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Acute and chronic observations of complete atrioventricular block in rats Daise N Q da Cunha, Vanessa G Pereira, Lukiya S C Favarato, Barbara S Okano, Ana P F Daibert, Betania S Monteiro, Marta R Araujo, Pablo H Carvalho, Robert L Hamlin and Ricardo J Del Carlo Lab Anim published online 23 April 2014 DOI: 10.1177/0023677214530905 The online version of this article can be found at: http://lan.sagepub.com/content/early/2014/04/08/0023677214530905

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Lab Anim OnlineFirst, published on April 23, 2014 as doi:10.1177/0023677214530905

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Acute and chronic observations of complete atrioventricular block in rats

Laboratory Animals 0(0) 1–13 ! The Author(s) 2014 Reprints and permissions: sagepub.co.uk/ journalsPermissions.nav DOI: 10.1177/0023677214530905 la.sagepub.com

Daise N Q da Cunha1, Vanessa G Pereira1, Lukiya S C Favarato1, Barbara S Okano1, Ana P F Daibert1, Betania S Monteiro2, Marta R Araujo1, Pablo H Carvalho1, Robert L Hamlin3 and Ricardo J Del Carlo1

Abstract The mechanisms of production, and gross, microscopic and electrocardiograhic findings of surgically-induced complete heart block (CHB) in the adult rat are presented. This is an effective in vivo model for establishing alternative methods to electronic pacemakers and for providing detailed information aimed at replacement, reduction and refinement of the technique. Sternal thoracotomy was employed to identify the epicardial fat pad by the aortic root, used as a landmark for cauterization of the atrioventricular (AV) node. Stable CHB was produced in 60 rats with a 70% survival rate. The best survival rate was observed in 8-week-old animals weighing 221 27.6 g. Heart rate before cauterization was 387 55 bpm, reduced after cauterization to 126 40 bpm in the survival and to 65 19 bpm in the non-survival groups. At 30 days findings were: elevated left ventricular end-diastolic pressure (21 5.4 mmHg, P < 0.05); maximal rate of rise of left ventricular pressure (LVP) during isovolumetric contraction (2192 235 mmHg/s, P < 0.05); maximal rate of decrease of LVP (1658 191 mmHg/s, P < 0.05); isovolumetric relaxation constant (5.7 0.8 ms, P < 0.05) with wet-to-dry lung–weight ratio (78.1 0.4, P < 0.05); heart weight/body weight (0.6 0.1, P < 0.05); heart volume (1.8 0.3 mL, P < 0.05); longitudinal diameter (20.2 1.91 mm, P < 0.05); and transversal diameter (17.0 1.4 mm, P < 0.05) with supported dilated cardiomyopathy which culminated in chronic heart failure. CHB hearts had increased preload and replacement of myofibrils by collagen. CHB was achieved reproducibly by cauterization of the rat AV node and/or His bundle. This led to electrophysiological, hemodynamic, and structural remodeling, and could be useful in long-term cardiac remodeling assessments and potential therapy development.

Keywords models, animals, atrioventricular node, bradychardia, heart block, methods

Complete heart block (CHB) is a condition in which the wave of excitation is not conducted from the atria to the ventricles,1 leading to severe bradychardia and acute heart failure.2,3 It may be inherited, but it is generally acquired as a result of myocardial infarction, cardiac surgery, drug toxicity, or degenerative, infectious and idiopathic diseases.4 Animal models of CHB are especially important for establishing alternatives to electronic pacemakers, such as gene therapy, cellular therapy and biological pacemakers.5 Additionally, this model reproduces the

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Veterinary Department, Federal University of Viçosa, Viçosa, Brazil 2 Animal Sciences, Vila Velha University, Vila Velha, Brazil 3 Veterinary Biosciences Department, The Ohio State University, Columbus, Ohio, USA Corresponding author: Daise Nunes Queiroz da Cunha, Veterinary Department, Federal University of Viçosa, Av Ph Rolfs, s/n, Minas Gerais State, 36570-000, Brazil. Email: [email protected]



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clinical circumstances of torsades de pointes,6 a condition favored by bradycardia as produced by CHB and by drugs that prolong QT duration.7 Therefore, this animal model is especially appropriate for the understanding of hemodynamics, electrophysiology, metabolic adaptations, volume overload, and atrioventricular (AV) desynchronization secondary to the bradycardia caused by CHB. Efforts to obliterate the AV node emerged in the 20th century, when Fre´de´ricq crushed the AV node with forceps after a right atriotomy in dogs.2 CHB was produced by cryoablation,8 section of the AV nodal junction,9 and electrocauterization10 during thoracotomy. Fisher et al.11 introduced a new method in dogs, in which no thoracotomy was needed; instead a catheter was used to inject formalin into the AV node. Thereafter, techniques using electrical ablation,12 laser,13 radiofrequency ablation,14,15 and cryoablation16 were used. In addition to these techniques, CHB was also produced by injecting caustic substances through open and closed chests.17,18 Different methods of AV node obliteration were introduced, such as ethanol infusion in the AV nodal artery,19 and gene transfer.20 The goal of this study was to present the description of a reproducible method of producing CHB in rats and its repercussion on the heart, presenting detailed information aimed at replacement, reduction and refinement of the technique.

Animals Animals were housed in cages, maintained at 23 C, 12 h light/dark cycle, and received food and water ad libitum. All procedures were approved by the Ethics Committee on Animal Experimentation (protocol number 14/2011) which had oversight of the facility in which the studies were conducted. The principles observed in this study are specified in the European Convention for the Protection of Vertebrate Animals and in the National Research Council Guide for the Care and Use of Laboratory Animals. Sixty-eight male adult Wistar rats (Central Vivarium, Biological Health Science Center of the federal University of Viçosa, Brazil), age from 6 to 9 weeks, and weighing 170 to 272 g were included in the study. The animals were weighed just prior to surgery, and at 15 and 30 days after. Sixty rats were allocated to two groups (30 rats/ group). CHB was induced, and rats were euthanized at 15 and 30 days after surgery (groups 15 and 30 days, respectively). Eight animals were used for cardiopulmonary morphophysiological evaluations, for which four animals underwent CHB (CHB group) and four were submitted to thoracotomy (Sham group).

Materials and methods Surgical procedures Antibiotic (enrofloxacin, 10 mg/kg intraperitoneally once a day) was administered preoperatively. The animals were placed individually in an induction chamber, and anesthesia was induced with 3% isoflurane and 100% oxygen at a constant flow rate of 1 L/min. Anesthesia was performed using a BioLITE system (BioTex Inc, Houston, TX, USA) to illuminate the trachea with a fiberoptic stylet inserted within an intravenous catheter (16–20 Ga, 2.54 cm length) adapted as an endotracheal tube. The animals were placed on intermittent positive pressure ventilation (Inspira Advanced Safety Single Animal Pressure/Volume Controlled Ventilators; Harvard Apparatus, Holliston, MA, USA) at a frequency of 50 to 60/min, an inspiratory pressure of 16 to 18 cmH2O, and an inspiratory/expiratory ratio of 1:1. Anesthesia was maintained with 1–2% isoflurane, diluted in 98–99% oxygen. The animal was positioned in dorsal recumbency on a polyethylene box, such that the hindlimbs were in direct contact with the dispersive pad/electrode of the electrocautery (BP 150; EMAI, SP, Brazil) and the skin was prepared for surgery. During the procedure, the body temperature was maintained at 37.5 1.1ºC by heat transfer from heated water bags placed inside the box used for containment. Median sternotomy provided access to the thoracic cavity while leaving the manubrium and the xiphoid intact, facilitating closure of the thorax. The thymus was retracted, and a partial pericardiotomy exposed the base of the heart. The right atrium (RA) was gently retracted using a sterilized cotton swab (Johnson & Johnson, SP, Brazil), and the groove between the RA and the aortic root was identified. This maneuver exposed the landmarks for the epicardial approach to the AV node. A fat pad was consistently found between the aortic root and the medial wall of the RA (Figure 1). This fat corresponded, on the outside aspect of the aortic root, to the commissure between the right and non-coronary leaflets of the aortic valve and served as a reference point for the precise location of the AV node. For ablation of the AV node, an acupuncture needle (0.25 mm 30 mm; Huan Qiu, Shanghai, China) was used. Previously, this needle was bent at a 90 angle 2 mm from the tip (Figure 1) to ensure a sel-limited insertion into the myocardium and to prevent entry into the lumen. The needle was introduced through the fat pad at approximately 1 mm dorsal from the aortic root and parallel to the aorta towards the apex of the heart. The latter approach ensured that the needle stayed in the intramural space. These maneuvers produced momentary interruption of the AV node



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Aorta Le atrium

Thymus

Right atrium

Fat pad at aorc root Le ventricle apex

Acupuncture needle Diaphragm

Figure 1. Schematic drawing showing the landmarks for locating the atrioventricular (AV) node and the acupuncture needle (bent at 90 ) used to make contact with the AV node and the active electrode of the electrocautery.

which confirmed the needle was properly placed for the ablation. The AV block observed on the electrocardiogram (ECG) and the lack of bleeding confirmed that the needle was placed correctly within the myocardium and within the AV node. Immediately after, the active electrode was placed in contact with the needle, activated in coagulation mode, and the power was set at 2, ablating the AV node. ECGs were acquired before opening the thorax, and before and after ablation/cauterization. Due to interference with the ECG, leads were removed before activation of the electrocautery, but were repositioned immediately to document the production of CHB. If the AV block was first or second degree, or transient, additional cauterizations were applied. The duration of electrocauterization and the number of attempts were registered. After producing the AV block, the animals were monitored by ECG until closure of the thorax (5 2.5 min). Induction to CHB was considered successful when AV dissociation by block was persistent (Figure 2a and b). Chronic CHB was determined in those cases where blockage persisted up to the 15th and 30th days after induction (Figure 2c and d). P waves, QRS complexes, and any lack of association between them were documented by ECG. Just prior to complete chest closure using 4-0 mononylon, the lungs were fully expanded for 2 s to minimize pneumothorax and to restore negative intrathoracic pressure. The skin was sutured using 4-0 mononylon (Johnson & Johnson, Norbi, Saõ Paulo, Brazil). The

endotracheal tube was constantly aspirated to avoid accumulation of secretions and possible airway obstruction. It was removed after spontaneous breathing was observed prior to regaining consciousness. During recovery from anesthesia the animals were placed in a custom-built wooden box with ambient temperature controlled to remain between 37 and 39 C. All the rats were housed in individual cages and recovery was uneventful. Tramadol was used for post-surgical analgesia, 4 mg/kg, given subcutaneously every 6 h for 24 h. Atropine was administered subcutaneously at 1 mg/kg to animals with pulmonary secretion during recovery. Cotton swabs were used to absorb eventual bleeding and for measurement of volume loss through hemorrhage during the surgery. Volume loss was estimated by subtracting the weight of a dry cotton swab from each soaked cotton swab. The soaked swab weighed approximately 0.227 mg. A milliliter of blood equates to 1.06 g (or mL of blood) (http://en.wikipedia.org/ wiki/Blood_alcohol_content) and, thus, 0.227 mg is equivalent to 0.240 mL of blood. Volumes were replaced with sodium chloride 0.9% solution intraperitoneally after closure of the thorax.

Electrocardiographic evaluation On the 15th day (group 15 days) and 30th day (group 30 days), the animals were lightly anesthetized with isoflurane for ECG acquisition. Good quality lead II ECG signals were acquired for at least 2 min.



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Ventricular (QRS) rate/frequency was averaged from a 10 s period of the ECG trace and used for comparisons between the groups. The animals were then euthanized by anesthetic overdose.

Cardiopulmonary morphophysiological evaluation Thirty days after surgery, a group of eight animals were lightly anesthetized for ECG acquisition and left ventricle (LV) catheterization. Of these rats, four had CHB and the other four were sham-operated. The anesthetic plane was deepened to a surgical plane for thoracotomy to be performed. The LV was catheterized with a catheter (22 gauge) fluid filled with heparinized saline (50 UI/mL). The catheter was inserted through the epicardium into the LV apex and was connected to a pressure transducer for LV pressure (LVP) recordings using a data acquisition system (Powerlab, model MLT0380; ADInstruments, Colorado Springs, CO, USA). Five minutes were allowed for hemodynamic stabilization before LVP and ECG were registered at a sample rate of 1000 kHz. Heart rate (HR, bpm), LV systolic pressure (LVSP, mmHg), LV end-diastolic pressure (LVEDP, mmHg), maximal rates of rise (mmHg/ms), and fall (mmHg/ms) of pressure, time constant of fall of isovolumetric pressure (ms) were derived from the LVP curve. For this, five subsequent cardiac cycles free of artifacts were averaged. After LVP recordings the animals were euthanized by isoflurane overdose. During necropsies the degree of

tissue adherence at the surgical site was graded (grade 0: no adherence; grade 1: RA adhered to cauterization area of the AV node; grade 2: thymus or pericardium, or both adhered to the heart; grade 3: lung and thymus adhered to the heart). The hearts were removed, rinsed in normal saline and gently dried for immediate measurement. Heart weight was acquired using a precision balance, and longitudinal and transverse diameters were measured using a caliper ruler. Heart weight-to-body weight (BW) ratios were calculated. Heart volume (HV) was estimated by the water displacement technique, where the heart was placed within a beaker filled with a known volume of water. The water volume displaced was equivalent to the volume of the organ. The hearts were dissected immediately below the AV valves, and fixed in 10% formalin solution for 48 h for usual histopathological processing. Four serial cuts containing both the RV and LV were made. Two sections were stained with Picrosirius Red21 and observed under polarized light microscopy (Olympus BX 53). Four images from each region, RV and LV, were captured with 10 objective, using a digital camera (Olympus DP 73) utilizing the imaging software Cellsens 1.7 (Olympus, Center Valley, PA, USA). The images were analyzed with Image-Pro Plus 4.5 software (Media Cybernetics, Silver Spring, MD, USA). The ‘intensity range selection’ tool was used to determine the colors for the regions of interest. Thick fibers observed as an orange to red hue under polarized light were considered to be collagen type I and mature; and thin fibers, with a green-bluish hue were collagen

(a)

(b)

(c)

(d)

Figure 2. Representative electrocardiograms (ECGs) from rats at (a) baseline, (b) immediately, (c) 15, and (d) 30 days after atrioventricular (AV) node cauterization. Heart rates were (a) 384; (b) 131; (c) 140 and (d) 99. Arrows indicate blocked p waves.



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type II21 and immature.22 The area occupied by the different types of fibers was subtracted from the total area of the image and the results were presented as a percentage of the total area. The cell types were evaluated by hematoxylin and eosin (HE) stained slides (n ¼ 2/animal). To determine whether rats with CHB had developed congestive heart failure, it was hypothesized that pulmonary edema would form in these individuals. Edematous lungs are heavier,23 so wet-to-dry weights were measured to investigate the hypotheses that chronic heart failure (CHF) was present. Immediately after excision and dissection of adjacent tissues the lungs were weighed wet. The lungs were then dried in an oven (MA035; Marconi, SP, Brazil) at 65 C for 7 h and weighed again. Wet-to-dry lung–weight ratios and wet-todry lung–weight ratios indexed to body weight (W/ DBW) were calculated as indicated: W/D ¼ [(W D)/ W], and W/D BW ¼ [(W D)/W]/BW, and posteriorly compared between the groups (CHB versus Sham).

Statistical analysis Differences in age, BW, HR, bleeding, total electrocautery time, numbers of attempts at electrocauterization, and adherence grades were determined by nonparametric analysis using Wilcoxon signed-rank test.24 Data were presented descriptively (Table 1). All variables were tested for normality of distribution (Lilliefors) and for homoscedasticity (Cochran). Mean differences of BW and HR were determined by analysis of variance (ANOVA) and the post-hoc Tukey test, requiring a P < 0.05 for significance. The degree of tissue adherence at the surgical site was determined using a frequency distribution analysis. For analyzing the differences in W/D, W/D BW, heart dimensions (weight, volume, longitudinal and transverse diameters), and hemodynamic parameters between the two groups (CHB versus Sham), the non-parametric Mann-Whitney U-test was used. Differences in collagen content were determined by a two-tailed t-test for

pairwise comparisons between groups and the regions of the heart, and a P < 0.05 was required for significance.

Results CHB was successfully obtained in all animals. AV block instability, when present, could be observed immediately after cauterization and until thoracic closure, in which instances new energy was applied until establishment of CHB. The number of attempts was 2.5 2.28, comprising a total duration of attempts of 17.5 16.8 s (Table 1). A maximal duration of 10 s was established for each electrocauterization attempt. In the 30% of animals that had CHB and did not survive the procedure (n ¼ 18), a greater number of attempts (4.5 2) and with prolonged total duration of electrocauterization (38.8 22.4 s) (P < 0.001) occurred (Table 1). Electrocardiography performed at baseline demonstrated that arrhythmias were not present. On the other hand, ECG findings on day 15 and on day 30 revealed a variety of arrhythmias, as shown in Figure 3. The most frequent rhythm was third degree AV block, present in 18 cases on day 15 and in 16 cases on day 30. Other aberrancies were associated with third degree AV block, with examples presented in Figure 3. These were junctional premature depolarizations with re-entry producing bigeminal rhythm with distorted AV conduction; ventricular premature depolarizations (VPDs); junctional depolarizations in addition to ventricular ectopic depolarizations and junctional beats produced by bigeminal rhythm; and bigeminal rhythm resulting from re-entry from the ventricular or from the AV junction with aberrancy. High grade second degree AV block was observed on day 15, but not on day 30. Variations of the sinus rhythms were present on both days 15 and 30. Sinus rhythms were always associated with some abnormality: bundle branch block; sinus rhythm with first degree AV block with interventricular conduction disturbance (IVCD) combined with

Table 1. Variables that determined the outcome of the atrioventricular node ablation in rats. Groups

Survived (n ¼ 42)

Dead (n ¼ 18)

Variables

Median (IQR)

Max

Min

Median (IQR)

Max

Min

P value

Age (weeks) Body weight (g) (baseline) Hemorrhage (mL) Number of attempts Duration of electrocauterization (s)

8 217.5 0.44 2 10

9 272 1.78 11 74

6 170 0.11 1 2

6 189.5 1.11 4 37.5

8 212 4.11 10 100

6 159 0.55 2 5

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