Stable labeled microspheres to measure perfusion: validation of a neutron activation assay technique.

Neutron activation is an accurate analytic method in which trace quantities of isotopes of interest in a sample are activated and the emitted radiation is measured with high-resolution detection equipment. This study demonstrates the application of neutron activation for the measurement of myocardial perfusion using stable isotopically labeled microspheres. Stable labeled and standard radiolabeled microspheres (15 microm) were coinjected in an in vivo rabbit model of myocardial ischemia and reperfusion. Radiolabeled microspheres were detected with a standard gamma-well counter, and stable labeled microspheres were detected with a high-resolution Ge detection after neutron activation of the myocardial and reference blood samples. Regional myocardial blood flow was calculated from the deposition of radiolabeled and stable labeled microspheres. Both sets of microspheres gave similar measurements of regional myocardial blood flow over a wide range of flow with a high linear correlation (r = 0.95-0.99). Neutron activation is capable of detecting a single microsphere in an intact myocardial sample while providing simultaneous quantitative measurements of multiple isotope labels. This high sensitivity and capability for measuring perfusion in intact tissue are advantages over other techniques, such as optical detection of microspheres. Neutron activation also can provide an effective method for reducing the production of low-level radioactive waste generated from biomedical research. Further applications of neutron activation offer the potential for measuring other stable labeled compounds, such as fatty acids and growth factors, in conjunction with microsphere measured flow, providing the capability for simultaneous measurement of regional metabolism and perfusion.

The effect of ischemic injury on the cardiac transport of Tc-99m N-NOET in the isolated rabbit heart.

BACKGROUND: Bis (N-ethoxy, N-ethyl dithiocarbamato) nitrido technetium-99m (V) (TcN-NOET) is a neutral lipophilic myocardial perfusion agent. The effect of ischemic injury on the cardiac transport of TcN-NOET and thallium-201 was determined in isolated rabbit hearts. METHODS AND RESULTS: The multiple indicator dilution method was used to determine the maximum (Emax) and net extraction (Enet, at 5 minutes) of TcN-NOET and TI-201 at control and after 10 minutes (n = 4) or 45 minutes (n = 4) of no-flow ischemia. After 10 minutes of ischemia the mean Emax for T1-201 was unchanged, 0.86 +/- 0.03 vs 0.85 +/- 0.02, whereas TI-201 Enet showed a small decrease from 0.46 +/- 0.03 to 0.40 +/- 0.03, P < .001. Forty-five minutes of ischemia mildly reduced Emax for TI-201 (0.87 +/- 0.04 to 0.74 +/- 0.04, P < .001) and severely reduced Enet (0.46 +/-0.03 vs 0.16 +/- 0.04, P < .001). Neither Emax nor Enet for TcN-NOET was significantly affected by 10 minutes of ischemia (0.54 +/- 0.04 vs 0.58 +/- 0.03 and 0.24 +/- 0.04 vs 0.26 +/- 0.04, respectively). However, severe ischemic injury caused significant reductions versus control in both Emax (0.59 +/- 0.06 vs 0.42 +/- 0.05, P < .001) and Enet (0.27 +/- 0.03 vs 0.18 +/- 0.05, P < .01). CONCLUSIONS: TcN-NOET is a new myocardial perfusion agent with moderate myocardial extraction. Although less sensitive than TI-201 to mild ischemic injury, TcN-NOET extraction and retention are decreased by severe ischemic injury, making uptake of TcN-NOET a possible marker of myocardial viability.

Rabbit heart can be "preconditioned" via transfer of coronary effluent.

Brief myocardial ischemia not only evokes a local cardioprotective or "preconditioning" effect but also can render remote myocardium resistant to sustained ischemia. We propose the following hypotheses: remote protection is initiated by a humoral trigger; brief ischemia-reperfusion will result in release of the humoral trigger (possibly adenosine and/or norepinephrine) into the coronary effluent; and transfer of this effluent to a virgin acceptor heart will elicit cardioprotection. To test these concepts, effluent was collected during normal perfusion from donor-control hearts and during preconditioning ischemia-reperfusion from donor-preconditioned (PC) hearts. After reoxygenation occurred and aliquots for measurement of adenosine and norepinephrine content were harvested, effluent was transfused to acceptor-control and acceptor-PC hearts. All hearts then underwent 40 min of global ischemia and 60 min of reperfusion, and infarct size was delineated by tetrazolium staining. Mean infarct size was smaller in both donor- and acceptor-PC groups (9% of left ventricle) than in donor- and acceptor-control groups (36% and 34%; P < 0.01). Protection in acceptor-PC hearts could not, however, be attributed to adenosine or norepinephrine. Thus preconditioning-induced cardioprotection can be transferred between rabbit hearts by transfusion of coronary effluent. Although adenosine and norepinephrine are apparently not responsible, these results suggest that remote protection is initiated by a humoral mechanism.

Ischemic preconditioning may be transferable via whole blood transfusion: preliminary evidence.

This research was designed to test the hypothesis that ischemic preconditioning can be transferred between animals via whole blood transfusion. Preconditioning at a distance refers to the reduction in myocardial infarct size seen when coronary artery occlusion is preceded by brief ischemic episodes of noncardiac tissue. Isolation of the trigger signal responsible for this effect may be useful in the diagnosis and treatment of acute coronary occlusive syndromes. Rabbits were paired by crossmatching blood samples prior to experimentation. Crossmatched pairs were placed into either preconditioned (P) or control sets. Rabbits in the preconditioned sets were further divided into donor (PD) and acceptor (PA) animals. PD animals underwent five episodes of circumflex and renal artery occlusion followed by reperfusion. Before and after each preconditioning episode, a whole blood exchange was performed between PD and PA animals. Alternatively, control rabbits underwent the same surgical procedures and time-sequenced transfusion without preconditioning. All animals then underwent prolonged circumflex occlusion (60 minutes) followed by reperfusion (30 minutes). The area of myocardium at risk (R) was determined by isotope-labeled microsphere injection. Infarct size (I) was determined by NBT staining. The percent infarct within the risk area (I/R) was then compared. The I/R was significantly lower in the PA (14.0% +/- 12.2) and PD (14.3% +/- 11.2) groups as compared with controls (61% +/- 20. 6). There was no significant difference between the tPA and TPD groups. In conclusion, the ischemic preconditioning effect can be transferred to nonpreconditioned animals via whole blood transfusion, suggesting a humoral mechanism for preconditioning at a distance.

Thallium reinjection versus standard stress/delay redistribution imaging for prediction of cardiac events.

OBJECTIVES: The purpose of this study was to compare thallium reinjection with standard stress/delay redistribution for the prediction of cardiac events. BACKGROUND: Although thallium reinjection enhances the detection of viable myocardium, its contribution to prognosis over stress/delay redistribution in a general referral population has not been clearly evaluated. METHODS: This retrospective analysis included 366 consecutive patients with coronary artery disease who underwent stress/delay redistribution imaging and thallium reinjection scintigraphy, with a mean follow-up of 33+/-12 months. RESULTS: Cardiac events occurred in 48 patients (40 deaths, 8 myocardial infarctions). Of the 366 original patients, 159 demonstrated ischemia by stress/delay redistribution, 107 showed ischemia by reinjection only, and 100 showed infarction only. Cardiac events occurred in 20 patients (12.6%) with stress/delay redistribution, 13 patients (12%) with ischemia detected by thallium reinjection only and 15 patients (15%) with infarction only. The size of the reversible thallium defect by either stress/delay redistribution imaging or reinjection scintigraphy did not predict cardiac events. Independent predictors of cardiac events included left ventricular cavity size, the size of the abnormal perfusion defect and patient age. CONCLUSIONS: Thallium reinjection does not contribute independent prognostic utility for cardiac events when compared with stress/delay redistribution. Left ventricular dilation and the size of the post-stress defect were predictors of cardiac events.

Direct detection of regional myocardial ischemia with technetium-99m nitroimidazole in rabbits.

UNLABELLED: Conventional perfusion scintigraphy assesses disparities in regional myocardial blood flow but does not directly detect hypoxic tissue. Nitroimidazoles labeled with positron-emitting radionuclides have recently shown promise as direct markers of myocardial hypoxia. This study evaluates a new 99mTc-labeled nitroimidazole of potential benefit in standard myocardial scintigraphy. METHODS: Technetium-99m-labeled nitroimidazole was administered to rabbits during the early reperfusion phase after 10 min (Group 1) or 60 (Group 2) min of coronary occlusion or after 10 min of a fixed coronary occlusion (Group 3). Tracer retention at 1 hr was assessed in relation to microsphere-determined blood flow during coronary occlusion and at tracer injection. The pattern of nitroimidazole retention on autoradiographs was then compared with the pattern of myocardial hypoperfusion defined by fluorescein photography to precisely define tracer localization. RESULTS: The retention of nitroimidazole in Group 1 rabbits (brief occlusion) was independent of both occlusion and reperfusion blood flow and was uniformly distributed on the autoradiographs. In contrast, nitroimidazole retention in Groups 2 and 3 increased with the severity of hypoperfusion during the occlusion phase and precisely delineated the ischemic zone on all autoradiographs. CONCLUSION: This 99mTc-labeled hypoxia-avid tracer delineates severe ischemia even after blood flow to the compromised myocardium has been restored. This class of compounds can potentially enhance the physiological assessment of patients with ischemic heart disease.

Correlations between uptake of technetium-99m Q12 and thallium-201: myocardial perfusion and viability in a model of acute coronary reperfusion.

UNLABELLED: To investigate whether Q12 uptake is affected by myocardial viability, as has been noted for 201Tl and sestamibi, we analyzed the initial and delayed distribution patterns of Q12 in a rat coronary artery occlusion-reperfusion model. METHODS: Animals were intubated and ventilated, and their arterial pressures were monitored. A left thoracotomy was performed. After a 1-hr occlusion and a 1-hr reperfusion of a major branch of the circumflex artery, 201Tl and Q12 were injected intravenously. Radiolabeled microspheres were used to document the areas of risk and reperfusion. The animals were killed at 5 min or 1 hr after administration of the diffusible tracers. Tracer distribution was determined by segmental tissue analysis, and tissue viability was determined by histochemical staining. RESULTS: Both the initial uptake and delayed retention of Q12 are sensitive to myocardial viability as shown by significantly lower uptake (28% +/- 8%) and retention (41% +/- 13%) of Q12 in the nonviable as compared to the viable segments (p < 0.001). In addition, the myocardial retention of Q12 was significantly less in the nonviable tissue when compared to the initial uptake (p < 0.01). CONCLUSION: The clinical implication of these observations suggests that initial and delayed imaging after Q12 administration would reflect both the initial regional blood flow pattern and myocardial viability. Also, delayed imaging of Q12 may reflect viability better than the initial imaging.

Myocardial uptake of 99mTc-tetrofosmin, sestamibi, and 201Tl in a model of acute coronary reperfusion.

BACKGROUND: To investigate whether tetrofosmin uptake is affected by myocardial viability as has been noted for 201Tl and sestamibi, we analyzed the initial and delayed distribution patterns of tetrofosmin in a rat coronary artery occlusion-reperfusion model. METHODS AND RESULTS: Animals were intubated and ventilated, and their arterial pressures were monitored. A left thoracotomy was performed. After 1-hour occlusion and 1-hour reperfusion of a major branch of the circumflex artery, 201Tl and either tetrofosmin or sestamibi were injected intravenously. Radiolabeled microspheres were used to document the area at risk and reperfusion. Five minutes or 1 hour after administration of the diffusible tracers, the animals were killed. Tracer distribution was determined by use of segmental tissue analysis, and tissue viability was determined by use of histochemical staining. Both the initial and delayed retention of tetrofosmin were sensitive to myocardial viability, as shown by significantly lower uptake (30 +/- 14%) and retention (24 +/- 12%) of tetrofosmin in the nonviable segments compared with the viable segments. In addition, the initial myocardial distribution of tetrofosmin was similar to that noted for 201Tl, but after 1 hour of tracer circulation, the tetrofosmin tissue distribution appeared unchanged compared with the initial regional blood flow distribution. This is in direct contrast to our present observations of significant 201Tl redistribution and some changes in sestamibi distribution as well. CONCLUSIONS: The clinical implication of these observations suggests that initial and delayed imaging after tetrofosmin administration would reflect both the initial regional blood flow pattern and myocardial viability.

Teboroxime is a marker of reperfusion after myocardial infarction.

BACKGROUND: It has been shown that serial teboroxime imaging can rapidly assess coronary perfusion in viable myocardial distributions. However, the myocardial uptake of teboroxime after reperfusion of acutely infarcted myocardium has not been critically evaluated. The study object was to assess whether teboroxime uptake in acutely infarcted myocardium is linearly related to blood flow. METHODS AND RESULTS: Seventeen New Zealand rabbits underwent occlusion of the left circumflex coronary artery for 1 hour. The animals were reperfused for 2 hours and, just before they were killed, teboroxime was injected. The infarct was delineated by triphenyltetrazolium chloride staining. Normalized blood flow and myocardial teboroxime distribution in the infarcted myocardium was determined by gamma well counting. Ex vivo planar images of the left ventricle were also acquired. Transmural myocardial infarction was documented in all 17 rabbits. The mean infarct size +/- one standard deviation was 25.5% +/- 10.7% (range, 11.9% to 43.3%). There was a direct linear relationship between normalized reperfusion flow and myocardial teboroxime distribution in the infarct zone (r = 0.91). A direct linear relationship between defect size and normalized infarct zone reperfusion was also evident on the ex vivo planar studies (r = 0.70). CONCLUSION: This study shows that the initial uptake of teboroxime in acutely infarcted myocardium is linearly related to blood flow. Teboroxime has properties that are well suited for the early evaluation of infarct zone perfusion.

Comparison of iodine-125-BMIPP and thallium-201 in myocardial hypoperfusion.

UNLABELLED: Radiolabeled fatty acids such as 15-(p-iodophenyl)-3-R,S-methylpentadecanoic acid (BMIPP) have unique metabolic properties of potential use as myocardial perfusion tracers. Accordingly, we compared the in vivo pattern of uptake of BMIPP and 201Tl in hypoperfused rabbit myocardium. METHODS: Animals were intubated, ventilated and their arterial pressures monitored. A left thoracotomy was performed. After ligation of a major branch of the circumflex artery, an intravenous injection of BMIPP or BMIPP/201TI was given. Radiolabeled microspheres were used to document the area of risk. After the circulation period, the animals were killed. Tracer deposition within the hearts was determined by either dual-tracer autoradiography (Protocol I) or by segmental tissue analysis (Protocol II). RESULTS: Dual-tracer autoradiographic activity profiles for BMIPP were comparable to those of 201TI. Moreover, the two tracers yielded similar values for normal-to-defect contrast and defect size. The myocardial activity concentration of BMIPP for both protocols correlated strongly with coronary blood flow and compared favorably with 201TI. CONCLUSION: BMIPP and 201TI accurately delineate areas of hypoperfusion distal to a coronary occlusion. Therefore, differences in the myocardial distribution of BMIPP and 201TI in clinical studies may be related to cellular fatty acid metabolism.

A method to evaluate tracer kinetics in small laboratory animals using a series of thermoluminescent dosimeters.

Miniature detector probes have previously been used in large animal models to investigate myocardial 201Tl clearance kinetics. The results of these studies helped develop clinical imaging protocols that greatly improved the accuracy of thallium scintigraphy. However, miniature detector probes are too large to be used in small animals. Thus, if a method could be developed to measure regional time activity curves in small animals, it would provide a cost-effective alternative to both experiments in large animals and/or multiple experiments at varying time points that can produce results only by postmortem analysis of several animals. Accordingly, we developed a method to measure a regional time activity curve of a tracer in rabbits by using a series of thin thermoluminescent dosimeters [CaF2 (dopant) TLDs, 1 mm thick] placed on the surface of the myocardium. Background contributions associated with high blood pool activity are modeled and then subtracted from the initial TLD response. To validate and illustrate this method, thallium kinetics were determined for nonischemic rabbit myocardium (n = 6). Myocardial thallium concentration decreased monoexponentially with a mean half-time equal to 396 +/- 141 min. Arterial blood activity decreased triexponentially with a final half-time of 243 +/- 73 min. No significant difference was found when the myocardial half-time was compared to the final arterial half-time. These findings are consistent with previous work using a cadmium telluride probe in a canine model. Therefore, TLD analysis can provide a cost-effective, reliable, and reproducible method to measure regional myocardial clearance kinetics.

Myocardial uptake of thallium 201 and technetium 99m-labeled sestamibi after ischemia and reperfusion: comparison by quantitative dual-tracer autoradiography in rabbits.

BACKGROUND: Myocardial scintigraphy with 99mTc-labeled sestamibi (99mTc-sestamibi) or 201Tl is used to assess regional perfusion in acute coronary syndromes associated with metabolic or functional abnormalities, such as acute coronary thrombosis with reperfusion and ischemia at rest. However, the initial uptake of these agents may be affected by a recent ischemic insult because the myocardial retention of these tracers depends on cellular metabolism. METHODS AND RESULTS: Accordingly, 99mTc-sestamibi and 201Tl were injected simultaneously in rabbits after transient brief (10 to 15 minutes, group I) or prolonged (45 to 60 minutes, group II) coronary occlusion. Accumulated subendocardial and subepicardial 99mTc-sestamibi and corresponding 201Tl activity were determined from autoradiographs of 30 microns short-axis slices comounted with serial tissue standards. Circumferential 99mTc-sestamibi and 201Tl activity profiles closely overlapped in both groups. The initial global and segmental myocardial activity per unit blood flow within the ischemic zone did not differ from unity for either tracer regardless of the duration of the ischemic insult. The initial myocardial uptake of both 99mTc-sestamibi and 201Tl after an acute ischemic insult reflected predominantly coronary blood flow, independent of myocardial viability. CONCLUSIONS: Thus this study supports the use of both 99mTc-sestamibi and 201Tl as perfusion probes in acute coronary syndromes characterized by acute occlusion and reperfusion.

A method of simultaneous dual-radionuclide cardiac imaging with technetium 99m and thallium 201. I: Analysis of interradionuclide crossover and validation in phantoms.

BACKGROUND: Simultaneous dual-radionuclide technetium 99m/thallium 201 scintigraphy can potentially produce perfectly aligned stress and rest images in less time than conventional protocols. However, interradionuclide crossover limits diagnostic accuracy. Accordingly, we evaluated 99mTc and 201Tl crossover in line and heart phantoms. METHODS AND RESULTS: 99mTc crossover in the 201Tl imaging window constituted as much as one half of the counts in the 99mTc window, varied significantly with attenuation, and was spatially incoherent. 201Tl crossover was relatively small, less variable, and spatially similar to the primary image. Based on these findings, the following simultaneous dual-radionuclide 99mTc/201Tl method was developed, and validated in line and heart phantoms. The 99mTc source is imaged first into dual 201Tl/99mTc windows, followed by 201Tl administration and dual-radionuclide imaging. The single-radionuclide 99mTc image in the 201Tl window is count-normalized for acquisition time and then subtracted from the dual-radionuclide 201Tl image to specifically correct for 99mTc crossover. Image quality of the corrected dual-radionuclide 201Tl images approached their single-radionuclide counterparts. Correction for 201Tl crossover was relatively unimportant. CONCLUSION: Simultaneous dual-radionuclide 99mTc/201Tl myocardial scintigraphy is feasible with 99mTc crossover correction specific to each acquisition. The proposed dual-radionuclide 99mTc/201Tl method and the principles on which it is based can be applied to a broad range of dual-radionuclide pairs.

Effect of triphenyl tetrazolium chloride staining on the distribution of radiolabeled pharmaceuticals.

Myocardial tissue is routinely exposed to the vital stain triphenyl tetrazolium chloride (TTC) to delineate infarction in conjunction with myocardial isotope research. However, it is unknown whether TTC has a direct effect on tracer deposition. We evaluated this possibility in rabbit hearts injected with either teboroxime, sestamibi or 201Tl. The hearts were excised and treated as follows: (1) TTC or normal saline was perfused through the heart and the residual activity monitored; (2) hearts were sliced into 0.5-cm thick sections, counted and incubated in either TTC or normal saline for 10 min then recounted; and (3) the circumflex artery was ligated postmortem and TTC perfused. Autoradiographic images were produced from 30-microns slices to depict any disparity in activity concentration from the selective perfusion of TTC. Both perfusion and incubation by TTC resulted in a significant activity loss of both 201Tl and sestamibi, but not teboroxime, compared to normal saline. An independent octanol extraction experiment measured the change in the partition coefficient of labeled teboroxime and sestamibi induced by the addition of TTC. TTC was shown to liberate the radiolabel from sestamibi, but not from teboroxime. We conclude that histochemical staining techniques involving TTC can alter the distribution of radiolabeled pharmaceuticals. As a result, experiments using TTC with 201Tl and sestamibi should be interpreted cautiously.

Teboroxime, sestamibi and thallium-201 as markers of myocardial hypoperfusion: comparison by quantitative dual-isotope autoradiography in rabbits.

The scintigraphic assessment of myocardial hypoperfusion depends on the ability of imaging agents to delineate flow disparities. Accordingly, we compared the differential uptake of teboroxime, sestamibi and 201Tl in normal and hypoperfused myocardium using quantitative dual isotope autoradiography. Rabbits with acute coronary occlusions (n = 29) received dual isotope injections of teboroxime 201Tl or sestamibi 201Tl or single isotope tracer injections. A group of sham-operated controls (n = 15) received teboroxime and/or 201Tl. Multiple 30-mu short axis slices were collected from each heart and mounted on x-ray film along with tissue standards to independently generate separate 99mTc and 201Tl autoradiographs. Teboroxime and sestamibi produced greater normal-to-defect activity contrast than 201Tl in each dual isotope heart (range 8.4-48.9 [teboroxime] versus 2.6-12.3 [201Tl], p < 0.02 and 4.5-10.4 [sestamibi] versus 3.6-7.3 [201Tl], p < 0.03). Similar profiles were obtained in the single isotope hearts. Teboroxime produced larger autoradiographic defects than 201Tl in the dual-isotope hearts [16.0% +/- 5.6% (teboroxime) versus 12.9% +/- 5.3% (201Tl) of the LV, p < 0.02]. We conclude that the 99mTc-based perfusion agents teboroxime and, to a lesser extent, sestamibi, delineate hypoperfused myocardium more clearly than 201Tl. Teboroxime detects the largest area of hypoperfusion and may provide the most accurate assessment of myocardium at risk.