressure decreases below 90 mmHg. Hemodynamics are restored with dobutamine infusion (from 2.5 &#181;g/kg/min to 7.5 &#181;g/kg/min). PV-loops and echocardiography are performed during each condition. Each condition requires around 40 minutes for induction, hemodynamic stabilization and data acquisition. Out of 9 animals, 2 died immediately after pulmonary embolism and 7 completed the protocol, which illustrates the learning curve of the model. The model induced a 3-fold increase in mean pulmonary artery pressure. The PV-loop analysis showed that ventriculo-arterial coupling was preserved after volume loading, decreased after acute pulmonary embolism and was restored with dobutamine. Echocardiographic acquisitions allowed to quantify right ventricular parameters of morphology and function with good quality. We identified right ventricular ischemic lesions in the model. The model can be used to compare different treatments or to validate non-invasive parameters of right ventricular morphology and function in the context of ARHF.

We present a protocol to induce and phenotype an acute right heart failure in a large animal model with chronic pulmonary hypertension. This model can be used to test therapeutic interventions, to develop right heart metrics&#160;or to improve the understanding of acute right heart failure pathophysiology.

The development of acute right heart failure (ARHF) in the context of chronic pulmonary hypertension (PH) is associated with poor short-term outcomes. The ...

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 ...