Comparative Study On The Models Of Sick Sinus Syndrome In Rats

background:Sick sinus syndrome(Sick sinus syndrome,SSS)is a common arrhythmia disease in clinic.At present,the treatment of SSS is mainly the implantation of a pacemaker,and the resulting medical expenses have also increased the burden of medical insurance.This study intends to compare various rat sick sinus syndrome models to study the structural and functional changes and related mechanisms of the sinus node,aiming to explore the influencing factors of sinus node dysfunction,and hope to be clinically sick sinus Nodal syndrome finds targets for intervention and treatment.Objective:1.To clarify the anatomical location and morphological characteristics of the rat sinoatrial node(Sinoatrial node,SAN),make SD rat SSS models by various methods,and observe the changes in heart rate,SAN morphology and structure of SSS rats,and the tissues in the SAN area.Fibrosis and the expression of Hyperpolarisation activated cyclic nucleotide gated channel 4(HCN4)activated by the hyperpolarization of the sinus node specific marker.2.Compare the success rate and mortality of multiple modeling methods of sick sinus syndrome,and the effect of pre-and post-modeling on the expression of membrane clock and calcium clock pacing related genes,in order to provide a theoretical basis for the clinical treatment of sick sinus syndrome.Methods:1.Take 250-300 g male SD rat sinoatrial node tissue,atrial tissue around the sinus node,and ventricular tissue,and use HE staining and Masson staining to observe the cell morphology and fibrosis of the surrounding tissues in the sinoatrial node area.Western Blot was used to detect the protein expression of Hyperpolarisation activated cyclic nucleotide gated channel 4(HCN4)and Connexin 43(CX43)in the three tissues.2.Establish a sick sinus syndrome model group:(1)control group(n=25);(2)20%sodium hydroxide wet compress group(n=30);(3)10% sodium hydroxide intravenous injection group(n=27);(4)40% formaldehyde Wet compress group(n=25);(5)Sino-atrial node ischemia-reperfusion injury group(n=23);(6)Ivabradine gavage group(n=25),(7)Sino-atrial node artery ligation group(n=20),to detect the changes in heart rate of rats before and after modeling in each group;HE staining and Masson staining were used to detect cell morphological changes and fibrosis in the sinoatrial node area of each group,and to compare the success rate,mortality and superiority of modeling.WB was used to detect the expression of HCN4 protein in sinoatrial node tissues in each group.3.PCR technology was used to detect the membrane clock ion channel genes(Hyperpolarisation activated cyclic nucleotide gated channel 4(HCN4)(encoding HCN4 ion channel)and hyperpolarization activated cyclic nucleotide gated channel 4(HCN4)of each group of sinoatrial node tissues.Activated cyclic nucleotide gated channel 1(Hyperpolarisation-activated cyclic nucleotide gated channel 1,HCN1)(encoding HCN1 ion channel),sodium voltage-gated channel alpha subunit 5(SCN5A)(Encoding Nav1.5sodium ion channel),Potassium inwardly rectifying channel subfamily J member 5 Kcnj5(encoding inward rectifying potassium channel Kir3.4),calcium voltage-gated channel subunit α 1C(Calcium voltage-gated channel subunit alpha1 C Cacna1c)(encoding L-type calcium channel Cav1.2),calcium voltage-gated channel subunit alpha1 H(Calcium voltage-gated channel subunit alpha1 H Cacna1h)(encoding T-type calcium channel Cav3.2))and calcium clock ion channel genes(ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2 Atp2a2)(encoding sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2 Atp2a2),ryanodine receptor 2(Ryanodine receptor)Ry R2)(encoding ryanodine receptor)m RNA expression.Results:1.The atrial and ventricular tissues in the sinoatrial node area and the area around the sinus node were taken.The HE staining results showed that the sinus node cell morphology in the sinoatrial node area was similar to that of atrial myocytes.The extracellular matrix in the sinus node area was slightly looser.The boundary between the atrial node and atrial muscle is unclear;Masson staining showed no obvious fibrosis in the sinus node area.Western Blot was used to detect the expression of HCN4 protein,and the results showed that HCN4 in the sinoatrial node area was significantly higher than that of the atrial tissue and ventricles surrounding the sinus node(P<0.05),with a small amount of expression in the peripheral atrial area and almost no expression in the ventricular area;CX43 showed Compared with the atrial and ventricular tissues surrounding the sinus node,the expression in the sinus node area was significantly lower(P<0.05);2.In 20% sodium hydroxide wet compress method,10% sodium hydroxide intravenous injection method,40% formaldehyde wet compress method and ischemia-reperfusion injury method,the heart rate of rats in each group was after modeling and after 1 week and 2 weeks After the heart rate is significantly slowed down and the heart rate is lower than 30% before modeling,it is considered that the modeling is successful..The results of HE staining showed that the sinoatrial node cells were reduced and the interstitial tissue was proliferated,and the results of Masson staining showed that the sinoatrial node area was obviously fibrotic.Western Blot showed that the expression of HCN4 in the sinus node area was significantly reduced by the four modeling methods(P<0.05),so the four methods can successfully make the sick sinus syndrome model,and the 20% sodium hydroxide wet compress method The success rate is the highest(62%),and the success rate of ischemia-reperfusion injury is the lowest(23%).Ivabradine gavage can cause the heart rate to slow down,HE staining shows no decrease in sinoatrial node cells,and no interstitial hyperplasia,Masson staining,no fibrosis in the sinoatrial node,and no changes in HCN4 expression in WB and PCR.Decreased heart rate is the main clinical manifestation of SSS,so it can be considered that ivabradine can make a rat SSS model.Sinus artery ligation can cause the heart rate of SD rats to slow down,but the heart rate of the rats is completely restored one week after modeling.HE staining does not show cell reduction and interstitial hyperplasia,but neovascularization in the sinus node area is visible,so this method Cannot make SSS model.3.In this study,20% sodium hydroxide wet compress method,10% sodium hydroxide intravenous injection method,40% formaldehyde wet compress method and ischemia-reperfusion injury method were used to establish the model.PCR was used to detect the membrane clock and calcium clock of the sinoatrial node tissue.Related pacing genes are significantly reduced.Conclusion:1.Five methods: 20% sodium hydroxide wet compress method,10% sodium hydroxide intravenous injection method,40% formaldehyde wet compress method,ischemia-reperfusion injury method,and ivabradine gavage method can produce stable sick sinus.In the rat model of nodal syndrome,ivabradine gavage has the highest success rate,followed by 20% sodium hydroxide wet compress.In addition,the sinus node artery ligation method can reduce the heart rate of rats,but the heart rate is completely restored after 1 week,so the SSS model cannot be successfully made.2.After modeling with the above four traditional methods,the expressions of media clock-related pacing genes in rat sinoatrial node tissue: HCN4,HCN1,SCN5 A,Kcnj5,Cacna1 c,Cacna1h,and calcium clock-related pacing genes Atp2a2,Ry R2 were significantly reduced.This indicates that the dysfunction of membrane clock and calcium clock ion channels has a great correlation with SSS.