Name: surgical placement of catheters for long-term cardiovascular exercise testing in swine
Uploaded: 2016-02-09T15:00:00
Description: Watch this Scientific Journal Video about Surgical Placement of Catheters for Long-term Cardiovascular Exercise Testing in Swine at JoVE.com

This study was supported by Netherlands Heart Foundation grant 2000T038 (to D.J. Duncker) grant 2000T042 (to D. Merkus), European Commission FP7-Health-2010 grant MEDIA-261409 (to D.J. Duncker and D. Merkus), Netherlands CardioVascular Research Initiative: the Dutch Heart Foundation, the Dutch Federation for University Medical Centers, the Netherlands Organisation for Health Research and Development and the Royal Netherlands Academy of Sciences &#160;CVON- ARENA CVON 2011-11 (to D.J. Duncker), CVON-PHAEDRA CVON2012-08 ( to D.&#160;Merkus) &#160;and CVON-RECONNECT CVON 2014-11 (to D.J. Duncker and D. Merkus), Sophia Foundation (to D. de Wijs-Meijler, D. Merkus and I.K.M. Reiss).

Procedures involving animal subjects have been approved by the Animal Care Committee at Erasmus Medical Center Rotterdam (NL). Swine with weights between 6 and 80 kg have been successfully instrumented using this protocol.
1. Adaptation of the Animals to Human Handling
<ol>
	<li>After arrival in the facility, house the animals solitarily but enable them to interact with each other.</li>
	<li>Accustomize swine to human handling and transportation from the animal facility to the experimental laboratory, by handlin...

<ol>
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The present study describes the surgery for chronical instrumentation of swine as well as the protocol for exercising the instrumented swine on a motor-driven treadmill while measuring hemodynamics and taking blood samples for measurement of oxygen content in arterial, mixed venous and coronary venous blood.
Critical Steps within the Protocol 
There are several critical steps within the protocol that start already during the intubation procedure. Thiopental (2.1.5) is a respiratory depres...

Adequate cardiopulmonary function is essential to supply the body with oxygen and nutrients, particularly during conditions of increased metabolic demand such as during exercise1. The cardiopulmonary response to exercise is characterized by a number of adaptations in cardiac function, i.e., an increase in heart rate, contractility and stroke volume, and microvascular function, i.e., vasodilation in the vascular beds supplying exercising muscles as well as in the pulmonary vasculature, and vasoconstriction in the vascular beds supplying the gastrointestinal system as well as inactive muscles1. Impaired exercise capacity is an early hallmark of cardiopulmonary dysfunction, and cardiopulmonary exercise testing is used as an effective method to delineate between cardiac dysfunction, vascular dysfunction and/ or pulmonary dysfunction in patients with impaired exercise capacity2. Early cardiopulmonary dysfunction is difficult to diagnose, particularly in animal models, as cardiopulmonary function is often measured invasively, requiring anesthesia, with many anesthetic agents possessing cardiodepressive properties3.
Chronic instrumentation allows for measurement of cardiopulmonary function in the awake state, and when the animals are fully adjusted to the laboratory conditions measurements can be obtained under quiet resting conditions without the effects of anesthesia and acute surgical trauma. Furthermore, when the animals are appropriately trained, measurements can also be obtained during graded treadmill exercise4,5. More specifically, left and right ventricular function can be assessed and related to myocardial perfusion, while regulation of vasomotor tone in the coronary, systemic and pulmonary microcirculation can be determined. The use of fluid-filled catheters allows measurement of pressure as well as taking blood samples without imposing additional stress on the animals. Another advantage of studying chronically instrumented animals is that cardiopulmonary exercise testing can be repeated allowing the use of an animal as its own control, either during disease development or during chronic treatment, thereby increasing statistical power and hence limiting the number of animals required for a study.
Cardiopulmonary anatomy of swine closely resembles that of humans and it is possible to induce various forms of cardiopulmonary disease, such as diabetes 6, myocardial infarction 7, pulmonary hypertension 8,9 and pacing-induced heart failure10,11. Moreover, the size of swine allows chronic instrumentation, and repeated blood sampling of sufficient quantity to analyze not only blood gases, but also to perform neurohumoral measurements and/or to search for biomarkers of disease.
This protocol describes the surgery used to chronically instrument swine as well as the protocol for exercising the swine on a motor-driven treadmill.

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			<td height="21" style="height:21px;width:496px;">3-way stopcocks</td>
			<td style="width:212px;">B. Braun</td>
			<td align="right" style="width:179px;">16496</td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;">Perfusor lines PVC (DEHP-free) 150cm/2.6ml&#160;</td>
			<td>B. Braun</td>
			<td align="right" style="width:179px;">8722960</td>
			<td>Used for fluid filled catheters</td>
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			<td height="21" style="height:21px;">&#8220;python &#8220; silicontubing</td>
			<td>Rubber BV</td>
			<td>1757 ID 1mm, OD 2mm</td>
			<td>Used for fluid filled catheters</td>
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			<td height="21" style="height:21px;width:496px;">Sodium Chloride 0.9%</td>
			<td style="width:212px;">Baxter</td>
			<td style="width:179px;">TKF7124</td>
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			<td height="21" style="height:21px;width:496px;">Suction device</td>
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			<td></td>
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			<td height="21" style="height:21px;">Slim-Line electrosurgical pencil with 2 buttons</td>
			<td>ERBE ELEKTROMEDIZIN GMBH</td>
			<td>20190-066</td>
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			<td>Vererinary Technics Int.</td>
			<td>11.02.47</td>
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			<td style="width:212px;">3M</td>
			<td style="width:179px;">1818FS</td>
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			<td height="21" style="height:21px;width:496px;">surgical hat</td>
			<td style="width:212px;">Klinidrape</td>
			<td align="right" style="width:179px;">621301</td>
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			<td>Molnlycke Health Care</td>
			<td align="right" style="width:179px;">97027809</td>
			<td>Surgical drape, gauze pads, syringes, beaker etc</td>
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			<td style="width:212px;">Alcon</td>
			<td style="width:179px;">288-28282-01</td>
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			<td>Meda Pharma BV</td>
			<td>RVG08939</td>
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			<td>Meda Pharma BV</td>
			<td>RVG01331</td>
			<td></td>
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			<td height="21" style="height:21px;width:496px;">Cuffed Endotracheal tube</td>
			<td style="width:212px;">Emdamed</td>
			<td style="width:179px;"></td>
			<td>size depends on animal size</td>
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			<td height="21" style="height:21px;width:496px;">Breathing filter Hyrdo therm 3HME</td>
			<td style="width:212px;">Intersurgical</td>
			<td align="right" style="width:179px;">1560000</td>
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			<td style="width:212px;">Kawe Germany</td>
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			<td height="21" style="height:21px;width:496px;">Manual resuscitator- Combibag</td>
			<td style="width:212px;">Weinmann</td>
			<td>6515-12-313-5596</td>
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			<td height="21" style="height:21px;width:496px;">Perivascular flow probe 3PS</td>
			<td style="width:212px;">Transonic</td>
			<td style="width:179px;"></td>
			<td>For coronary artery; Size 2.5-4 mm depending on animal size</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:496px;">Confidence flow probe</td>
			<td style="width:212px;">Transonic</td>
			<td style="width:179px;"></td>
			<td>For aorta/pulmonary artery, 16-20 mm; size depends on animal size</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:496px;">Venflon-Venisystem 20Gx 32 mm</td>
			<td style="width:212px;">BD</td>
			<td align="right" style="width:179px;">393224</td>
			<td>For coronary venous catheter</td>
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			<td height="21" style="height:21px;width:496px;">Blunt Needle 18G</td>
			<td style="width:212px;"></td>
			<td style="width:179px;"></td>
			<td>For coronary venous catheter</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:496px;">Tygon Tubing</td>
			<td style="width:212px;">Rubber BV</td>
			<td>2802 ID 0.8mm (1/32&#8217;&#8217;), OD 2.4mm (3/32&#8217;&#8217;)</td>
			<td>For coronary venous catheter</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Suction Handle 17 cm 6 6/8 &#34; Coupland 18/8 martinit with tube connector</td>
			<td>KLS Martin Group</td>
			<td>18-575-24</td>
			<td></td>
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			<td height="21" style="height:21px;width:496px;">Scalple blade&#160;</td>
			<td style="width:212px;"></td>
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			<td></td>
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			<td height="21" style="height:21px;">Scalpel Handle 13.5 cm 5 3/8 &#34; Stainless Steel solid</td>
			<td>KLS Martin Group</td>
			<td>10-100-04</td>
			<td></td>
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			<td height="21" style="height:21px;">Vascular Forceps 20.2 cm 8 &#34; De Bakey Stainless Stee</td>
			<td>KLS Martin Group</td>
			<td>24-388-20</td>
			<td>&#177; 14 cm</td>
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			<td height="21" style="height:21px;">Dressing Forceps 17 cm 6 6/8 &#34; Cushing Stainless Steel</td>
			<td>KLS Martin Group</td>
			<td>12-189-17</td>
			<td>&#177; 18 cm</td>
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			<td height="21" style="height:21px;">halsted-musquito straight 12.5cm - 5&#34;</td>
			<td>Rudolf Medical</td>
			<td>RU-3100-13</td>
			<td>&#177; 12 cm</td>
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			<td height="21" style="height:21px;">halsted-musquito curved 12.5cm - 5&#34;</td>
			<td>Rudolf Medical</td>
			<td>RU-3101-12</td>
			<td>&#177; 12 cm</td>
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			<td height="21" style="height:21px;">Dissecting and Ligature Forceps 13 cm 5 1/8 &#34; Gemini Stainless Steel</td>
			<td>KLS Martin Group</td>
			<td>13-451-13</td>
			<td>&#177; 12 cm</td>
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			<td height="21" style="height:21px;">Dissecting and Ligature Forceps 18.5 cm 7 2/8 &#34; Schnidt Stainless Steel</td>
			<td>KLS Martin Group</td>
			<td>13-363-18</td>
			<td></td>
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			<td height="21" style="height:21px;">Rib Retractor Finochietto, Baby Aluminium -</td>
			<td>KLS Martin Group</td>
			<td>24-162-01</td>
			<td></td>
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			<td height="21" style="height:21px;">suture forceps Mayo-Hegar 3mm 18cm - 7&#34;</td>
			<td>Rudolf Medical</td>
			<td>RU-6050-18</td>
			<td></td>
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			<td height="21" style="height:21px;">Metchenbaum blunt curved 14,5cm - 5(3/4)&#34;</td>
			<td>Rudolf Medical</td>
			<td>RU-1311-14M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Retrector farabeuf 12cm - 4 (3/4)&#34;</td>
			<td>Rudolf Medical</td>
			<td>RU-4497-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Towel forceps schr&#228;del curved 9cm - 3,5&#34;</td>
			<td>Rudolf Medical</td>
			<td>RU-3550-09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">surgical scissors blunt 13cm - 5&#34;</td>
			<td>Rudolf Medical</td>
			<td>RU-1001-13</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gauzes Cutisoft 10 x 10 cm 4-ply</td>
			<td>BSN Medical</td>
			<td>45846-00</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Gauzes Cutisoft 5 x 5 cm 4-ply</td>
			<td>BSN Medical</td>
			<td>45844-00</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:496px;">Flowmeter -CM2 / SF2 - 2gas (O2 and Air)</td>
			<td>UNO BV</td>
			<td align="right" style="width:179px;">180000008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tec 7 Vaporizer</td>
			<td>Datex-Ohmeda</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:496px;">Acederm wound spay</td>
			<td style="width:212px;">Ecuphar NV</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:496px;">Vaseline Album</td>
			<td style="width:212px;">Bufa</td>
			<td align="right" style="width:179px;">165313</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:496px;">silkam 3-0 Natural silk, non-absorbable</td>
			<td>B. Braun</td>
			<td>F 1134043</td>
			<td>sutures for placement of catheters</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:496px;">silkam 2-0 Natural silk, non-absorbable</td>
			<td>B. Braun</td>
			<td>F 1134051</td>
			<td>sutures for muscular approximation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:496px;">dagrofil 3-0 Polyester, non-absorbable</td>
			<td>B. Braun</td>
			<td>C 0842478</td>
			<td>sutures for fluid fille catheters after tunneling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vicryl rapide 3-0, 1x45 cm FS2, V2930G</td>
			<td>Daxtrio medische producten</td>
			<td>15560</td>
			<td>sutures for electrical catheters after tunneling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:496px;">Vitafil 6 USP</td>
			<td style="width:212px;">SMI</td>
			<td align="right" style="width:179px;">6080</td>
			<td>Ties</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:496px;">Syringes</td>
			<td style="width:212px;"></td>
			<td style="width:179px;"></td>
			<td>10 ml and 2.5 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:496px;">Heparin LEO (heparin sodium)&#160;</td>
			<td>LEO Pharma A/S</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:496px;">Zoletil</td>
			<td style="width:212px;">Virbac</td>
			<td style="width:179px;"></td>
			<td>tiletamine / zolazepam</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:496px;">Sedazine</td>
			<td style="width:212px;">AST farma</td>
			<td align="right" style="width:179px;">108855</td>
			<td>xylazine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:496px;">Temgesic</td>
			<td style="width:212px;">RB Pharmaceuticals</td>
			<td align="right" style="width:179px;">5429</td>
			<td>buprenorphine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:496px;">Tensogrip</td>
			<td style="width:212px;">BSN Medical</td>
			<td style="width:179px;">71522-00</td>
			<td>elastic vest</td>
		</tr>
	</tbody>
</table>

surgical placement of catheters for long-term cardiovascular exercise testing in swine

This protocol describes the surgical procedure to chronically instrument swine and the procedure to exercise swine on a motor-driven treadmill. Early cardiopulmonary dysfunction is difficult to diagnose, particularly in animal models, as cardiopulmonary function is often measured invasively, requiring anesthesia. As many anesthetic agents are cardiodepressive, subtle changes in cardiovascular function may be masked. In contrast, chronic instrumentation allows for measurement of cardiopulmonary function in the awake state, so that measurements can be obtained under quiet resting conditions, without the effects of anesthesia and acute surgical trauma. Furthermore, when animals are properly trained, measurements can also be obtained during graded treadmill exercise.
Flow probes are placed around the aorta or pulmonary artery for measurement of cardiac output and around the left anterior descending coronary artery for measurement of coronary blood flow. Fluid-filled catheters are implanted in the aorta, pulmonary artery, left atrium, left ventricle and right ventricle for pressure measurement and blood sampling. In addition, a 20 G&#160;catheter is positioned in the anterior interventricular vein to allow coronary venous blood sampling.
After a week of recovery, swine are placed on a motor-driven treadmill, the catheters are connected to pressure and flow meters, and swine are subjected to a five-stage progressive exercise protocol, with each stage lasting 3 min. Hemodynamic signals are continuously recorded and blood samples are taken during the last 30 sec of each exercise stage.
The major advantage of studying chronically instrumented animals is that it allows serial assessment of cardiopulmonary function, not only at rest but also during physical stress such as exercise. Moreover, cardiopulmonary function can be assessed repeatedly during disease development and during chronic treatment, thereby increasing statistical power and hence limiting the number of animals required for a study.

Exercise up to 5 km/hr resulted in a doubling of cardiac output from 4.3 &#177; 0.3 to 8.5 &#177; 0.7 L/min which was principally accomplished by an increase in heart rate from 137 &#177; 7 to 256 &#177; 8 beats per min in combination with a small increase in stroke volume from 32 &#177; 2 to 36 &#177; 3 ml (Figure 3). The increase in stroke volume was facilitated by an increase in left ventricular contractility, as evidenced by an increase in the maximum of the first der...

Watch this Scientific Journal Video about Surgical Placement of Catheters for Long-term Cardiovascular Exercise Testing in Swine at JoVE.com

Surgical Placement of Catheters for Long-term Cardiovascular Exercise Testing in Swine

Here we present a protocol to assess cardiopulmonary function in awake swine, at rest and during graded treadmill exercise. Chronic instrumentation allows for repeated hemodynamic measurements uninfluenced by cardiodepressive anesthetic agents.

This protocol describes the surgical procedure to chronically instrument swine and the procedure to exercise swine on a motor-driven treadmill. Early ...

The overall goal of this surgery is to chronically instrument animals to allow for hemodynamic measurements during cardiopulmonary stress testing in alert animals with and without cardiovascular disease. This method can help answer key questions in the cardiovascular field, such as how metabolic syndrome affects left ventricular function and perfusion therby contributing to the development of heart failure with preserved ejection fraction. Two key advantages of this technique are that hemodynamic measurements and blood samples are obtained from awake animals and the technique can be performed at rest and during cardiovascular stress such as exercise.The implications of this technique can extend towards early diagnosis of pulmonary hypertension because pulmonary microvascular function, as well as right ventricular function and perfusion can be measured at well defined stages after introduction of the disease. Visual demonstration of this technique is essential. It is difficult to learn how to place a flow probe around a coronary artery due to vasospasm of this vessel.Also, placing the flow probe around aorta is difficult due to location of this vessel with respect to the thoracotomy. Demonstrating the procedure will be Annemarie Verzijl, a technician from our laboratory. After preparing the animal for surgery, make an incision in the skin, starting one centimeter caudal to the left inferior angle of the scapula and cut downward to the left axilla.Following the incision, use diathermy to cauterize blood vessels in the skin. Next, using the cutting modality of the diathermy, cut though the serratus muscle and pectoralis major muscle. Continue using diathermy to cauterize blood vessels in the muscle layer.Now, using blunt dissection, carefully divide the intercostal muscle of the 4th left intercostal space. Finish this using a mosquito clamp. Then expose the costal surface of the left lung, which is covered with visceral and parietal pleura.Next, enter the pleural cavity by carefully piercing both layers of the pleura and tearing them open. Improve the exposure with a thoracic retractor by separting the edges of the wound and ribs then forcefully pushing the tissues apart. To expose the heart and great vessels, push the left lung caudally and wedge it in place with wet gauze.Begin this portion of the surgery with a blunt dissection to remove about 2 square centimeters of connective tissue surrounding the descending thoracic aorta. Next, place a three stitch purse string suture in the aortic wall using non-absorbable USP 3-0 braided silk suture. Then penetrate the aortic vessel wall through the suture with a stainless steel, 16-gauge needle.Following the needle, insert the tip of the fluid-filled catheter into the aorta. Then pull the purse string suture firmly together and tie it off. To secure the catheter, wind the excess suture around the catheter above the ring three times, tie it off, and add a new stitch about 1 centimeter cranial to its insertion site.Now connect the fluid-filled catheter to the calibrated pressure transducer connected to a computer. Thus, monitor the mean arterial pressure during the subsequent steps. Next, without damaging the phrenic nerve, open the pericardium with a crossed cut.Beneath the cut, expose the ascending aorta and aortic arch with a Farabeuf retractor by identifying the pulmonary artery and gently pulling it caudally. With the aorta exposed, make a 1 centimeter cut in the connective tissue between the ascending aorta and the pulmonary artery using Metzenbaum scissors. Now attach the flow probe.first get the rubber band around the vessel by using a suture lead. And secondly, attach the flow probe measurement device to the rubber band. Thirdly, connect the flow probe to the computer and fourthly, check the cardiac output signal to confirm the probe is properly positioned.The next step is to expose and dissect the proximal part of the left anterior descending coronary artery. First, lift the tissue with forceps. And then, using Metzenbaum scissors, make a small cut in the tissue.Then carefully tease away tissues from the artery using a cotton swab. And to ensure the coronary artery is completely dissected, pass a small, straight-angled mosquito clamp underneath it. When vasospasm of the coronary artery occurs, spray with 10%lidocane to relax the vessel.Using the same technique, place fluid-filled catheters in the pulmonary artery, right ventricle, left ventricle, and left atrium. All the catheters should now be connected to the computer. Then make a stitch parallel to the anterior interventricular coronary vein with a suture connected to the coronary venous catheter.Following the stitch, puncture the vein with the 20-gauge needle of the coronary venous catheter and insert the cannula of the catheter intravenously. Then secure the catheter with the existing stitch. Remove the needle and connect to the extension line.Now place a coronary flow probe around the previously dissected left anterior descending coronary artery. With the probe placed, check the signal of the coronary flow on the computer to confirm that the flow probe is correctly placed. The shape of the coronary flow signal should be similar to that shown here.Begin this phase of the surgery by making a 1.5 centimeter incision about 8 centimeters caudal and parallel to the first incision. Then using a large, curved mosquito clamp, subcutaneously lead the drain from the pleural cavity through the 6th intercostal space to this incision. Connect the drain to the suction device to remove remaining fluid from the thorax.Next, use a large, curved mosquito clamp to tunnel the flow probes individually through the 3rd left intercostal space and the muscle above the rib. Tunnel the fluid-filled catheters through either the 3rd or the 5th left intercostal space. Firstly, clamp off the fluid-filled catheters.Next, remove the three-way stopcock to minimize the piercing area. And thirdly, pierce the intercostal muscle. Now, fix the flow probes and the fluid-filled catheters with non-absorbable USP 2-0 braided silk.Use a purse string suture on the intercostal muscle. This sutre will also prevent air leakage after reinstating negative intrathoracic pressure. Next, make three incisions in the skin about 2 centimeters to the left and parallel to the vertebral column.Make these three incisions 3 centimeters long and 3 centimeters apart from each other. Now pierce a trocar beneath the left latissimus dorsi muscle from the rostral incision site to the incisions on the back. Then using the trocar, tunnel the flow probes and fluid catheters to the back.Once tunneling is complete, place stopcocks on the fluid-filled catheters and remove the clamp. Withdraw some blood to remove the clots and air bubbles. Then fill the fluid-filled catheters with heparin at 1, 000 units per milliliter, and fill the coronary venous catheters with heparin at 5, 000 units per milliliter.Close the thorax by pulling together the ribs of the 4th intercostal space at two separate sites. Use non-absorbable USP 6 braided polyester. Then close the serratus muscle and pectoralis major muscle with the running stitch.Close the skin with the running subcuticular suture using non-absorbable USP 2-0 braided silk. Then sutre the incisions on the dorsal side between the catheters. First, tie a knot directly onto the skin to close the incision.Then fixate the catheters to the suture with another knot about 1 centimeter from the skin. Suture the flow probes with polyglactin suture so that the flow probe wire is not cut by the suture. Then carefully remove the drain while applying pressure to the cranial side of the incsion so as to reinstate negative pressure in the pleural cavity.Now complete the surgery by closing the incision with a purse string suture and sealing the wound with petroleum jelly. Follow the text protocol on how to recover the animal and how to conduct the treadmill experiment. Exercising swine to move up to 5 kilometers per hour resulted in a doubling of cardiac output.A tripling of body oxygen consumption was observed. This was due to the increase in cardiac output coupled with an increase in hemoglobin concentration and an increase in body oxygen extraction. This was principally accomplished by an increase in heart rate with a small increase in stroke volume.The increase in stroke volume was facilitated by an increase in left ventricular contractility as evidenced by an increase in the maximum of the 1st derivative of left ventricular pressure together with an increased rate of relaxation of the left ventricle and an increase in left atrial pressure, which is the filling pressure of the left ventricle. Systemic vasodilation occurred as evidenced by an increase in systemic vascular conductance and a decrease in systemic vascular resistance. This accommodated the increase in cardiac output almost completely so that mean aortic pressure increased only slightly.Further interpretations of several other parameters are provided in the text protocol. After watching this video, you should have a good understanding of how to chronically instrument animals to make hemodynamic measurements in subsequent cardiovascular experiments.

surgical-placement-catheters-for-long-term-cardiovascular-exercise

Research

JoVE Journal

Medicine

Minimal Invasive Surgical Procedure of Inducing Myocardial Infarction in Mice

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last decades. For clarification of the underlying mechanisms and the development of new therapeutic strategies, the availability of valid animal models are mandatory. Since we need new insights into pathomechanisms of cardiovascular diseases under in vivo conditions to combat myocardial infarction, the validity of the animal model is a crucial aspect. However, protection of animals are highly relevant in this context. Therefore, we establish a minimally invasive and simple model of myocardial infarction in mice, which assures a high reproducibility and survival rate of animals. Thus, this models fulfils the requirements of the 3R principle (Replacement, Refinement and Reduction) for animal experiments and assure the scientific information needed for further developing of therapeutical strategies for cardiovascular diseases.

A highly reproducible model for myocardial infarction in mice with minimal invasive manipulations is described. The model can be easily performed, resulting in a high reproducibility and survival rate. Thus, the described model will reduce the number of required animals as requested by the 3R principle (Replacement, Refinement and Reduction).

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last ...

Testing the Efficacy of Pharmacological Agents in a Pericardial Target Delivery Model in the Swine

To date, many pharmacological agents used to treat or prevent arrhythmias in open-heart cases create undesired systemic side effects. For example, antiarrhythmic drugs administered intravenously can produce drops in systemic pressure in the already compromised cardiac patient. While performing open-heart procedures, surgeons will often either create a small port or form a pericardial cradle to create suitable fields for operation. This access yields opportunities for target pharmacological delivery (antiarrhythmic or ischemic preconditioning agents) directly to the myocardial tissue without undesired side effects.
We have developed a swine model for testing pharmacological agents for target delivery within the pericardial fluid. While fully anesthetized, each animal was instrumented with a Swan-Ganz catheter as well as left and right ventricle pressure catheters, and pacing leads were placed in the right atrial appendage and the right ventricle. A medial sternotomy was then performed and a pericardial access cradle was created; a plunge pacing lead was placed in the left atrial appendage and a bipolar pacing lead was placed in the left ventricle. Utilizing a programmer and a cardiac mapping system, the refractory period of the atrioventricular node (AVN), atria and ventricles was determined. In addition, atrial fibrillation (AF) induction was produced utilizing a Grass stimulator and time in AF was observed. These measurements were performed prior to treatment, as well as 30 min&#160;and 60 min&#160;after pericardial treatment. Additional time points were added for selected studies. The heart was then cardiopleged and reanimated in a four chamber working mode. Pressure measurements and function were recorded for 1 hr after reanimation. This treatment strategy model allowed us to observe the effects of pharmacological agents that may decrease the incidence of cardiac arrhythmias and/or ischemic damage, during and after open-heart surgery.

We have developed a swine model for the target delivery of pharmacological agents within the pericardial space/fluid. Using this approach, the relative benefits of administered agents on induced atrial fibrillation, relative refractory periods and/or ischemic protection can be investigated.

To date, many pharmacological agents used to treat or prevent arrhythmias in open-heart cases create undesired systemic side effects. For example, ...

Long-term Blood Pressure Measurement in Freely Moving Mice Using Telemetry

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. Indeed, research focusing on the discovery of new potential therapeutic targets using genetically altered mice requires a reliable, long-term assessment of the systemic arterial pressure variation. Currently, the gold standard for obtaining long-term measurements of blood pressure in ambulatory mice uses implantable radio-transmitters, which require&#160;artery cannulation. This technique eliminates the need for tethering, restraining, or anesthetizing the animals which introduce stress and artifacts during data sampling. However, arterial blood pressure monitoring in mice via catheterization can be rather challenging due to the small size of the arteries. Here we present a step-by-step guide to illustrate the crucial key passages for a successful subcutaneous implantation of radio-transmitters and carotid artery cannulation in mice. We also include examples of long-term blood pressure activity taken from freely moving mice after a period of post-surgery recovery. Following this procedure will allow reliable direct blood pressure recordings from multiple animals simultaneously.

The goal of this protocol is to assess systemic blood pressure in conscious freely moving mice using implantable radio-telemetry devices.

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. ...

Tachycardia-Induced Cardiomyopathy As a Chronic Heart Failure Model in Swine

A stable and reliable model of chronic heart failure is required for many experiments to understand hemodynamics or to test effects of new treatment methods. Here, we present such a model by tachycardia-induced cardiomyopathy, which can be produced by rapid cardiac pacing in swine.
A single pacing lead is introduced transvenously into fully anaesthetized healthy swine, to the apex of the right ventricle, and fixated. Its other end is then tunneled dorsally to the paravertebral region. There, it is connected to an in-house modified heart pacemaker unit that is then implanted in a subcutaneous pocket. 
After 4 - 8 weeks of rapid ventricular pacing at rates of 200 - 240 beats/min, physical examination revealed signs of severe heart failure - tachypnea, spontaneous sinus tachycardia, and fatigue. Echocardiography and X-ray showed dilation of all heart chambers, effusions, and severe systolic dysfunction. These findings correspond well to decompensated dilated cardiomyopathy and are also preserved after the cessation of pacing.
This model of tachycardia-induced cardiomyopathy can be used for studying the pathophysiology of progressive chronic heart failure, especially hemodynamic changes caused by new treatment modalities like mechanical circulatory supports. This methodology is easy to perform and the results are robust and reproducible.

Here, we present a protocol to produce tachycardia-induced cardiomyopathy in swine. This model represents a potent way to study the hemodynamics of progressive chronic heart failure and the effects of applied treatment.

A stable and reliable model of chronic heart failure is required for many experiments to understand hemodynamics or to test effects of new treatment ...

Harvest of Endothelial Cells from the Balloon Tips of Swan-Ganz Catheters after Right Heart Catheterization

A variety of pathologies lead to pulmonary hypertension (PH), which is defined as a mean pulmonary artery pressure exceeding 25 mmHg at rest. To further diagnose and manage PH, patients undergo repeated right heart catheterizations (RHC) wherein a Swan-Ganz catheter is advanced into a branch of the pulmonary artery and a balloon is inflated to wedge the catheter tip. This article illustrates a protocol whereby pulmonary artery endothelial cells (PAECs) may be harvested from the balloon tips of Swan-Ganz catheters after RHC, and purified with an anti- CD146 affinity column technique to purify putative PAECs. These cells might be used to provide an in situ snapshot of the biological state of the pulmonary vasculature endothelium to complement hemodynamic measurements obtained during RHC. Harvested and purified PAECs may be used for either cell culture or for subsequent analytical assays such as flow cytometery.

This protocol describes the removal and purification of pulmonary artery endothelial cells (PAECs) that adhere to the balloon tips of Swan-Ganz catheters while they are wedged in the pulmonary artery during right heart catheterization and remain adherent to the deflated balloon following removal from the patient's body.

A variety of pathologies lead to pulmonary hypertension (PH), which is defined as a mean pulmonary artery pressure exceeding 25 mmHg at rest. To further ...

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the treatment of many hematopoietic diseases in humans, as their use offers stable transgene expression due to the vector&#39;s ability to integrate into the host genome. This method demonstrates the application of ex vivo gene therapy of hepatocytes to a large animal model of hereditary tyrosinemia type I. This process consists of 1) isolation of primary hepatocytes from the autologous donor/recipient animal, 2) ex vivo gene delivery via hepatocyte transduction with a lentiviral vector, and 3) autologous transplant of corrected hepatocytes via portal vein injection. Success of the method generally relies upon efficient and sterile removal of the liver resection, careful handling of the excised specimen for isolation of viable hepatocytes sufficient for re-engrafting, high-percentage transduction of the isolated cells, and aseptic surgical procedures throughout to prevent infection. Technical failure at any of these steps will result in low yield of viable transduced hepatocytes for autologous transplant or infection of the donor/recipient animal. The pig model of human type 1 hereditary tyrosinemia (HT-1) chosen for this approach is uniquely amenable to such a method, as even a small percentage of engraftment of corrected cells will lead to repopulation of the liver with healthy cells based on a powerful selective advantage over native-diseased hepatocytes. Although this growth selection will not be true for all indications, this approach is a foundation for expansion into other indications and allows for manipulation of this environment to address additional diseases, both within the liver and beyond, while controlling for exposure to viral vector and opportunity for off-target toxicity and tumorigenicity.

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

This protocol is intended to describe porcine hepatocyte isolation and ex vivo gene delivery to cure models of metabolic diseases via autologous cell transplantation. Although this particular model enjoys unique advantages that favor successful therapy, the application is a relevant foundation to address additional diseases and indications.

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the ...

Conducting Maximal and Submaximal Endurance Exercise Testing to Measure Physiological and Biological Responses to Acute Exercise in Humans

Regular physical activity has a positive effect on human health, but the mechanisms controlling these effects remain unclear. The physiologic and biologic responses to acute exercise are predominantly influenced by the duration and intensity of the exercise regimen. As exercise is increasingly thought of as a therapeutic treatment and/or diagnostic tool, it is important that standardizable methodologies be utilized to understand the variability and to increase the reproducibility of exercise outputs and measurements of responses to such regimens. To that end, we describe two different cycling exercise regimens that yield different physiologic outputs. In a maximal exercise test, exercise intensity is continually increased with a greater workload resulting in an increasing cardiopulmonary and metabolic response (heart rate, stroke volume, ventilation, oxygen consumption and carbon dioxide production). In contrast, during endurance exercise tests, the demand is increased from that at rest, but is raised to a fixed submaximal exercise intensity resulting in a cardiopulmonary and metabolic response that typically plateaus. Along with the protocols, we provide suggestions on measuring physiologic outputs that include, but are not limited to, heart rate, slow and forced vital capacity, gas exchange metrics, and blood pressure to enable the comparison of exercise outputs between studies. Biospecimens can then be sampled to assess cellular, protein, and/or gene expression responses. Overall, this approach can be easily adapted into both short- and long-term effects of two distinct exercise regimens.

To assess the influence of exercise intensity on physiologic and biologic responses, two different exercise testing protocols were utilized. Methods outlining exercise testing on a cycle ergometer as an incremental maximal oxygen consumption test and endurance, steady state submaximal endurance test are described.

Regular physical activity has a positive effect on human health, but the mechanisms controlling these effects remain unclear. The physiologic and biologic ...

Integration of Brain Tissue Saturation Monitoring in Cardiopulmonary Exercise Testing in Patients with Heart Failure

Cerebral hypo-oxygenation during rest or exercise negatively impacts the exercise capacity of patients with heart failure with reduced ejection fraction (HF). However, in clinical cardiopulmonary exercise testing (CPET), cerebral hemodynamics is not assessed. NIRS is used to measure cerebral tissue oxygen saturation (SctO2) in the frontal lobe. This method is reliable and valid and has been utilized in several studies. SctO2 is lower during both rest and peak exercise in patients with HF than in healthy controls (66.3 &#177; 13.3% and 63.4 &#177; 13.8% vs. 73.1 &#177; 2.8% and 72 &#177; 3.2%). SctO2 at rest is significantly linearly correlated with peak VO2 (r = 0.602), oxygen uptake efficiency slope (r = 0.501), and brain natriuretic peptide (r = -0.492), all of which are recognized prognostic and disease severity markers, indicating its potential prognostic value. SctO2 is determined mainly by end-tidal CO2 pressure, mean arterial pressure, and hemoglobin in the HF population. This article demonstrates a protocol that integrates SctO2 using NIRS into incremental CPET on a calibrated bicycle ergometer.

This protocol integrated near-infrared spectroscopy into conventional cardiopulmonary exercise testing to identify the involvement of the cerebral hemodynamic response in exercise intolerance in patients with heart failure.

Cerebral hypo-oxygenation during rest or exercise negatively impacts the exercise capacity of patients with heart failure with reduced ejection fraction ...

Novel Percutaneous Approach for Deployment of 3D Printed Coronary Stenosis Implants in Swine Models of Ischemic Heart Disease

Minimally invasive methods for creating models of focal coronary narrowing in large animals are challenging. Rapid prototyping using three-dimensionally (3D) printed coronary implants can be employed to percutaneously create a focal coronary stenosis. However, reliable delivery of the implants can be difficult without the use of ancillary equipment. We describe the use of a mother-and-child coronary guide catheter for stabilization of the implant and for effective delivery of the 3D printed implant to any desired location along the length of the coronary vessel. The focal coronary narrowing was confirmed under coronary cineangiography and the functional significance of the coronary stenosis was assessed using gadolinium-enhanced first-pass cardiac perfusion MRI. We showed that reliable delivery of 3D printed coronary implants in swine models (n = 11) of ischemic heart disease can be achieved through repurposing mother-and-child coronary guide catheters. Our technique simplifies the percutaneous delivery of coronary implants to create closed-chest swine models of focal coronary artery stenosis and can be performed expeditiously, with a low procedural failure rate.

We describe a novel, cost-effective, and efficient technique for percutaneous delivery of three-dimensionally printed coronary implants to create closed-chest swine models of ischemic heart disease. The implants were fixed in place using a mother-and-child extension catheter with high success rate.

Minimally invasive methods for creating models of focal coronary narrowing in large animals are challenging. Rapid prototyping using three-dimensionally ...

Analyzing Long-Term Electrocardiography Recordings to Detect Arrhythmias in Mice

Arrhythmias are common, affecting millions of patients worldwide. Current treatment strategies are associated with significant side effects and remain ineffective in many patients. To improve patient care, novel and innovative therapeutic concepts causally targeting arrhythmia mechanisms are needed. To study the complex pathophysiology of arrhythmias, suitable animal models are necessary, and mice have been proven to be ideal model species to evaluate the genetic impact on arrhythmias, to investigate fundamental molecular and cellular mechanisms, and to identify potential therapeutic targets.
Implantable telemetry devices are among the most powerful tools available to study electrophysiology in mice, allowing continuous ECG recording over a period of several months in freely moving, awake mice. However, due to the huge number of data points (&gt;1 million QRS complexes per day), analysis of telemetry data remains challenging. This article describes a step-by-step approach to analyze ECGs and to detect arrhythmias in long-term telemetry recordings using the software, Ponemah, with its analysis modules, ECG Pro and Data Insights, developed by Data Sciences International (DSI). To analyze basic ECG parameters, such as heart rate, P wave duration, PR interval, QRS interval, or QT duration, an automated attribute analysis was performed using Ponemah to identify P, Q, and T waves within individually adjusted windows around detected R waves. 
Results were then manually reviewed, allowing adjustment of individual annotations. The output from the attribute-based analysis and the pattern recognition analysis was then used by the Data Insights module to detect arrhythmias. This module allows an automatic screening for individually defined arrhythmias within the recording, followed by a manual review of suspected arrhythmia episodes. The article briefly discusses challenges in recording and detecting ECG signals, suggests strategies to improve data quality, and provides representative recordings of arrhythmias detected in mice using the approach described above.

Here we present a step-by-step protocol for a semiautomated approach to analyze murine long-term electrocardiography (ECG) data for basic ECG parameters and common arrhythmias. Data are obtained by implantable telemetry transmitters in living and awake mice and analyzed using Ponemah and its analysis modules.

Arrhythmias are common, affecting millions of patients worldwide. Current treatment strategies are associated with significant side effects and remain ...