Exercise Test for Evaluation of the Functional Efficacy of the Pig Cardiovascular System

Summary

The present protocol describes a large animal exercise test model to assess the functional capacity of the cardiovascular system for evaluating the efficiency of new therapies in the preclinical setting. It is comparable to a clinical exercise test.

Abstract

Despite the progress in treatments, cardiovascular diseases are still one of the biggest causes of mortality and morbidity worldwide. Gene therapy-based therapeutic angiogenesis is a promising approach for treating patients with significant symptoms, despite optimal pharmacological therapy and invasive procedures. However, many promising cardiovascular gene therapy techniques have failed to accomplish expectations in clinical trials. One explanation is a mismatch between preclinical and clinical endpoints used to measure efficacy. In animal models, the emphasis has usually been on easily quantifiable endpoints, such as the number and area of the capillary vessels calculated from histological sections. Apart from mortality and morbidity, endpoints in clinical trials are subjective, such as exercise tolerance and quality of life. However, the preclinical and clinical endpoints likely measure different aspects of the applied therapy. Nevertheless, both types of endpoints are required to develop successful therapeutic approaches. In clinics, the main goal is always to alleviate patients' symptoms and improve their prognosis and quality of life. To achieve better predictive data from preclinical studies, endpoint measurements must be better matched to those in clinical studies. Here, we introduce a protocol for a clinically relevant treadmill exercise test in pigs. This study aims to: (1) provide a reliable exercise test in pigs that can be used to evaluate the safety and functional efficacy of gene therapy and other novel therapies, and (2) better match the endpoints between preclinical and clinical studies.

Introduction

Chronic cardiovascular diseases are significant causes of mortality and morbidity worldwide^1,2. Although current treatments are effective for the majority of patients, many still cannot benefit from the current therapies due to, for example, diffuse chronic disease or comorbidities. In addition, in some patients, cardiac symptoms are not relieved by the available treatments, and their cardiovascular disease progresses despite optimal medical therapy³. Thus, there is a clear need to develop novel treatment options for severe cardiovascular diseases.

During the past several years, new molecular pathways and ways to manipulate these targets have been discovered, making gene therapy, cell therapy, and other novel therapies a realistic option for treating severe cardiovascular diseases⁴. However, after promising preclinical results, many cardiovascular applications have failed to fulfill expectations in clinical trials. In spite of the poor efficacy in clinical trials, several trials have established good safety profiles of novel therapies^5,6,7,8,9. Thus, bringing new cardiovascular therapies to patients will require improved approaches and better preclinical models, study settings, and endpoints in preclinical studies that can predict clinical efficacy.

In animal models, the emphasis has usually been on easily quantifiable endpoints, such as the number and area of capillary vessels calculated from histological sections or parameters from left ventricle imaging at rest and under pharmacological stress. In clinical trials, many endpoints have been more subjective, such as exercise tolerance or symptom relief⁴. Thus, it is likely that the endpoints in preclinical studies and clinical trials measure different aspects of the applied therapy. For example, an increase in the quantity of blood vessels does not always correlate with better perfusion, cardiac function, or exercise tolerance. Nevertheless, both types of endpoints are required to develop successful therapeutic approaches¹⁰. Still, the main goal is always to alleviate symptoms and to improve the patient's prognosis and quality of life. To achieve this, endpoint measurements must be better matched between preclinical and clinical studies⁴.

Cardiorespiratory fitness reflects the ability of the circulatory and respiratory systems to provide oxygen during sustained physical activity, and thus it quantifies the functional capacity of an individual. Functional capacity is a key prognostic marker as it is a strong independent predictor for the risk of cardiovascular and all-cause mortality¹¹. Improvements in cardiorespiratory fitness are associated with a reduced risk of mortality¹². Exercise tests are suitable for evaluating aerobic performance and treatment responses in cardiovascular diseases. Depending on the availability, tests are performed on a bicycle ergometer or a treadmill. A gradual increase in the workload per minute is usually used, and abrupt increases are avoided; this leads to a linear physiological response. The most important variables in the exercise tests include the total exercise time, metabolic equivalents (METs) achieved, heart rate, and changes on an electrocardiogram (ECG) line between the QRS complex (Q, R, and S waves) and T-wave (ST segment). Clinical stress tests have low costs and are easily accessible¹³. For these reasons, stress tests, such as the 6 min walking test, have been widely used in clinics and should also be used in the preclinical evaluation of new therapies.

To our knowledge, there are no well-described large animal models for evaluating the functional efficacy of gene therapy or other novel therapies. Therefore, the clinically relevant exercise test provides an excellent perspective for evaluating the efficiency of these new therapies in the preclinical setting.

Protocol

All experiments are approved by the Animal Experiment Board of the University of Eastern Finland. This protocol describes a clinically relevant treadmill exercise test for pigs to evaluate the safety and efficacy of novel therapies for heart diseases. Female domestic pigs weighing 25-80 kg were used for the present study. The animals were obtained from a commercial source (see Table of Materials).

1. Setting up the running track

1. Set up the running track so that animals can only move one way. Use gates and hatches to prevent the animals from moving back. The floor plan of the running track is shown in Figure 1, and an example of a running track is in Figure 2.
2. Ensure the treadmill (see Table of Materials) has enough space to allow incline changes.
3. Ensure the treadmill has an adjustable width to prevent the animal from turning during the run.
4. Use transparent plastic to make the front wall of the treadmill. This prevents the animal from running away from the treadmill, but still allows the animal to see through the wall.
   NOTE: It is essential that the animals can see through the front wall, as our experience suggests that pigs are more motivated to run if they see their fellow pigs on the other side of the wall.
5. Place an ECG monitor and defibrillator (see Table of Materials) next to the treadmill.
   NOTE: Fatal arrhythmias may occur during the stress test, especially if the pig has myocardial ischemia^14,15,16.
6. Ensure the running track includes a water point where the animals can drink and cool off after the run.

2. Acclimation period of the pigs before the test

1. House the animals for 2 weeks before starting the experiments.
2. During the 1st week of acclimation, ensure the animals get accustomed to their handlers and new housing environment, excluding the running track.
3. During the 2nd week of the acclimation period, ensure the animals get accustomed to the running track.
4. Start accustoming so that the animals get familiar with the running track. First, keep all the gates open, so the animals can walk freely on the track and explore the environment.
5. When the animals are more familiar with the track, turn on the treadmill and let the animal run for short periods at a time, such as 7 min. The length of the running times must be extended daily.
   ​NOTE: Remember to reward the animals during the acclimation period. For example, the pigs were rewarded with unsalted popcorn in the present study.

3. The exercise test

NOTE: Pigs should be fasted at least 2 h before the exercise test or given only a tiny portion of food before the run.

1. Turn on the treadmill and set the incline to 5%-10%.
2. As soon as the animal is on the treadmill, start the treadmill with a starting speed of 2 km/h.
3. Increase the speed by 0.5 km/h every 60 s until 5 km/h is reached. The total running time is 15 min.
4. In case the animal cannot run the entire time at the maximum speed, perform the steps below.
   1. If the pig is not running as fast as a selected speed, gently push it from the back, as this may give the animal the feeling that it needs to run faster without slowing down.
   2. Try gently pushing the animal a maximum of three times; after that, slow down the speed by 0.5 km/h at a time until the pig can handle the speed. Do not slow down to below 2 km/h.
   3. If the animal refuses to run even at a slow speed, turn off the treadmill and stop the test.

4. ECG monitoring during the exercise test

1. Place ECG electrodes (see Table of Materials) in anatomical locations which have minimal movement during the run, such as scapulas or the chest.
   NOTE: Use ECG electrodes designed for exercise tests to achieve better adhesion to the skin. Remember to shave hair from the area where the ECG electrodes will be placed.
2. Record the heart rate changes during the run.
   ​NOTE: Our experience suggests that ST segment analyses are often complicated due to movement and other artifacts. Rhythm monitoring can also be done using an implantable loop recorder or pacemaker.

5. Data collection

1. Record the run distance, total time, and speed every time the speed is changed.
   NOTE: Modern treadmills may collect a lot of other data, so it is essential to familiarize yourself with the treadmill manual to utilize the equipment's full potential.
2. Note possible changes in animal behavior, such as limping.
   NOTE: If needed, contact a veterinarian and make sure that the animal receives necessary analgesia. Remove the animal from future exercises until it fully recovers.

6. Post-procedural care

1. Ensure the animal has access to the water point.
2. Reward the animal, for example, with treats or toys.
3. Monitor the animal for 30 min after the run for possible adverse effects.

Results

One must have experience working with large animals to succeed with this protocol. Researchers need to be able to evaluate whether an animal stops running due to fatigue or lack of motivation. Recording the speed and distance may help to evaluate this, as usually, animals lacking motivation stop running totally, whereas fatigued animals keep running after slowing down the speed (Figure 3). If necessary, the protocol can be repeated the next day if the results seem unreliable.

Discussion

This large animal exercise test mimics the test used in clinics, reducing the gap in endpoints between the preclinical studies and clinical trials. It can be applied to evaluate the efficacy of new treatments for severe cardiovascular diseases, such as arteriosclerosis obliterans, heart failure, and ischemic heart diseases. The time points applied in this protocol may vary depending on the tested treatment. This protocol has been standardized based on a long experience of working with large animals and can be used to eva...

Materials

Name	Company	Catalog Number	Comments
Defibrillator	Zoll M series	TO9K116790	All portable defribrillators will work
Defibrillator pads	Philips	M3713A	All pads work, as long as the pads are compatible with the defibrillator
ECG electrodes	Several providers		Prefer ECG electrodes designed for exercise tests
Loop recorder	Abbott Oy	DM3500	Optional for rhythm monitoring
Patient monitor	Schiller Argus LCM Plus	7,80,05,935	All portable ecg monitors will work
Pigs	Emolandia Oy
Treadmill	NordicTrack		All treadmills with adjustable incline and speed are suitable for the exercise test.  The treadmill should be as long and wide as possible.
Ultrasound system	Philips EPIQ 7 ultrasound
Various building materials	Several providers		For building fences, ramps and gates according to the Figure 1 and Figure 2
Various treats for the animals