A study of conduction system fluorescence imaging by anterograde perfusion with fluorescent dyes -labeled antibody in <i>ex vivo</i> rat hearts_Chinese Journal of Clinical Thoracic and Cardiovascular Surgery

Authors：

REN Yifei ¹ , YU Kai ² ,  GAN Changping ¹

1. Department of Cardiovascular Surgery, West China Hospital, Sichuan University, Chengdu, 610041, P. R. China;
2. Department of Cardiovascular Surgery, Guang'an People's Hospital, Guang'an, 638000, Sichuan, P. R. China;

Corresponding author：

GAN Changping, Email: ganchangping@hotmail.com

Keywords：

Cardiac conduction system; arrhythmia; biological imaging

DOI：

10.7507/1007-4848.202311043

Video：

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Objective To study feasibility of retrograde infusion of hyperpolarization-activated cyclic nucleotide-gated channel protein 4 (HCN4) and connexin fluorescent dye (Alexa Fluor 633)-labeled antibodies through the aorta to image the cardiac conduction system (CCS) in rat hearts. The optimal dosage, infusion time and photochemical stability of fluorescent dyes were also studied. Methods Ex vivo rat heart anterograde perfusion models were established in 33 male SPFSD rats, and the primary and secondary antibody solutions were injected sequentially. The atrioventricular junction was observed and the fluorescence intensity of the area was recorded when the perfusion reaches the scheduled time. We set five dose gradients (3 rats per gradient), 5 perfusion time gradients (three rats per gradient) and 10 LED continuous illumination time gradients in 3 rats under specific dose and perfusion time, the fluorescence intensities of the region were observed and recorded. Standard immunofluorescence stained paraffin sections and frozen sections were prepared for histological comparison. Res ults A HCN4 red fluorescence signal aggregation region was observed in the atrioventricular junction, which was identified as the AVN structure based on HCN4/Cx43 semi-quantitative fluorescence intensity analysis and histological comparison. With increasing antibody perfusion time, both AVN and background fluorescence intensity showed no statistically significant difference. The ratio of AVN to background fluorescence intensity also increased with the increasing antibody perfusion time. When the illumination time of AVN was prolonged, the fluorescence intensity of both AVN and background showed a downward trend but no statistically significant difference. Conclusion The anterograde perfusion with fluorescent dye (Alexa Fluor 633)-labeled antibody can successfully image the AVN of the CCS in ex vivo rat hearts under stereoscopic fluorescence microscopy. Increasing the antibody dose results in different AVN imaging effects. The imaging effect of AVN improves with an increase antibody perfusion time. Even after long-term (8 h) exposure to light, Alexa Fluor 633 can still maintain a certain level of AVN image.

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2.	Sanchez-Quintana D, Cook AC, Macias Y, et al. The atrioventricular conduction axis revisited for the 21st century. J Cardiovasc Dev Dis, 2023, 10(11): 471.
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14.	Kaiser GA, Waldo AL, Beach PM, et al. Specialized cardiac conduction system. Improved electrophysiologic identification technique at surgery. Arch Surg, 1970, 101(6): 673-676.
15.	Huang C, Sachse FB, Hitchcock RW, et al. Sensitivity and specificity of cardiac tissue discrimination using fiber-optics confocal microscopy. PLoS One, 2016, 11(1): e0147667.
16.	Turner MA, Lwin TM, Amirfakhri S, et al. The use of fluorescent anti-CEA antibodies to label, resect and treat cancers: A review. Biomolecules, 2021, 11(12): 1819.
17.	Hu Z, Fang C, Li B, et al. First-in-human liver-tumour surgery guided by multispectral fluorescence imaging in the visible and near-infrared-Ⅰ/Ⅱ windows. Nat Biomed Eng, 2020, 4(3): 259-271.
18.	Pei G, Liu Y, Liu Q, et al. The safety and feasibility of intraoperative near-infrared fluorescence imaging with indocyanine green in thoracoscopic sympathectomy for primary palmar hyperhidrosis. Thorac Cancer, 2020, 11(4): 943-949.
19.	Raina A, Tan NY, Fatunde OA, et al. Case report: A case of perinodal atrial tachycardia and review of the relevant clinical anatomy surrounding the retroaortic node. Front Cardiovasc Med, 2023, 10: 1143409.
20.	Peischard S, Möller M, Disse P, et al. Virus-induced inhibition of cardiac pacemaker channel HCN4 triggers bradycardia in human-induced stem cell system. Cell Mol Life Sci, 2022, 79(8): 440.
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25.	Öztürk E, Kafalı HC, Tanıdır İC, et al. Early postoperative arrhythmias in patients undergoing congenital heart surgery. Turk Gogus Kalp Damar Cerrahisi Derg, 2021, 29(1): 27-35.
26.	Haga T, Akamine Y, Yamamoto H, et al. Association between post-procedure intracardiac pressures and the use of temporary epicardial pacing wires after pediatric cardiac surgery. Pediatr Cardiol, 2020, 41(2): 366-371.

1. Nooma K, Saga T, Iwanaga J, et al. A novel method with which to visualize the human sinuatrial node: Application for a better understanding of the gross anatomy of this part of the conduction system. Clin Anat, 2020, 33(2): 232-236.
2. Sanchez-Quintana D, Cook AC, Macias Y, et al. The atrioventricular conduction axis revisited for the 21st century. J Cardiovasc Dev Dis, 2023, 10(11): 471.
3. Cabrera JÁ, Anderson RH, Macías Y, et al. Variable arrangement of the atrioventricular conduction axis within the triangle of Koch: Implications for permanent his bundle pacing. JACC Clin Electrophysiol, 2020, 6(4): 362-377.
4. Yamamoto K, Miwa S, Yamada T, et al. Managing postoperative atrial fibrillation after open-heart surgery using transdermal β1 blockers. J Cardiothorac Surg, 2023, 18(1): 103.
5. Mondal A, Yoo M, Tuttle S, et al. Cost of pacing in pediatric patients with postoperative heart block after congenital heart surgery. JAMA Netw Open, 2023, 6(11): e2341174.
6. Romer AJ, Tabbutt S, Etheridge SP, et al. Atrioventricular block after congenital heart surgery: Analysis from the Pediatric Cardiac Critical Care Consortium. J Thorac Cardiovasc Surg, 2019, 157(3): 1168-1177.
7. Sachse FB, Johnson J, Cottle B, et al. Toward detection of conduction tissue during cardiac surgery: Light at the end of the tunnel? Heart Rhythm, 2020, 17(12): 2200-2207.
8. Waddingham PH, Behar JM, Roberts N, et al. Post-operative cardiac implantable electronic devices in patients undergoing cardiac surgery: A contemporary experience. Europace, 2021, 23(1): 104-112.
9. Wiggins NB, Chong DT, Houghtaling PL, et al. Incidence, indications, risk factors, and survival of patients undergoing cardiac implantable electronic device implantation after open heart surgery. Europace, 2017, 19(8): 1335-1342.
10. Johnson JK, Cottle BK, Mondal A, et al. Localization of the sinoatrial and atrioventricular nodal region in neonatal and juvenile ovine hearts. PLoS One, 2020, 15(5): e0232618.
11. Kaza AK, Hitchcock R, Mondal A, et al. Systematic mapping of conduction tissue regions during congenital heart surgery. Ann Thorac Surg, 2022, 114(4): 1500-1504.
12. Xia R, Vlcek J, Bauer J, et al. Whole-mount immunofluorescence staining, confocal imaging and 3D reconstruction of the sinoatrial and atrioventricular node in the mouse. J Vis Exp, 2020, (166).
13. Aiello VD. Understanding the morphology of the specialized conduction tissues in congenitally malformed hearts. World J Pediatr Congenit Heart Surg, 2015, 6(2): 239-249.
14. Kaiser GA, Waldo AL, Beach PM, et al. Specialized cardiac conduction system. Improved electrophysiologic identification technique at surgery. Arch Surg, 1970, 101(6): 673-676.
15. Huang C, Sachse FB, Hitchcock RW, et al. Sensitivity and specificity of cardiac tissue discrimination using fiber-optics confocal microscopy. PLoS One, 2016, 11(1): e0147667.
16. Turner MA, Lwin TM, Amirfakhri S, et al. The use of fluorescent anti-CEA antibodies to label, resect and treat cancers: A review. Biomolecules, 2021, 11(12): 1819.
17. Hu Z, Fang C, Li B, et al. First-in-human liver-tumour surgery guided by multispectral fluorescence imaging in the visible and near-infrared-Ⅰ/Ⅱ windows. Nat Biomed Eng, 2020, 4(3): 259-271.
18. Pei G, Liu Y, Liu Q, et al. The safety and feasibility of intraoperative near-infrared fluorescence imaging with indocyanine green in thoracoscopic sympathectomy for primary palmar hyperhidrosis. Thorac Cancer, 2020, 11(4): 943-949.
19. Raina A, Tan NY, Fatunde OA, et al. Case report: A case of perinodal atrial tachycardia and review of the relevant clinical anatomy surrounding the retroaortic node. Front Cardiovasc Med, 2023, 10: 1143409.
20. Peischard S, Möller M, Disse P, et al. Virus-induced inhibition of cardiac pacemaker channel HCN4 triggers bradycardia in human-induced stem cell system. Cell Mol Life Sci, 2022, 79(8): 440.
21. Gómez-Torres F, Ruíz-Sauri A. Morphometric analysis of the His bundle (atrioventricular fascicle) in humans and other animal species. Histological and immunohistochemical study. Vet Res Commun, 2021, 45(4): 319-327.
22. Landra F, Marallo C, Santoro A, et al. Moderator band and ventricular tachycardia: Structural or functional substrate? J Cardiovasc Dev Dis, 2023, 10(4): 159.
23. Shi X, Zhang S, Liu Y, et al. Spatial distribution and network morphology of epicardial, endocardial, interstitial, and Purkinje cell-associated elastin fibers in porcine left ventricle. Bioact Mater, 2022, 19: 348-359.
24. Demchenko AP. Photobleaching of organic fluorophores: Quantitative characterization, mechanisms, protection. Methods Appl Fluoresc, 2020, 8(2): 022001.
25. Öztürk E, Kafalı HC, Tanıdır İC, et al. Early postoperative arrhythmias in patients undergoing congenital heart surgery. Turk Gogus Kalp Damar Cerrahisi Derg, 2021, 29(1): 27-35.
26. Haga T, Akamine Y, Yamamoto H, et al. Association between post-procedure intracardiac pressures and the use of temporary epicardial pacing wires after pediatric cardiac surgery. Pediatr Cardiol, 2020, 41(2): 366-371.

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Latest ArticlesA study of conduction system fluorescence imaging by anterograde perfusion with fluorescent dyes -labeled antibody in ex vivo rat hearts

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