Name: a model of reverse vascular remodeling in pulmonary hypertension due to left heart disease by aortic debanding in rats
Uploaded: 2022-03-01T14:45:00
Description: Watch this Scientific Journal Video about A Model of Reverse Vascular Remodeling in Pulmonary Hypertension Due to Left Heart Disease by Aortic Debanding in Rats at JoVE.com

This research was supported by grants of the DZHK (German Centre for Cardiovascular Research) to CK and WMK, the BMBF (German Ministry of Education and Research) to CK in the framework of VasBio, and to WMK in the framework of VasBio, SYMPATH, and PROVID, and the German Research Foundation (DFG) to WMK (SFB-TR84 A2, SFB-TR84 C9, SFB 1449 B1, SFB 1470 A4, KU1218/9-1, and KU1218/11-1).

All procedures were performed following the &#34;Guide for the Care and Use of Laboratory Animals&#34; (Institute of Laboratory Animal Resources, 8th edition 2011) and approved by the local governmental animal care and use committee of the German State Office for Health and Social Affairs (Landesamt f&#252;r Gesundheit und Soziales (LaGeSO), Berlin; protocol no. G0030/18). First, congestive heart failure was surgically induced in juvenile Sprague-Dawley rats ~100 g body weight (bw) (see Table of Materials</strong...

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Here, a detailed surgical technique for aortic debanding in a rat AoB model is reported that can be utilized to investigate the reversibility of PH-LHD and the cellular and molecular mechanisms that drive reverse remodeling in the pulmonary vasculature and the RV. Three weeks of aortic constriction in juvenile rats results in PH-LHD evident as increased LV pressures, LV hypertrophy, and concomitantly increased RV pressures and RV hypertrophy. Aortic debanding at week 3 post-AoB was able to unload the LV and fully reverse...

Heart failure is the leading cause of death in developed countries and is expected to increase by 25% over the next decade. Pulmonary hypertension (PH) - a pathological increase of blood pressure in the pulmonary circulation - affects approximately 70% of patients with end-stage heart failure; the World Health Organization classifies PH as pulmonary hypertension due to left heart disease (PH-LHD)1. PH-LHD is initiated by impaired systolic and/or diastolic left ventricular (LV) function that results in elevated filling pressure and passive congestion of blood into the pulmonary circulation2. Albeit initially reversible, PH-LHD gradually becomes fixed due to active pulmonary vascular remodeling in all compartments of the pulmonary circulation, i.e., arteries, capillaries, and veins3,4. Both reversible and fixed PH increase RV afterload, initially driving adaptative myocardial hypertrophy but ultimately causing RV dilatation, hypokinesis, fibrosis, and decompensation that progressively lead to RV failure1,2,5,6. As such, PH accelerates disease progression in heart failure patients and increases mortality, particularly in patients undergoing surgical treatment by implantation of left ventricular assist devices (LVAD) and/or heart transplantation7,8,9. Currently, no curative therapies exist that could reverse the process of pulmonary vascular remodeling, so fundamental mechanistic research in appropriate model systems is needed.
Importantly, clinical studies show that PH-LHD as a frequent complication in patients with aortic stenosis can improve rapidly in the early post-operative period following aortic valve replacement10. Analogously, high (&#62;3 Wood Units) pre-operative pulmonary vascular resistance (PVR) that was, however, reversible on nitroprusside was sustainably normalized after heart transplantation in a 5-year follow-up study11. Similarly, an adequate reduction of both reversible and fixed PVR and improvement of RV function in LHD patients could be realized within several months by unloading the left ventricle using implantable pulsatile and non-pulsatile ventricular assist devices12,13,14. Currently, the cellular and molecular mechanisms that drive reverse remodeling in the pulmonary circulation and RV myocardium are unclear. Yet, their understanding may provide important insight into physiological pathways that may be therapeutically exploited to reverse lung vascular and RV remodeling in PH-LHD and other forms of PH.
A suitable preclinical model that adequately replicates the pathophysiological and molecular features of PH-LHD can be used for translational studies in pressure overload-induced congestive heart failure due to surgical aortic banding (AoB) in rats4,15,16. In comparison to similar heart failure due to pressure overload in the murine model of transverse aortic constriction (TAC)17, banding of the ascending aorta above the aortic root in AoB rats does not produce hypertension in the left carotid artery as the banding site is proximal of the outflow of the left carotid artery from the aorta. As a result, AoB does not cause left-sided neuronal injury in the cortex as is characteristic for TAC18, and which may affect the study outcome. Compared to other rodent models of surgically induced PH-LHD, rat models in general, and AoB in particular, prove to be more robust, reproducible and replicate the remodeling of the pulmonary circulation characteristic for PH-LHD patients. At the same time, perioperative lethality is low19. Increased LV pressures and LV dysfunction in AoB rats induce PH-LHD development, resulting in elevated RV pressures&#160;and RV&#160;remodeling. As such, the AoB rat model has proven extremely useful in a series of previous studies by independent groups, including ourselves, to identify pathomechanisms of pulmonary vascular remodeling and test potential treatment strategies for PH-LHD4,15,20,21,22,23,24,25.
In the present study, the AoB rat model was utilized to establish a surgical procedure of aortic debanding to study mechanisms of reverse remodeling in the pulmonary vasculature and the RV. Previously, myocardial reverse remodeling models such as aortic debanding in mice26 and rats27 have been developed to investigate the cellular and molecular mechanisms regulating the regression of left ventricular hypertrophy and test potential therapeutic options to promote myocardial recovery. Moreover, a limited number of earlier studies have explored the effects of aortic debanding on PH-LHD in rats and showed that aortic debanding might reverse medial hypertrophy in pulmonary arterioles, normalize the expression of pre-pro-endothelin 1 and improve pulmonary hemodynamics27,28, providing evidence for the reversibility of PH in rats with heart failure. Here, the technical procedures of the debanding surgery are optimized and standardized, e.g., by applying a tracheotomy instead of endotracheal intubation or by using titanium clips of a defined inner diameter for aortic banding instead of polypropylene sutures with a blunt needle26,27, thus providing for better control of the surgical procedures, increased reproducibility of the model and an improved survival rate.
From a scientific perspective, the significance of the PH-LHD debanding model does not solely lie in demonstrating the reversibility of the cardiovascular and pulmonary phenotype in heart failure, but more importantly, in the identification of molecular drivers that trigger and/or sustain reverse remodeling in pulmonary arteries as promising candidates for future therapeutic targeting.

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		<tr>
			<td>Amoxicillin</td>
			<td>Ratiopharm</td>
			<td>PC: 04150075615985</td>
			<td>Antibiotic</td>
		</tr>
		<tr>
			<td>Anti-BNP antibody</td>
			<td>Abcam</td>
			<td>ab239510</td>
			<td>Western Blotting</td>
		</tr>
		<tr>
			<td>Aquasonic 100 Ultrasound gel</td>
			<td>Parker Laboratories</td>
			<td>BT-025-0037L</td>
			<td>Echocardiography consumables</td>
		</tr>
		<tr>
			<td>Bepanthen</td>
			<td>Bayer</td>
			<td>6029009.00.00</td>
			<td>Eye ointment 
			eye ointment</td>
		</tr>
		<tr>
			<td>Carprosol (Carprofen)</td>
			<td>CP-Pharma</td>
			<td>401808.00.00</td>
			<td>Analgesic</td>
		</tr>
		<tr>
			<td>Clip holder</td>
			<td>Weck stainless USA</td>
			<td>523140S</td>
			<td>Surgical instruments</td>
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		<tr>
			<td>Fine scissors Tungsten carbide</td>
			<td>Fine Science Tools</td>
			<td>14568-12</td>
			<td>Surgical scissors</td>
		</tr>
		<tr>
			<td>Fine scissors Tungsten carbide</td>
			<td>Fine Science Tools</td>
			<td>14568-09</td>
			<td>Surgical scissors</td>
		</tr>
		<tr>
			<td>High-resolution imaging system</td>
			<td>FUJIFILM VisualSonics, Amsterdam, Netherlands</td>
			<td>VeVo 3100</td>
			<td>Echocardiography machine. Images were acquired with pulse-wave Doppler mode, M-mode and B-mode</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>CP-Pharma</td>
			<td>400806.00.00</td>
			<td>Anesthetic</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>CP-Pharma</td>
			<td>401650.00.00</td>
			<td>Anesthetic</td>
		</tr>
		<tr>
			<td>Mathieu needle holder</td>
			<td>Fine Science Tools</td>
			<td>12010-14</td>
			<td>Surgical instruments</td>
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			<td>Mechanical ventilator (Rodent ventilator)</td>
			<td>UGO Basile S.R.L.</td>
			<td>7025</td>
			<td>Volume controlled respirator</td>
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			<td>Metal clip</td>
			<td>Hemoclip</td>
			<td>523735</td>
			<td>Surgical consumables</td>
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			<td>Microscope</td>
			<td>Leica</td>
			<td>M651</td>
			<td>Manual surgical microscope for microsurgical procedures</td>
		</tr>
		<tr>
			<td>Millar Mikro-Tip pressure catheters</td>
			<td>ADInstruments</td>
			<td>SPR-671</td>
			<td>Hemodynamics assessment</td>
		</tr>
		<tr>
			<td>Moria Iris forceps</td>
			<td>Fine Science Tools</td>
			<td>11373-12</td>
			<td>Surgical forceps</td>
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		<tr>
			<td>Noyes spring scissors</td>
			<td>Fine Science Tools</td>
			<td>15013-12</td>
			<td>Surgical scissors</td>
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			<td>Povidone iodine/iodophor solution</td>
			<td>B/Braun</td>
			<td>16332M01</td>
			<td>Disinfection</td>
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		<tr>
			<td>PowerLab</td>
			<td>ADInstruments</td>
			<td>4_35</td>
			<td>Hemodynamics assessment</td>
		</tr>
		<tr>
			<td>Prolene Suture, 4-0</td>
			<td>Ethicon</td>
			<td>EH7830</td>
			<td>Surgical consumables</td>
		</tr>
		<tr>
			<td>Rib spreader (Alm selfretaining retractor blunt, 70 mm, 2 3/4&#8243;)</td>
			<td>Austos</td>
			<td>AE-BV010R</td>
			<td>Surgical instruments</td>
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		<tr>
			<td>Serrated Graefe forceps</td>
			<td>Fine Science Tools</td>
			<td>11052-10</td>
			<td>Surgical forceps</td>
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			<td>Silk Suture, 4-0</td>
			<td>Ethicon</td>
			<td>K871</td>
			<td>Surgical consumables</td>
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			<td>Skin disinfiction solution (colored)</td>
			<td>B/Braun</td>
			<td>19412M07</td>
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			<td>Spectra 360 Elektrode gel</td>
			<td>Parker Laboratories</td>
			<td>TB-250-0241H</td>
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			<td>Sponge points tissue</td>
			<td>Sugi</td>
			<td>REF 30601</td>
			<td>Surgical consumables</td>
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			<td>Sprague-Dawley rat</td>
			<td>Janvier Labs, Le Genest-Saint-Isle, France</td>
			<td></td>
			<td>Study animals</td>
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			<td>Tracheal cannula</td>
			<td></td>
			<td></td>
			<td>Outer diameter 2 mm</td>
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			<td>Xylazin</td>
			<td>CP-Pharma</td>
			<td>401510.00.00</td>
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a model of reverse vascular remodeling in pulmonary hypertension due to left heart disease by aortic debanding in rats

Pulmonary hypertension due to left heart disease (PH-LHD) is the most common form of PH, yet its pathophysiology is poorly characterized than pulmonary arterial hypertension (PAH). As a result, approved therapeutic interventions for the treatment or prevention of PH-LHD are missing. Medications used to treat PH in PAH patients are not recommended for treatment of PH-LHD, as reduced pulmonary vascular resistance (PVR) and increased pulmonary blood flow in the presence of increased left-sided filling pressures may cause left heart decompensation and pulmonary edema. New strategies need to be developed to reverse PH in LHD patients. In contrast to PAH, PH-LHD develops due to increased mechanical load caused by congestion of blood into the lung circulation during left heart failure. Clinically, mechanical unloading of the left ventricle (LV) by aortic valve replacement in aortic stenosis patients or by implantation of LV assist devices in end-stage heart failure patients normalizes not only pulmonary arterial and right ventricular (RV) pressures but also PVR, thus providing indirect evidence for reverse remodeling in the pulmonary vasculature. Using an established rat model of PH-LHD due to left heart failure triggered by pressure overload with subsequent development of PH, a model is developed to study the molecular and cellular mechanisms of this physiological reverse remodeling process. Specifically, an aortic debanding surgery was performed, which resulted in reverse remodeling of the LV myocardium and its unloading. In parallel, complete normalization of RV systolic pressure and significant but incomplete reversal of RV hypertrophy was detectable. This model may present a valuable tool to study the mechanisms of physiological reverse remodeling in the pulmonary circulation and the RV, aiming to develop therapeutic strategies for treating PH-LHD and other forms of PH.

First, successful aortic debanding was confirmed by transthoracic echocardiography performed before and after the debanding procedure in AoB animals (Figure 6). To this end, the aortic arch was assessed in&#160;parasternal long axis&#160;(PLAX) B-mode view. The position of the clip on the ascending aorta in AoB animals and its absence after the Deb surgery was visualized (Figure 6A,B). Next, aortic blood flow was evaluated by pulsed-wave Doppler...

Watch this Scientific Journal Video about A Model of Reverse Vascular Remodeling in Pulmonary Hypertension Due to Left Heart Disease by Aortic Debanding in Rats at JoVE.com

A Model of Reverse Vascular Remodeling in Pulmonary Hypertension Due to Left Heart Disease by Aortic Debanding in Rats

The present protocol describes a surgical procedure to remove ascending-aortic banding in a rat model of pulmonary hypertension due to left heart disease. This technique studies endogenous mechanisms of reverse remodeling in the pulmonary circulation and the right heart, thus informing strategies to reverse pulmonary hypertension and/or right ventricular dysfunction.

Pulmonary hypertension due to left heart disease (PH-LHD) is the most common form of PH, yet its pathophysiology is poorly characterized than pulmonary ...

Our protocol describes a surgical procedure to remove ascending aortic banding in a rat model of pulmonary hypertension due to left heart disease, which leads to the reversal of pulmonary hypertension. This technique presents a valuable tool to study mechanisms of physiological reverse remodeling in the pulmonary circulation and the right ventricle. It can help in developing strategies for treating pulmonary hypertension.After the surgical instruments are prepared, place an anesthetized rat in a supine position on a sterilized surgical table. Then, fix the animal's abdomen and limbs with an adhesive tape, and disinfect animal skin with povidone-iodine/iodophor solution. Note scars and sutures from the primary surgical aortic banding, or AoB, surgery.After ensuring adequate depth of anesthesia with toe pinching, remove the sutures from the primary AoB surgery. Then, perform a tracheotomy by making a 7-to 10-millimeters-long cervical midline incision with fine scissors. With the help of a pair of blunt forceps, dissect the cervical soft tissue to expose the infrahyoid muscles.Then, by splitting the muscles in the midline, visualize the trachea. When done, make a two-millimeter trachea incision between two cartilaginous rings using angled Noyes spring scissors to insert the tracheal cannula of outer diameter two millimeters into the trachea. Then, secure the cannula with a 4-0 silk suture.Connect the other end of the tracheal cannula to a mechanical ventilator while keeping the dead space minimum. Keep perioperative lung ventilation at a respiratory rate of 90 breaths per minute at a tidal volume of 8.5 milliliters per kilogram of body weight. For the aortic debanding, make a 20-millimeter-long skin incision between the second and third ribs using fine scissors.With the help of smaller surgical scissors, spread and cut the muscles layer by layer, followed by making a 10-millimeter lateral incision along the intercostal space between the second and third rib. Use a rib spreader to expand the intercostal space between the second and third rib, to create a surgical window. With the help of blunt forceps, separate the thymus from the heart and conduit arteries to visualize the aorta with the clip.Hold the clip with the help of the forceps, followed by removing the connective tissue around the clip to expose the aorta. Open the clip with a needle holder, and remove the clip from the thoracic cavity. Before closing the chest, open up lung atelectasis and ensure adequate lung recruitment without over-distention with mechanical ventilation, with a tidal volume of 9.5 milliliters per kilogram body weight for 10 minutes, and return to a tidal volume of 8.5 milliliters per kilogram body weight.Later, close the deep muscles by a simple interrupted suture using 4-0 silk, and connect the upper muscles with a simple continuous suture. Then, close the skin with a simple continuous suture. Disconnect the tracheal cannula from the ventilation machine while observing the rat for spontaneous breathing.If the animal fails to breathe spontaneously upon disconnection, reconnect the ventilator and continue ventilating for an additional five minutes before repeating the procedure. After spontaneous breathing is reestablished, remove the cannula from the trachea and clean the liquid around the trachea with sponge points. Close the trachea with a simple suture using 6-0 Prolene, and close the infrahyoid muscles in a simple interrupted suture using 4-0 silk.Connect the skin in a simple continuous suture, followed by cleaning and disinfecting the muscles and the skin in the process with povidone-iodine/iodophor solution. After completing the surgical procedure, move a single animal to a recovery cage with supplemental oxygen and an infrared lamp, and place the oxygen mask close to the rat's snout. In the study, the position of the clip on the ascending aorta in the AoB animals and its absence after the debanding, or Deb, surgery was visualized.Also, aortic blood flow was evaluated by pulsed-wave Doppler imaging before and after the clip in AoB animals prior to the debanding and in corresponding aortic segments after the Deb surgery. The results showed a marked attenuation of the blood flow gradient in line with the functional debanding. At week five, the Deb rats expressed brain natriuretic peptide, or BNP, at levels comparable to the sham animals, indicating the reversal of left ventricle, or LV, failure by the aortic debanding.The evaluation of LV function by transthoracic echocardiography revealed an increased LV ejection fraction and LV volume in the Deb animals compared to the AoB rats. The debanding surgery performed at week three after AoB resulted in a significant reduction of left ventricular systolic pressure and LV hypertrophy in comparison to the AoB animals. Compared to the AoB rats at week three and week five, the Deb animals also showed a significant reduction in right ventricular systolic pressure and right ventricular hypertrophy, demonstrating a successful reversal of pulmonary hypertension due to left heart disease.The most critical steps in this procedure are removal of the clip and the recruitment of the lung after the aortic debanding. These steps, when correctly performed, increase animal survival.

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