Valery Kupriyanov

Relative Contributions of Hemoglobin and Myoglobin to Near-Infrared Spectroscopic Images of Cardiac Tissue:

Near-infrared (NIR) spectroscopic imaging is emerging as a unique tool for intra-operative assessment of myocardial oxygenation, but quantitative interpretation of the images is not straightforward. One confounding factor specific to muscle tissue (both skeletal and cardiac) is that the visible/NIR absorbance spectrum of myoglobin (Mb), an intracellular O2 storage protein, is virtually identical to that of hemoglobin (Hb). As a consequence, the relative contributions of Mb and Hb to the NIR spectra measured in vivo for blood perfused muscle tissue cannot be determined from the measured spectra alone. To estimate the relative contributions of Mb and Hb to NIR spectra and spectroscopic images, isolated pig hearts were perfused first with a Hb-free blood substitute (Krebs-Henseleit buffer; KHB) and then with a 50/50 KHB/blood mixture, with spectroscopic images acquired at each step. Tissue Mb levels were estimated directly from the measurements during KHB perfusion, and total (Mb+Hb) levels were estimated from the images acquired during 50/50 blood/KHB perfusion. Myoglobin accounted for 63 ± 11% of the total heme content during perfusion with the 50/50 mixture (implying that Mb would contribute 46% of the combined (Mb+Hb) NIR profile during whole blood perfusion), confirming that Mb contributes substantially to near-infrared absorbance spectra of blood perfused cardiac tissue.

Hemoglobin plus myoglobin concentrations and near infrared light pathlength in phantom and pig hearts determined by diffuse reflectance spectroscopy

To noninvasively determine absolute concentrations of hemoglobin (Hb) plus myoglobin (Mb) in cardiac tissue by means of regular near infrared (NIR) light diffuse reflectance measurements, a first derivative approach was applied. The method was developed to separately calculate oxygenated and deoxygenated [Hb+Mb] as well as an effective pathlength, which NIR light passes through in the tissue between optodes. Applying a cotton wool-based phantom, which mimics muscle tissue, it was shown that the intensity of the pseudo-optical density first derivative depends linearly on both oxygenated and deoxygenated Hb concentration, thereby validating the Lambert-Beer law in the range of 0 to 0.25 mM tetrameric Hb. A high correlation (R=0.995) was found between concentrations of Hb loaded onto the phantom and those determined spectrophotometrically, thereby verifying the first derivative method validity. The efficiency of the method was tested using in vivo pig hearts prior to and after ischemia initiated experimentally by left anterior descending artery branches occlusion. The results showed that the total [Hb+Mb] was 0.9-1.2 mM heme, the average tissue oxygen saturation was approximately 70% (which reduced to nearly 0% after occlusion), and the NIR (700-965 nm) light pathlength was 2.3 mm (differential pathlength factor [DPF]=2.7-2.8) in a living heart tissue.

Three-dimensional 87Rb NMR imaging and spectroscopy of K+ fluxes in normal and postischemic pig hearts†

K+ uptake rates were measured in the anterior (An) and posterior (Pos) LV walls of pig hearts before and after regional ischemia and reperfusion using Rb+ as a K+ congener and 3D 87Rb NMR imaging and spectroscopy as detection methods. The hearts were perfused by the Langendorff method with Krebs‐Henseleit (KH) buffer and loaded with Rb+ (4.7 mM, Rb‐KH) after 120‐min ischemia and 60‐min reperfusion. A second protocol involved Rb+ loading prior to ischemia. Ischemia was produced by occlusion of the left anterior descending artery, which after 110 min of reperfusion resulted in infarction in the An wall (24 ± 6% of the LV mass) determined by triphenyltetrazolium chloride staining. At the end of reperfusion pressure‐rate product and oxygen consumption rate decreased to 58 ± 10 and 74 ± 4% of their preischemic values, respectively. Phosphocreatine, ATP, and intracellular pH (pHi), measured by 31P NMR spectroscopy in the infarcted area, decreased to 59 ± 17, 32 ± 6%, and 6.7 ± 0.36 (from 7.05 ± 0.13), respectively. Serial 87Rb images were acquired according to both protocols. Rate constants (k × 103, min−1), relative amount of intracellular Rb+ (A, %) and relative fluxes (F = kA, %/min) for the An and Pos walls were determined from the images. Before ischemia, F and k were comparable in the Pos and An walls. Ischemia + reperfusion decreased F in the An wall (from 4.4 ± 0.3 to 1.4 ± 0.85) due to a decrease in A (20 vs. 73) and increased F in Pos wall (from 3.2 ± 0.6 to 6.6 ± 0.23) due to an increase in k (from 42 ± 3 to 93 ± 6). The intensities of the Rb images correlated with the Rb+ content measured in tissue samples. Magn Reson Med 44:83–91, 2000. Published 2000 Wiley‐Liss, Inc.

Noninvasive assessment of cardiac ischemic injury using87Rb and23Na MR imaging,31P MR, and optical spectroscopy

The aim of the study was to compare and analyze different noninvasive indices of cell damage in the isolated pig heart model of regional ischemia. We used 23Na and 87Rb MR imaging to evaluate Na+/K+ balance, 31P MR spectroscopy to measure energetics, and optical spectroscopy to assess oxymyoglobin (MbO2). Hearts were subjected to 120‐min occlusion of the left anterior descending artery and were then reperfused for 120 min. Reperfusion resulted in an increase in 23Na (37 ± 18% of the posterior wall) and decrease in 87Rb (55 ± 15%) image intensities, partial recovery of PCr, ATP, the total phosphates, and MbO2 in the anterior wall. The above changes are consistent with the irreversible cell damage in the anterior wall, confirmed by lack of staining with triphenyltetrazolium chloride. Changes in Na+ and Rb+ in the infarct area inversely correlated and their ratio is a more sensitive index of cell injury than either of them alone. Magn Reson Med 44:899–908, 2000. Published 2000 Wiley‐Liss, Inc.

Magnetic resonance imaging tracking of alginate beads used for drug delivery of growth factors at sites of cardiac damage.

Alginate-based beads labeled with contrast agent and loaded with vascular growth hormones were used for site-specific chronic delivery of hormones at the site of myocardial damage in a porcine model. Position of the beads within the pericardium could be monitored by MRI for optimal hormone delivery due to the presence of contrast agent. The beads facilitate the slow release of cytochrome c, myoglobin and methemoglobin used as protein models of growth factors. This application allows for site-specific delivery of hormones while the incorporated contrast agent in the beads provides a tool for MRI tracking in chronic studies.

MRI studies of cryoinjury infarction in pig hearts: ii. Effects of intrapericardial delivery of adipose-derived stem cells (ADSC) embedded in agarose gel

The purpose was to assess effects of intrapericardially deposited adipose‐derived stem cells (ADSC) as a source of angiogenic factors on cryoinjury infarction. To enhance this effect and reduce incorporation of ADSC into tissue, the cells were immobilized in agarose gel patches transplanted onto cryoinjured epicardium. In domestic pigs (15–20 kg) the left ventricular (LV) anterior wall of exposed hearts was cryoinjured using aluminum rod (Φ=25 mm) cooled in liquid nitrogen. Sterilized circular patches made of agarose gel were placed in a nylon bag and sutured to cryoinjured epicardium. In 4 pigs, the patches contained 650,000 human ADSCs; in control animals patches were cell‐free (n,=,2) or no patches were implanted (n,=,2). Cine and T1‐weighted MRI was performed in vivo weekly (4 weeks) after injury using a 3 T imager. Following baseline imaging, a double bolus of gadopentate dimeglumine was injected (GdDTPA, 0.05 and 0.15 mmol/kg) and serial short axis images were acquired. In the 4‐week ADSC‐treated group, 2 pigs were assessed using GdDTPA and 2 pigs were assessed using MnCl2 (70 micromol/kg/14 min). In all pigs, 5 × 106 NIR fluorescent microspheres (15 µm FMS, 645/680 nm) were injected into the hearts, which were excised, sliced and examined with fluorescence imaging for FMS content. Triphenyltetrazolium chloride (TTC) staining was used to determine necrotic areas. Four‐week infarction areas were hypokinetic and appeared hyperintensive on Gd‐enhanced MR images, hypointensive on Mn‐enhanced images, and were TTC‐negative. First‐pass Gd enhancement kinetics was faster in the infarct area of ADSC‐treated hearts: 152 ± 89 vs 54 ± 5.3% of normal in control (p = 0.03). Accordingly, FMS fluorescence was much higher in the treated infarcts (144 ± 59% of remote, n = 4) relative to control hearts (58 ± 13%, n = 4), which correlated with 3 times higher microvascular density in treated hearts. LV wall thickening was partially restored by ADSC treatment. ADSC‐containing patches attached to cryoinjured epicardium greatly improved perfusion and microvascular density of scar tissue. Copyright

Visualization and grading of regional ischemia in pigs in vivo using near-infrared and thermal imaging.

Reductions in regional coronary flow result in tissue deoxygenation and decrease in surface temperature, changes detectable by near-infrared spectroscopic (NIRS) and thermal imaging, respectively. In anesthetized open-chest pigs, an inflatable occluder and flow probe were placed around the left anterior descending artery. Gated NIRS and nongated thermal images were acquired at baseline, partial (17% and 50%), and complete occlusion and reflow. At each step, dobutamine was infused (10 microg.min(-1).kg(-1)) for 7-9 min to increase blood pressure and flow. Changes in the oxygen saturation parameter, rate of indocyanine green flow tracer passage, and the surface temperature were correlated with the measured left anterior descending artery flow. Location and sizes of the areas of reduced oxygenation, indocyanine green uptake, and temperature were similar. Decrease in the coronary flow to 50% and 17% of baseline resulted in progressive decrease in the above parameters, whereas increase in flow from 75% to approximately 250% achieved by dobutamine and reactive hyperemia did not significantly change them. Dobutamine increased total and epicardial flow in ischemic areas and increased subepicardial oxygenation. NIRS and thermal imaging provide epicardial maps of oxygen saturation and perfusion that reveal ischemic areas. Combination of these techniques may be useful in the coronary artery bypass graft (CABG) surgery setting.

Cardioselective sulfonylthiourea HMR 1098 blocks mitochondrial uncoupling induced by a KATP channel opener, P-1075, in beating rat hearts

We investigated effects of blockade of cardiac ATP-sensitive potassium channels (KATP) with a novel cardioselective sulfonylthiourea, HMR 1098, on metabolic uncoupling caused by a potent KATP opener, P-1075, in Langendorff-perfused rat hearts. We used (1) 87Rb-NMR to detect activation-deactivation of sarcolemmal KATP, (2) 31P-NMR to monitor high-energy phosphates, (3) oxygen uptake measurements to monitor cellular respiration, and (4) myocardial optical absorbance measurements at 603 nm to follow changes in cytochrome c oxidase redox state. Activation of sarcolemmal KATP by P-1075 (5 microM) and a mitochondrial uncoupler 2,4-dinitrophenol (DNP) (50 microM) stimulated Rb+ efflux from the hearts by 130% and 60%, respectively. HMR 1098 (5 and 30 microM) blocked activation of sarcolemmal KATP in situ. HMR 1098 also prevented cardiac arrest and mitochondrial uncoupling induced by P-1075, such as (a) depletion of phosphocreatine and ATP by 40%, (b) two-fold decrease in venous oxygen, and (c) reduction of cytochrome c oxidase (demonstrated by an increase in 603 nm optical absorbance). The metabolic effects of P-1075 can be readily explained by activation of putative mitochondrial KATP. We concluded that blockade of mitochondrial uncoupling by HMR 1098 included an inhibiting effect of HMR 1098 on sarcolemmal and mitochondrial KATP in beating rat hearts.

Characterization of cryoinjury-induced infarction with manganese-and gadolinium-enhanced MRI and optical spectroscopy in pig hearts

PURPOSE To investigate progression of cryoinjury in pigs using contrast-enhanced magnetic resonance imaging (MRI) as well as optical spectroscopy and imaging. METHODS Cryoinjury was produced in 16 pigs in vivo and investigated using Gd-and Mn-enhanced MRI, optical imaging/spectroscopy and histology in acute and chronic setting up to 4 weeks after the injury. RESULTS (1) Acute cryoinjury resulted in formation of a lesion with a severely reduced rate of sub-epicardial indocyanine green (intravascular optical flow tracer) passage. In vivo late Gd-enhanced MRI showed a approximately 10 mm deep hypointense area that was surrounded by a hyperintense rim while ex vivo Mn-enhanced MRI (MEMRI) detected a homogenous hypointense zone. Histological and spectroscopic examination revealed embolic erythrocytes blockages within the cryolesion with a thin necrotic rim neighboring the normal myocardium. (2) Chronic 4-week cryoinjury was characterized by uniform Gd-enhancement, whereas MEMRI revealed reduced Mn(2+)enhancement. Histological examination showed replacement of the cryoinjured myocardium by scar tissue. CONCLUSIONS Acute cryoinjury resulted in formation of a no-reflow core embolized by erythrocytes and surrounded by a rim of necrotic tissue. Upon injury progression, the no-reflow zone shrunk and was completely replaced with scar tissue by 4 weeks after injury.

Assessment of optical path length in tissue using neodymium and water absorptions for application to near-infrared spectroscopy

Quantitative analysis of blood oxygen saturation using near-IR spectroscopy is made difficult by uncertainties in both the absolute value and the wavelength dependence of the optical path length. We introduce a novel means of assessing the wavelength dependence of path length, exploiting the relative intensities of several absorptions exhibited by an exogenous contrast agent (neodymium). Combined with a previously described method that exploits endogenous water absorptions, the described technique estimates the absolute path length at several wavelengths throughout the visible/near-IR range of interest. Isolated rat hearts (n = 11) are perfused separately with Krebs-Henseleit buffer (KHB) and a KHB solution to which neodymium had been added, and visible/near-IR spectra are acquired using an optical probe made up of emission and collection fibers in concentric rings of diameters 1 and 3 mm, respectively. Relative optical path lengths at 520, 580, 679, 740, 800, 870, and 975 nm are 0.41+/-0.13, 0.49+/-0.21, 0.90+/-0.09, 0.94+/-0.01, 1.00, 0.84+/-0.01, and 0.78+/-0.08, respectively. The absolute path length at 975 nm is estimated to be 3.8+/-0.6 mm, based on the intensity of the water absorptions and the known tissue water concentration. These results are strictly valid only for the experimental geometry applied here.