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Try out PMC Labs and tell us what you think. Learn More. There is controversy regarding the superiority of carvedilol C over metoprolol M in congestive heart failure. We hypothesized that C is superior to M in chronic ischemic cardiomyopathy because of its better anti-inflammatory and pro-angiogenic effects. In order to test our hypothesis we used a chronic canine model of multivessel ischemic cardiomyopathy where myocardial microcatheters were placed from which interstitial fluid was collected over time to measure leukocyte count and cytokine levels.
Tissue was examined for immunohistochemistry, oxidative stress, and capillary density. At 3 months both rest and stress wall thickening were better in C compared to the other groups. At the end of 3 months of treatment endsystolic wall stress also decreased the most in C. Myocardial interstitial fluid showed greater attenuation of leukocytosis with C compared to M, which was associated with less fibrosis and oxidative stress.
C also had higher Iso of a mbf for Elizabeth Pennsylvania level and capillary density. In conclusion, in a chronic canine model of multivessel ischemic cardiomyopathy we found 3 months of C treatment resulted in better resting global and regional function as well as better regional function at stress compared to M. These changes were associated with higher myocardial levels of the anti-inflammatory cytokine IL and less myocardial oxidative stress, leukocytosis, and fibrosis. Capillary density and MBF were almost normalized.
Thus in the doses used in this study, C appears to be superior to M in a chronic canine model of ischemic cardiomyopathy from beneficial effects on inflammation and angiogenesis. Further studies are required for comparing additional doses of these drugs. Whereas ly they were thought to exert their beneficial effects solely through anti-adrenergic or anti-ischemic [ 1320 ] mechanisms, it has now become increasingly clear that they also have important anti-inflammatory properties, which can offer independent salutary effects in CHF [ 24 ].
Almost all clinical trials evaluating BB have included patients with both ischemic and non-ischemic cardiomyopathy. Furthermore, a sizeable proportion of patients with ischemic cardiomyopathy have had prior myocardial infarction MI. animal studies of BB in ischemia have mostly utilized infarct models where the myocardial effects of BB have been studied at one point in time at autopsy [ 21303137 ]. Furthermore, a large of patients with ischemic cardiomyopathy do not have a prior MI.
These patients either have repetitive stunning severe coronary stenosis with reduced coronary flow reserve leading to repeated episodes of myocardial ischemia during stress or hibernation [reduced resting myocardial blood flow MBF from severe coronary narrowing] [ 5681415 ]. There has been no investigation on the regional myocardial effects of BB in an animal model that mimics one or both of these two latter situations.
In the present study, we hypothesized that BB have a beneficial role in a canine model of chronic ischemic cardiomyopathy through their anti-inflammatory and proangiogenic effects and carvedilol may be superior to metoprolol in this regard. In order to test our hypothesis we gave BB therapy to dogs with chronic ischemic cardiomyopathy produced by placing ameroid constrictors on the proximal portions of the left anterior descending LAD and left circumflex LCx coronary arteries and their major branches [the right coronary artery does not supply the left ventricular LV myocardium in the dog].
Over time the constrictors occlude the coronary arteries producing chronically ischemic myocardial segments with either stunning or hibernation, but no necrosis. We have described the features of this model ly [ 231134 ], and have tested it for various therapies aimed at reducing chronic myocardial ischemia and CHF [ 1819 ]. For this study, animals were chronically instrumented to measure left atrial and aortic pressures and radiolabeled microsphere-derived MBF. Regional LV systolic function was measured at regular intervals using 2-dimensional echocardiography 2DE.
In addition, microcatheters were placed within the myocardium from which interstitial fluid could be collected periodically over time to measure leukocyte count and cytokine levels that reflect myocardial inflammation. Of these, seven underwent sham surgery dissection of coronary arteries and insertion of microcatheters only and the remaining 36 animals were instrumented to create chronic ischemic cardiomyopathy. They were given 75 mg of aspirin daily for 3 days prior to surgery and Gentamicin 80 mg and Cefazolin 1 g immediately prior to surgery, and Cefazolin alone daily through post-operative day 3.
Surgery was performed under sterile conditions. The animals were intubated and anesthesia was maintained with a Iso of a mbf for Elizabeth Pennsylvania of 1—1. Minute volume was set between 5.
Heart rate and electrocardiogram were monitored throughout the operation. A small incision was made in the right groin and a 6F indwelling catheter Cook Instruments, Bloomington, IN was inserted into the femoral artery and secured in place with silk ties. The catheter was flushed with a dilute solution of heparin Sololak Laboratories, Elk Grove Village, IL and capped off with a rubber injection port. It was then tunneled beneath the skin in the groin to allow subsequent transcutaneous access for arterial pressure monitoring, as well as withdrawal of samples for blood gas and radiolabeled microsphere-derived MBF analyses.
The groin incision was then closed in layers. A left lateral thoracotomy was performed in the fourth intercostal space and the heart was suspended in a pericardial cradle. The proximal portions of the LAD and LCx were dissected free from surrounding tissues, and any large proximal branches of these arteries were similarly dissected. Three or more appropriately sized 1—3. LV function was assessed by epicardial 2DE after placement of each ameroid constrictor to ensure that no deterioration in regional systolic function occurred.
Four microcatheters two in the LAD and 2 in the LCx regions were inserted in the myocardium at the low-papillary muscle level using a gauge catheter.
They were threaded through approximately 3 cm of myocardium before retrieving them again on the epicardial surface of the heart. In this manner, a portion of the catheter containing multiports see below was embedded in the myocardium with the two ends extruding out. Epicardial 2DE was performed to confirm that the entire lengths of the catheters were within the myocardium without entering the LV cavity. Both free ends of the microcatheters were then tunneled beneath the dorsal skin to allow subsequent access for myocardial interstitial fluid collection. A 6F indwelling catheter was inserted in the left atrium and secured in placed with prolene sutures.
After flushing with a dilute heparin solution, its end was capped off with a rubber injection port and buried beneath the abdominal skin to allow subsequent transcutaneous injections of radiolabeled microspheres. The chest was closed in layers, and the animal was revived and transferred to a heated and oxygenated observation area.
Pain medications were administered as needed.
The animals were examined at least twice a day for the follow-up period of 6 months and treated for heart failure as evidenced by tachypnea, poor appetite, and reluctance to engage with handlers or infection, if required. Microcatheters were constructed by creating 40 multiports in a 1 cm segment of a cm Micro-Renathane catheter 0. The gauze was used to anchor the catheter to the epicardium at the time of insertion into the myocardium. The microcatheters were gas sterilized.
Interstitial fluid leukocyte count was measured using an automated analyzer Sysmex XE, Kobe, Japan and then confirmed using a hemocytometer. The Bio-Plex suspension array system was used to quantify cytokine levels from myocardial interstitial fluid and plasma collected from venous blood. The samples were incubated with antibody-coupled be, washed, incubated with biotinylated detection antibody, washed again, and then incubated with streptavidin—phycoerythrin and read on a microplate-based bioassay system.
In order to maximize the accuracy, all samples were measured in duplicate at the same time. Because the duration of the protocol was 5—6 months we used radiolabeled microspheres with long half-lives and adequate separation of the energy windows to measure regional MBF. Iso of a mbf for Elizabeth Pennsylvania each dog four of the following five [with their energy windows keVand half-lives days ] microspheres were used: Ce, —, 33; Sn, —, ; Ru, —, 39; 95 Nb, —, 35; and, 46 Sc, —1, The microspheres with longer half-lives were injected earlier in the protocol to allow enough counts in the sample on post-mortem analysis.
This dose of radiolabeled microspheres allows at least 1, microspheres to be counted in each gram of normal tissue and at least microspheres in each gram of ischemic tissue. Reference samples were withdrawn from the femoral artery over s with a constant rate withdrawal pump Harvard Apparatus, Model The post-mortem heart slices see latercorresponding to the mid-papillary muscle 2DE short-axis image, were cut into 16 wedge-shaped pieces.
Each piece was further divided into epicardial, mid-myocardial, and endocardial portions. Corrections were made for activity spill-over from one window to the next using a set of simultaneous equations. For arterial pressure measurements, tubing primed with heparinized normal saline was connected at one end to a pressure transducer model A, Hewlett Packard, Palo Alto, CA and at the other end to the arterial catheter. This transducer and the electrocardiographic port were connected to a multi-channel recorder model C, Hewlett-Packardwhich in turn was connected to a computer.
Apart from the intraoperative 2DE described above, all data were acquired with the dog lying on its left side and imaging performed from the right thorax. Although the examination was performed using multiple views, only three short-axis views were recorded on each examination. Care was taken to acquire the same views every time in an individual dog. For each animal, the short-axis view displaying the maximal degree of global LV dysfunction at any time during follow-up was identified.
All wall thickening WT measurements were then made at this single level usually the mid-papillary muscle. An entire systolic contraction sequence from end-diastole to end-systole was selected from each examination and the images were transferred to a custom-deed offline image analysis system. In brief, 8—12 epicardial and endocardial targets were defined by the observer in each frame from end-diastole to end-systole. These points were then automatically connected using cubic-spline interpolation to derive the epicardial and endocardial contours.
In order to correct for cardiac systolic rotation, the junction of the posterior LV free wall and the right ventricular free wall was defined over the epicardium in each frame. The computer generated equidistant chords between the two contours starting at this point with each chord representing the shortest distance between the epicardial and endocardial contours.
The observer then identified the regions of the myocardium in which the chord lengths were averaged in each frame. The heart slice corresponding to the 2DE image was immersed in a solution of 1. Using this method, areas of viable myocardium stain brick red whereas infarcted areas do not take up the stain due to lack of enzymatic activity [ 12 ]. All analysis was performed by observers blinded to the source of tissue. Myocardial tissue was fixed in formalin and paraffin-embedded for histologic analysis. Endothelial cells of capillaries were identified using immunohistochemical staining with the antibody to von Willebrand factor.
All rinses were with tris-buffered saline TBSpH 7. The secondary antibody, biotinylated goat anti-rabbit diluted in blocking serum, Vectorwas applied to the tissue for 60 min at room temperature, and sections were incubated with avidin—biotin-peroxidase complex Vectastain Elite kit, Vector for 30 min.
The color reaction was visualized with diaminobenzidine DAB. Capillary density was expressed as the of capillaries per high power field. In sections with fibrosis, a representative area immediately adjacent to the fibrotic tissue was selected for analysis. The density of apoptotic myocytes was expressed as the of apoptotic myocytes per high power field.
Frozen myocardial tissue was homogenized and supernatant was collected.
The gradient mobile phase was delivered at a flow rate of 0. Individual compounds were monitored with multiple reactions monitoring MRM. Optimal instrument MRM parameters were obtained for the available standards using direct infusion. Data were acquired and analyzed using Analyst 1.
The standard curves were from 0 to 2, pg per sample and the limit of quantification was 50 pg. All except the sham dogs were ased randomly to one of three groups at the time of initial surgery: a placebo, b metoprolol treatment, or c carvedilol treatment. All animals underwent weekly 2DE to evaluate for development of LV systolic dysfunction. Severe LV dysfunction occurred 5. At this time a 3 month treatment protocol was initiated: non-sham animals were treated orally with either placebo, metoprolol These doses were selected to be comparable to an average human that is twice the weight of the dogs used.
At baseline and the Iso of a mbf for Elizabeth Pennsylvania of each month of treatment, hemodynamic data were collected, 2DE was performed, and intra-myocardial fluid and venous blood were collected with the dogs anesthetized and intubated. If target heart rate of — bpm was not achieved, intravenous atropine 0. At the conclusion of 3 months of therapy the animal was euthanized and the heart was removed for radiolabeled microsphere-derived blood flow analysis, histopathology, immunohistochemistry and oxidative stress analyses. Other than the seven sham dogs, 12 each were randomly ased to the three treatment groups out of which three died before and two after start of treatment in the placebo group, one before start of treatment in the metoprolol group and two before start of treatment in the carvedilol group.
No dog died after start of treatment in the two latter groups. Consequently the reported here pertain to the 7 sham dogs and 8, 11, and 10 dogs in the placebo, metoprolol, and carvedilol groups, respectively. As stated above, all measurements were made with the closed chest animals anesthetized and intubated. Resting heart rate was not different between any of the groups between pre-surgery, baseline development of severe LV dysfunctionand 1—3 months after treatment.
Table 1 lists data at baseline and 3 months later. The mean aortic pressure at rest measured in mm Hg via indwelling catheter was also not different at baseline between the groups.
However, it was higher at the end of 3 months in the placebo compared to other groups. It was also higher in the placebo group compared to baseline.Iso of a mbf for Elizabeth Pennsylvania
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Iso of a mbf for Elizabeth Pennsylvania