Transesophageal Atrial Burst Pacing for Atrial Fibrillation Induction in Rats

Summary

The present work describes an experimental protocol of transesophageal atrial burst pacing for efficient induction of atrial fibrillation (AF) in rats. The protocol can be used in rats with healthy or remodeled hearts, allowing the study of AF pathophysiology, identification of novel therapeutic targets, and evaluation of new therapeutic strategies.

Abstract

Animal studies have brought important insights into our understanding regarding atrial fibrillation (AF) pathophysiology and therapeutic management. Reentry, one of the main mechanisms involved in AF pathogenesis, requires a certain mass of myocardial tissue in order to occur. Due to the small size of the atria, rodents have long been considered 'resistant' to AF. Although spontaneous AF has been shown to occur in rats, long-term follow-up (up to 50 weeks) is required for the arrhythmia to occur in those models. The present work describes an experimental protocol of transesophageal atrial burst pacing for rapid and efficient induction of AF in rats. The protocol can be successfully used in rats with healthy or remodeled hearts, in the presence of a wide variety of risk factors, allowing the study of AF pathophysiology, identification of novel therapeutic targets, and evaluation of novel prophylactic and/or therapeutic strategies.

Introduction

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia encountered in clinical practice and its incidence and prevalence continue to increase dramatically worldwide¹. This arrhythmia affects up to 4% of the world population according to recent studies². However, given that paroxysmal AF can be asymptomatic and may therefore escape detection, the true prevalence of AF is likely to be much higher than that presented in the literature.

The pathophysiology of AF has been intensely studied. Nevertheless, the underlying mechanisms of this complex arrhythmia remain incompletely elucidated and this reflects in the limited therapeutic options, with questionable efficacy. Animal studies have brought important insights into our understanding regarding AF pathophysiology and therapeutic management. Reentry, one of the main mechanisms involved in AF pathogenesis³, requires a certain mass of myocardial tissue in order to occur. Thus, large animals have generally been preferred in AF studies, whereas, due to the small size of their atria, rodents have long been considered 'resistant' to AF. However, the use of large animals is hampered mostly by handling difficulties. Meanwhile, although spontaneous AF has been shown to occur in rats⁴, long-term follow-up (up to 50 weeks) is required for the arrhythmia to occur in those models⁵. Models that ensure rapid AF occurrence in small rodents have also been developed. Most often, those models use acute electrical stimulation, often in the presence of other favoring conditions, such as concomitant parasympathetic stimulation or asphyxia, to artificially induce AF^6,7. Although efficient, such models do not allow the evaluation of critical AF-related features, such as the progressive electrical, structural, autonomic, or molecular remodeling of the atria, nor the effects of conventional or non-conventional antiarrhythmic drugs on the atrial substrate or on the risk of ventricular pro-arrhythmia^8,9.

The present work describes an experimental protocol of long-term transesophageal atrial burst pacing for rapid and efficient induction of AF in rats. The protocol is suitable for both acute and long-term studies and can be successfully used in rats with healthy or remodeled hearts, in the presence of a wide variety of risk factors, allowing the study of AF pathophysiology, identification of novel therapeutic targets, and evaluation of novel prophylactic and/or therapeutic strategies.

Protocol

Procedures involving animal subjects were approved by the Ethics Committee of the University of Medicine, Pharmacy, Science and Technology "George Emil Palade" of Târgu Mureș, by the Romanian National Sanitary Veterinary and Food Safety Authority and complied with the International Council for Laboratory Animal Science guidelines (Directive 2010/63/EU).

1. Transesophageal atrial burst pacing protocol

1. Randomize adult male Wistar rats (200-400 g of bodyweight) into two groups: STIM and SHAM.
2. Anesthetize the animals.
   1. For induction, use 2.5% isoflurane, 4 L/min, 99.5% O₂.
   2. For maintenance, use a mixture of ketamine/medetomidine (75.0/0.5 mg/kg) administered intraperitoneally.
   3. Check the depth of the anesthesia by testing the corneal reflex (5% glucose solution) and the nociceptive withdrawal reflex (toe pinch). Monitor the respiratory rate (a fall of 50% is acceptable during anesthesia; normal rate is between 70-120 breaths/minute) and the body temperature using a rectal thermometer (normal temperature is between 96.5 - 99.5 °F or 35.9 - 37.5 °C).
      NOTE: Continue the procedure only after the effectiveness of anesthesia is confirmed. Monitor the depth of anesthesia periodically throughout the protocol. Repeat the intraperitoneal ketamine/medetomidine injection when needed.
   4. Apply an ophthalmic ointment to both eyes to prevent corneal damage.
3. Lay the animal in supine position and place it on a heating pad to maintain body temperature at ~37 °C.
4. Attach the three surface ECG electrodes to the rat limbs in a lead II configuration (Figure 1A).
   1. Place the negative electrode on the right forelimb.
   2. Place the positive electrode on the left hindlimb.
   3. Place the grounding electrode on the left forelimb.
   4. Secure the electrodes into position using thin elastic bracelet string cords.
5. Turn on the surface ECG recording and perform continuous ECG recording throughout the procedure (Figure 1B) using a commercial or a locally developed acquisition program¹⁰.
6. For electrical stimulation, use a 5-6 F quadripolar catheter connected to a microcontroller-based cardiac pacemaker¹⁰.
7. Once the animal is anesthetized, insert the catheter through the oral cavity, into the esophagus. Measure the distance between the upper incisors and the heart (assessed by palpation) to approximate the depth at which the catheter should be inserted into the esophagus.
   CAUTION: Be careful not to force the catheter as there is a risk of esophageal perforation.
8. Confirm the correct position of the stimulation catheter at the level of the atria as follows.
   1. Apply electrical stimulation at a frequency of 400 stimuli/minute (stimulus duration 6 ms).
   2. Check whether the ECG tracing shows constant capture of the atria (i.e., each electrical stimulus is followed by a narrow QRS complex) (Figure 2).
9. Determine the diastolic threshold—i.e., the lowest voltage required to obtain atrial capture (generally, between 10 V and 20 V).
   NOTE: Perform the following for the animals in the STIM group.
10. Once the correct position of the catheter is determined, set the stimulator to a frequency of 4,000 stimuli/minute (stimulus duration 6 ms), at a voltage 3 V above the diastolic threshold (Figure 3).
11. Apply to each animal 15 successive cycles of stimulation, 20 s each, with a free interval of 5 min between cycles¹¹. Depending on the study objectives, repeat the protocol for each rat for 10 days, at a rate of 5 days/week, at the same time on each day.
12. Check the effectiveness of the stimulation as follows.
    1. Identify the sinus node recovery time (SNRT), which appears at the end of the rapid pacing as a time interval that is longer than the cycle length recorded during sinus rhythm (Figure 4A) and represents the interval of time required for resumption of sinus rhythm after overdrive suppression ends.
       NOTE: Overdrive suppression represents the inhibition of sinus node activity by electrically stimulating the heart at a rate higher than the intrinsic rhythm.
    2. Identify the occurrence of the AF episode, which is defined here as the presence of three or more consecutive irregular, supraventricular beats (i.e., irregular ventricular response with narrow QRS complexes), with P-waves absent or replaced by small, distorted "f" waves (Figure 4B).
13. If the AF episode does not end spontaneously by the time the next stimulation cycle should be performed (i.e., by the end of the five free minutes between cycles), do not apply the next stimulation.
    1. Wait for another 5 min. If the AF episode still continues after those 10 min, end the protocol for that day.
       NOTE: If evaluation of the severity of electrically-induced AF is desired, longer ECG monitoring can be performed.
14. If severe bradycardia or asystole occurs at the end of the stimulation (i.e., due to electrical stimulation of the vagus nerve), end the protocol. If the electrical activity does not return to normal rapidly, perform external cardiac massage and administer atropine sulfate (0.05 mg/kg) intraperitoneally.
15. At the end of the procedure, reverse the anesthesia with atipamezole (1 mg/kg) administered intraperitoneally. House the rats individually in clean cages with supplemental heat and observe periodically until they are fully recovered. No other specific animal care is required at the end of the protocol.
16. Analyze the surface ECG tracings and determine the following.
    1. The inducibility of AF which is expressed in percentage (i.e., [number of stimulation cycles followed by AF episodes / total number of stimulation cycles applied] x 100).
    2. The duration of each AF episode.
    3. The presence of 'persistent' (i.e., >10 min) AF episodes.
       NOTE: Perform the following for the animals in the SHAM group.
17. For the rats in the SHAM group, follow steps 1.1 to 1.7 as described above, without applying any electrical stimulation.
18. Maintain the catheter into position for 80 min (i.e., the time required for completing the protocol in STIM rats) without applying any electrical stimulation, while continuously recording the surface ECG.
19. At the end of the procedure, reverse the anesthesia with atipamezole (1 mg/kg). No other specific animal care is required at the end of the protocol.
20. Analyze the surface ECG tracings and determine the parameters described in step 1.16.

Results

In a proof-of-concept study, 22 adult male Wistar rats (200-400 g) were randomly assigned into two groups: STIM (n = 15) and SHAM (n = 7). All animals were housed individually in polycarbonate cages, in a climate-controlled room (21-22 °C), having free access to water and dry food throughout the study. The transesophageal stimulation protocol described above was applied to all animals for 10 days, 5 days per week. All animals underwent the same protocol, except that the rats in the SHAM group did not receive active ...

Discussion

The present paper describes an experimental protocol of long-term transesophageal atrial burst pacing for rapid and efficient induction of AF in rats, suitable for both acute and long-term AF studies. The 10-day stimulation protocol described herein has been successfully used to develop a 'secondary spontaneous AF model' (i.e., a model in which, following a period of AF induction by electrical stimulation, AF develops spontaneously)¹⁰. However, the duration of the protocol can vary depending on th...

Materials

Name	Company	Catalog Number	Comments
Antisedan (Atipamezole Hydrochloride) 5mg / mL, solution for injection	Orion Corporation	06043/4004	for Rats use 1 mg / kg
Dormitor (Medetomidine Hydrochloride) 1 mg / mL, solution for injection	Orion Corporation	06043/4003	for Rats use 0.5 mg / kg
E-Z Anesthesia Single Animal System	E-Z Systems Inc	EZ-SA800	Allows the manipulation of one animal at a time
Isoflurane 99.9%, 100 mL	Rompharm Company	N01AB06
Ketamine 10%, 25 mL			for Rats use 75 mg / kg
Microcontroller-based cardiac pacemaker for small animals			Developed in our laboratory (See Reference number 10 in the manuscript)
Surface ECG recording system			Developed in our laboratory (See Reference number 10 in the manuscript)