Methods for the development and assessment of atrial fibrillation and heart failure dog models

Objective To report Medtronic experiences with the development of animal models for atrial fibrillation （AF） and chronic heart failure （CHF） using high-rate pacing for AF and microemboli for CHF. Methods For the AF model, an atrial lead was attached to a Medtronic SynergyTM neurostimulator, which was programmed to stimulate at 50 Hz in an on-off duty cycle. Atrial natfiuretic peptide （ANP）, brain natriuretic peptide （BNP） and N-terminal pro brain nalriuretic peptide （NT-proBNP） were assayed at select time points. For CHF model, a serial injection of 90 μm polystyrene microspheres at 62,400 beads/mL （Polybead, Polysciences, Inc.） was performed to induce global ischemia, either with weekly monitoring and embolization schedule （group 1, n = 25） or with biweekly monitoring and emboliation schedule （group 2, n = 36 ）. Echocardiograms were used along with ventriculograms and magnetic resonance imaging scans weekly to assess cardiac function and ANP, BNP and NT-proBNP were monitored. Results For the AF model, the days to sustained AF for four animals following surgery were 7, 25, 21 and 19, respectively; For the CHF model, the days to meet CHF endpoints were 116 in group 1 and 89 in group 2. For both AF and CHF models, NT-proBNP correlated well with the development of disease states. Conclusion Our experience for the development and assessment of AF and CHF dog models may help researchers who are in search for animal model for assessing the safety and efficacy of a device-based therapy.

Nicorandil pretreatment inhibits myocardial apoptosis and improves cardiac function after coronary microembolization in rats

Background Nicorandil （NIC） is a vasodilatory drug used to treat angina. However, its efficacy of cardioprotection in coronary mi- croembolization （CME） is largely unknown. This study was undertaken to determine whether nicorandil pretreatment could attenuate myo- cardial apoptosis and improve cardiac function after CME in rats. Methods Forty-five rats were randomly divided into a Sham group, a CME group and a CME ＋ NIC （NIC） group （n = 15 per group）. CME was established by injecting plastic microspheres （42 pm in diameter） into the left ventricle of the rats in all of the groups except the Sham group. The NIC group received nicorandil at 3 mg/kg per day for seven days before the operation. Cardiac fimction was assessed by echocardiography, the expression levels of cleaved caspase-9/8/3 were detected by Western blot, microinfarction area was measured by haematoxylin-basic fuchsin picric acid staining, and myocardial apoptosis was de- tected by TUNEL staining. Results Compared to that in the Sham group, cardiac fimction in the CME group was significantly decreased （P 〈 0.05）. However, compared to the CME group, the NIC group showed improved cardiac function （P 〈 0.05）. The expression levels of cleaved caspase-9/8/3 protein and myocardial apoptosis were dramatically increased in the CME group compared to those in the Sham group （P 〈 0.05）, while the NIC pretreatment group had significantly decreased expression levels of cleaved caspase-9/8/3 protein as well as a de- creased apoptotic rate （P 〈 0.05）. Conclusions NIC pretreatment inhibited CME-induced myocardial apoptosis and improved cardiac func- tion through blockade of the mitochondrial and death receptor-mediated apoptotic pathways.

Transcranial Doppler ultrasonography: From methodology to major clinical applications

Non-invasive Doppler ultrasonographic study of cerebral arteries [transcranial Doppler(TCD)] has been extensively applied on both outpatient and inpatient settings. It is performed placing a low-frequency(≤ 2 MHz) transducer on the scalp of the patient over specific acoustic windows, in order to visualize the intracranial arterial vessels and to evaluate the cerebral blood flow velocity and its alteration in many different conditions. Nowadays the most widespread indication for TCD in outpatient setting is the research of right to left shunting, responsable of so called "paradoxical embolism", most often due to patency of foramen ovale which is responsable of the majority of cryptogenic strokes occuring in patients younger than 55 years old. TCD also allows to classify the grade of severity of such shunts using the so called "microembolic signal grading score". In addition TCD has found many useful applications in neurocritical care practice. It is useful on both adults and children for day-to-day bedside assessment of critical conditions including vasospasm in subarachnoidal haemorrhage(caused by aneurysm rupture or traumatic injury), traumatic brain injury, brain stem death. It is used also to evaluate cerebral hemodynamic changes after stroke. It also allows to investigate cerebral pressure autoregulation and for the clinical evaluation of cerebral autoregulatory reserve.

Expression of p53 in myocardium following coronary microembolization inrats and its significance

Background Cardiomyocyte apoptosis is a primary cause for coronary microembolization （CME）-induced cardiac dysfunction, p53induces cell growth retardation and apoptosis through stress pathway. The present study investigated the mechanism of p53-induced myocar-dial apoptosis and cardiac dysfunction by activating the mitochondrion apoptotic pathway following CME. Methods Forty SD rats wereequally divided into microembolization （CME）, sham operation （sham）, CME＋siRNA-p53, and CME＋control-p53 groups. The CME ratmodel was established by injecting microembolization spheres via the left ventricle. Cardiac ultrasound, TUNEL, fluorescence quantitativePCR, and Western blot were used to assess the cardiac function indicators, cardiomyocyte apoptosis, and the expressions of mRNA and pro-tein in myocardial tissues, respectively. Results Echocardiography revealed a significantly reduced cardiac function of the CME group thanthe sham group while the CME-induced cardiac dysfunction was improved in the CME＋siRNA-p53 group. The indicators of myocardialapoptosis in the CME group increased significantly than the sham group; those of the CME＋siRNA-p53 group decreased significantly thanthe CME group. Fluorescence quantitative PCR and Western blot demonstrated that p53, Bbc3 （PUMA）, and cleaved caspase-3 expressionswere significantly increased, and BCL-2 expression was declined in myocardial tissues of the CME group compared to the sham group. Acontrasting result was observed in the CME＋siRNA-p53 group as compared to the CME group. Conclusions P53 is involved in theCME-induced cardiac dysfunction, which may up-regulate Bbc3 to activate BCL-2/caspase3 mitochondrial apoptotic pathway and inducemyocardial apoptosis. Inhibiting the p53 expression can effectively suppress this pathway, thereby reducing myocardial apoptosis and car-diac dysftmction.

Effect of metoprolol on myocardial apoptosis after coronary microembolization in rats

BACKGROUND： Coronary microembolization （CME） is a serious complication following percutaneous coronary intervention （PCI） in patients with acute coronary syndromes. The use of metoprolol before PCI can significantly protect ischemic myocardium from myocardial damage, but the function of metoprolol in the treatment of CME is not entirely clear. This study was to explore the effect and significance of metoprolol on myocardial apoptosis and caspase-3 activation after CME in rats. METHODS： Thirty rats were randomly divided into three groups including sham-operation （control group）, CME plus saline （CME group）, CME plus metoprolol （metoprolol group）, 10 rats for each group. The CME group was induced by injecting 3 000 polyethylene microspheres （42 tJm） into the left ventricle during a 10-second occlusion of the ascending aorta; the control group was injected with physiological saline instead of microembolization ball; the metoprolol or saline group was given three intravenous bolus injections before CME. Echocardiography, TUNEL staining, and Western blotting were used to evaluate cardiac function, proportion of apoptotic cells and activation of caspase-3 respectively at 6 hours after operation. RESULTS： Echocardiographic parameters displayed that the metoprolol group improved cardiac function significantly compared with the CME group （P〈0.05）. The myocardial apoptotic rate of the CME group as well as the contents of activated caspase-3 increased significantly （P〈0.05）, both of which were ameliorated significantly by metoprolol treatment （P〈0.05）. CONCLUSIONS： This study demonstrates that metoprolol can protect the myocardium during CME in rats by inhibiting apoptosis and improving cardiac function. These results suggest that the inhibition of apoptosis can be a potential therapeutic strategy for the treatment of CME.

Magnetic resonance imaging characterization of circumferential and longitudinal strain under various coronary interventions in swine

AIM:To compare the acute changes in circumferential and longitudinal strain after exposing a coronary artery to various interventions in swine.METHODS:Percutaneous balloon angioplasty catheter was guided to location aid device(LAD)under X-ray fluoroscopy to create different patterns of ischemic insults.Pigs(n=32)were equally divided into 4 groups:controls,90 min LAD occlusion/reperfusion,LAD microembolization,and combined LAD occlusion/microembolization/reperfusion.Three days after interventions,cine,tagged and viability magnetic resonance imaging(MRI)were acquired to measure and compare left and right circumferential strain,longitudinal strain and myocardial viability,respectively.Measurements were obtained using HARP and semi-automated threshold method and statistically analyzed using unpaired t-test.Myocardial and vascular damage was characterized microscopically.RESULTS:Coronary microemboli caused greater impairment in l left ventricular(LV)circumferential strain and dyssynchrony than LAD occlusion/reperfusion despite the significant difference in the extent of myocardial damage.Microemboli also caused significant decrease in peak systolic strain rate of remote myocardium and LV dyssynchrony.Cine MRI demonstrated the interaction between LV and right ventricular(RV)at 3 d after interventions.Compensatory increase in RV free wall longitudinal strain was seen in response to all interventions.Viability MRI,histochemical staining and microscopy revealed different patterns of myocardial damage and microvascular obstruction.CONCLUSION:Cine MRI revealed subtle changes in LV strain caused by various ischemic insults.It also demonstrated the interaction between the right and left ventricles after coronary interventions.Coronary microemboli with and without acute myocardial infarction(AMI)cause complex myocardial injury and ventricular dysfunction that is not replicated in solely AMI.

Coronary microembolization induced myocardial contractile dysfunction through a p38 mapk pathway

Background Cathepsin S and its endogenous inhibitor cystatin C are implicated in the pathogenesis of atherosclerosis,especially in the plaque destabilization and rupture leading to acute coronary syndrome.However, whether circulating cathepsin S and cystatin C also change in association with coronary plaque morphology is unknown yet. Methods Sprague-Dawley rats were randomly divided into three groups;Sham group,CME group and SB203580 group (n=10 per group).CME rats were produced by injection of 42μm microspheres into the left ventricle with occlusion of the ascending aorta.SB203580,a p38 MAPK inhibitor,was injected into femoral vein after finishing the injection of microspheres in SB203580 group.Left ventricular Ejection Fraction was determined by echocardiography.The level of phosphorylated and total P38 MAPK in myocardium was assessed by Western Blot.Results Left ventricular(LV) Ejection Fraction was depressed at 3 hours and until up to 12 hours in CME group.The increased p38 MAPK activation was observed in CME group.The administration of SB203580 partly inhibited the p38 MAPK activity and preserved cardiac contractile function.Conclusions p38 MAPK is significantly activated by CME and the inhibition of p38 MAPK can partly preserve cardiac contractile function.

Coronary Embolization and Myocardial Microinfarction: MR Imaging and Histopathologic Characterization

Magnetic resonance imaging (MRI) has been proven to reliably assess regional perfusion and left ventricular (LV) function of microembolized myocardium. The visibility of microinfarct on delayed enhancement MRI (DE-MRI) is limited and dependent on technical and biological issues. Furthermore, MRI underestimates total microinfarct size compared with microscopy. MRI studies revealed that the presence of microemboli in pre-existing acute infarct delays infarct healing and magnifies LV remodeling. Discrimination of acute from chronic microinfarct is based on presence of inflammatory cells, edema and scar tissue, respectively. These noninvasive findings highlight the importance of prognostic utility of MRI and warrant larger clinical studies or registries to evaluate the significance of presence of focal microinfarct. Serial microscopic studies revealed that intravascular microemboli migrate into the extravascular space and this migration process is a function of time. This phenomenon may limit the use of microemboli therapy in occluding hemorrhagic blood vessels or treating tumors. Despite current standard of care, existing methods and therapies do not prevent coronary embolization nor reverse their deleterious effects.

Clinical utilization of microembolus detection by transcranial Doppler sonography in intracranial stenosis-occlusive disease

Objective To discuss the clinical ultiliazation and significance of microembolus detection by transcranial Doppler （TCD） sonography in intracranial stenosis-occlusive disease. Data sources All related articles in this review were mainly searched from PubMed published in English from 1996 to 2012 using the terms of microembolic signal, transcranial Doppler, intracranial stenosis, stroke. Study selection Original articles and reviews were selected if they were related to the clinical utilization of microembolus detection in intracranial stenosis-occlusive disease. Results Intracranial stenosis is a significant cause of cerebral emboli, and microembolus detection by TCD sonography were widely used in exploring the mechanisms of ischemic stroke with intracranial stenosis （including the middle cerebral artery stenosis and the vertebral-basilar stenosis）, evaluating the prognosis of acute stroke, evaluating the therapeutic effects, and predicting the recurrent events of stroke. Conclusion Microembolus detection by TCD sonography plays an important role in the cerebral ischemic stroke patients with intracranial stenosis.

Efficacy of Dual Antiplatelet Therapy Combined with Naoxintong Capsules(脑心通胶囊) Following Coronary Microembolization Induced by Homologous Microthrombi in Rats

Objective： To evaluate the efficacy of dual antiplatelet therapy combined with Naoxintong Capsule （脑心通胶囊, NXTC） in a rat model of coronary microembolization （CME）. Methods： A total of 95 rats were randomly divided into 6 groups： control, sham-operation, CME model, NXTC, dual antiplatelet （clopidogrel and aspirin） intervention （DA）, and NXTC combined with DA （NDA） groups. The complete data in 69 rats were obtained. The number of CME, myocardial apoptosis rate, bleeding time, clotting time, and adensosine diphosphate （ADP）-induced platelet aggregation were assessed. Results： Compared with the CME group, the number of CME and myocardial apoptosis rates were significantly decreased in the NXTC, DA, and NDA groups （P〈0.01）. Compared with other intervention groups, the number of CME and myocardial apoptosis rates were the least in the NDA group （P〈0.01）, and the incidence of surgical bleeding was the highest in the DA group （P〈0.01）. Compared with the CME group, ADP-induced maximum platelet aggregation rate was significantly inhibited in the NXTC, DA, and NDA groups （P〈0.01）, both bleeding time and clotting time were significantly increased in the NXTC, DA, and NDA groups （P〈0.01）, while the above parameters were the highest in the DA group （P〈0.05）. Conclusion： The combination therapy of NXTC and DA enhanced the anti-CME effect of either therapy alone and reduced the risk of the DA therapy-associated bleeding, demonstrating an improved benefit/ risk ratio in the rat model of CME.

Shexiang Tongxin Dropping Pill(麝香通心滴丸)Reduces Coronary Microembolization in Rats via Regulation of Mitochondrial Permeability Transition Pore Opening and AKT-GSK3βPhosphorylation

Objective To investigate the protective effects of Shexiang Tongxin Dropping Pill(麝香通心滴丸,STDP)following sodium laurate-induced coronary microembolization(CME)in rats.Methods Forty rats were divided into 4 groups:the control(sham)group,CME group,low-dose STDP pretreatment group(20 mg·kg^(−1)·d^(−1)),and high-dose STDP pretreatment group(40 mg·kg^(−1)·d^(−1)).The rats were intragastric administrated with STDP 2 weeks before operation.Moreover,the histopathological alterations were observed using optical microscopy and transmission electron microscopy.Antioxidant biomarkers were analyzed by enzyme-linked immunosorbent assay.Mitochondrial functions including the mitochondrial permeability transition pore(mPTP)mtDNA copy number were determined and proteins of AKT/GSK3βwere analyzed by Western blot.Results The rats in the CME group showed a significant increase in the fibrinogen-like protein 2 expression level and mitochondrial dysfunction and a decrease in the expression level of antioxidant biomarkers(superoxide dismutase and catalase,P<0.01 for all).In contrast,the rats in the low-and high-dose STDP pretreatment groups showed a significant decrease in coronary microthrombi(P<0.05);moreover,STDP restored the antioxidant-related protein activities and mitochondrial function,inhibited mPTP opening,decreased AKT-Ser473 phosphorylation,and increased GSK3β-Ser9 phosphorylation(P<0.05 or P<0.01).Conclusion STDP may be useful for treatment of CME,possibly via regulation of mPTP opening and AKT/GSK3βphosphorylation.

Acute hyperenhancement on delayed contrast-enhanced magnetic resonance imaging is the characteristic sign after coronary microembolization

Background Detection of coronary microembolization is of clinical importance for patient management and prediction of long-term outcome. However, there are few studies of the changes of magnetic resonance imaging after coronary microembolization. This study was designed to investigate the imaging of the left ventricle using delayed contrast enhanced magnetic resonance imaging as well as the left ventricular ejection fraction after coronary microembolization in animal models.Methods Eight miniswine, of either sex （body weight 21-25 kg）, were used to make the coronary microembolization model. After coronary angiography, a 2.8F infusion catheter was placed in the left anterior descending artery with the tip located between the second and third diagonal branches. Microspheres with the diameter of 42μm and mean dosage of 1.2×10^5 were selectively infused into the left anterior descending artery. First pass and stressed first pass perfusion scan were performed after cine images were acquired. Then a second bolus of 0.15 mmol/kg gadolinium DTPA was given at a rate of 2 ml/s. Ten minutes later, delayed contrast enhanced magnetic resonance images of the left ventricular wall were evaluated. Serum changes of tumor necrosis factor a （TNF-α） were evaluated by enzyme-linked immunosorbent assay （ELISA）.Results Hypoenhancement was not observed at first pass perfusion at the anterior wall of the left ventricle. Hyperenhancements of the anterior-septal and anterior wall of the left ventricle was in evidence on delayed enhancement images 6 hours after microembolization and disappeared one week later. The characteristic change of coronary microembolization on delayed contrast enhanced magnetic imaging was non-enhanced regions within the hyperenhancement zone. Left ventricular ejection fraction measured by magnetic resonance imaging decreased significantly from 0.451±0.063 at baseline to 0.362±0.070 at the sixth hour （P 〈0.01）, and recovered to 0.431±0.053 one week later （P 〈0.01 vs 6th hour）. Compared with baseline values, the left ventricular end systolic volume enlarged significantly at 6th hour and at one week after microembolization （P 〈0.05 and P 〈0.01 respectively）. Serum TNF-α increased significantly at 6th hour （22.62±6.96） pg/ml compared with baseline （16.83±3.45） pg/ml （P 〈0.05） and it further increased to （27.44±3.97） pg/ml at one week after coronary microembolization and was significantly higher than that at baseline （P 〈0.01）.Conclusions On delayed contrast enhanced magnetic resonance imaging, hyperenhancement of the anterior-septal and anterior wall of the left ventricle show at 6th hour but not at one week after coronary microembolization. This might represent the characteristic imaging after coronary microembolization. The left ventricular ejection fraction decreased at 6th hour and recovered one week later after coronary microembolization. Although impairment of left ventricular function could be recovered at 1 week after coronary microembolization, the left ventricular remodeling process still continued in concert with continuously elevation of serum TNF-α.

IRAK1, TRAF6 and AUF1 are involved in myocardial inflammatory response after coronary microembolization in miniature swines

Background Coronary microembolization （CME） is characterized by distal microvascular occlusion. However, the inflammatory mechanisms and therapeutic targets of CME are largely unknown. Methods A total of 11 Guangxi Bama miniature swines were divided into two groups： sham （n = 5） and CME （n = 6）. Microspheres were injected into the left anterior descending artery of the CME group to make an animal model of CME. The expres- sions of microRNA-146a （miR-146a） and IRAK1, TRAF6, and AUF1 in the myocardium were detected by qPCR. Results In the CME group, microspheres, microinfarction, and inflammatory cell infiltration were found under an optical microscope. The expression levels of miR-146a were low in both groups. After CME, the expression levels of IRAK1, TRAF6, and AUF1 in the CME group were upregulated compared with those in the sham group （P 〈 0.01;P 〈 0.05;P 〈 0.05, respectively）. Conclusions AUF1, IRAK1 and TRAF6, but not miR-146a, could be involved, in myocardium inflammation following CME.