Eugene Gussakovsky

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.

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

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.

MRI study of cryoinjury infarction in pig hearts: i. Effects of intrapericardial delivery of bFGF/VEGF embedded in alginate beads

The aim of the study was the testing of sustained intrapericardial delivery of vascular growth factors (GFs) from alginate beads on cryoinjury size and perfusion. In domestic pigs (15–20 kg, n = 21), the left ventricular (LV) anterolateral wall of exposed hearts was cryoinjured using an aluminum rod (25 mm o.d.) cooled in liquid nitrogen. Alginate beads (d = 3.2 ± 0.2 mm), containing human recombinant basic fibroblast GF (bFGF, 50 µg) and vascular endothelial GF (VEGF, 50 µg) + heparin (50 µg) or heparin alone (Con, n = 5), were sutured to the cryoinjured epicardium (GF, n = 5; Con, n = 3 ) or pericardium (GF, n = 3; Con, n = 2), or no beads were implanted (n = 4). Four pigs were sham‐operated. Cine and T1‐weighted MRI was performed in vivo at ~2.5 h and 1, 2, 3 and 4 weeks after injury in a 3T imager. A double bolus of GdDTPA was injected (0.05 and 0.15 mmol/kg) and first‐pass and late enhancement kinetics were monitored. After 4‐week cryoinjury, following the injection of 5x106 15‐µm NIR fluorescent microspheres (FMS, 645/680 nm), hearts were sliced and examined with fluorescence imaging. Triphenyltetrazolium chloride (TTC) staining was used to determine infarct areas. Epicardial GF‐containing beads were encapsulated within the hypointense 3‐4‐week infarct tissue. This tissue had a 75% higher LV thickening index, a lower distribution volume for GdDTPA (0.44 ± 0.12 vs 0.68 ± 0.05, p = 0.02), and 25% faster first‐pass Gd kinetics relative to control infarctions. TTC staining revealed TTC‐positive islands in the core of treated infarcts, which showed higher FMS fluorescence relative to surrounding infarct tissue (0.64 ± 0.14 vs. 0.31 ± 0.14; p < 0.0001) and to control infarcts (0.37 ± 0.09, p < 0.05). GF‐beads attached to the pericardium were not effective. We conclude that sustained intrapericardial release of bFGF + VEGF from alginate beads attached to the epicardium facilitated vascular growth in the cryoinjured area. Copyright

Mapping the myoglobin concentration, oxygenation, and optical pathlength in heart ex vivo using near-infrared imaging.

A method that provides maps of absolute concentrations of oxygenated and deoxygenated myoglobin (Mb), its oxygenation, and its near-infrared (NIR) optical pathlength in cardiac tissue was developed. These parameters are available simultaneously. The method is based on NIR diffuse reflectance spectroscopic imaging and specific processing of the NIR images, which included a first derivative of the diffuse reflectance spectrum. Mb oxygenation, total Mb concentration, and NIR light pathlength were found to be in the range of 92%, 0.3 mM, and 12.5 mm, respectively, in beating isolated buffer-perfused and arrested pig hearts. The charge-coupled device camera enables sub-millimeter spatial resolution and spectroscopic imaging in 1.5 to 2.0 min. The technique is noninvasive and nondestructive. The equipment has no mechanical contact with the tissue of interest, leaving it undisturbed.

Assessment of Near-Infrared Path Length in Fibrous Phantom and Muscle Tissue

The first derivative of the pseudo-absorption spectrum of a water-loaded cotton wool (water–CW) phantom, which mimics muscle tissues, was used to determine the light path length in the near-infrared (NIR) region. The light path length increased as the density of the turbid medium decreased. It is independent of both water content in the range of 75–85% (by weight) and the diffuse reflecting reference used to determine the pseudoabsorbance. The path length determination procedure was verified by measurements of diffuse reflectance in chicken breast tissue for which the path length of 1.8 mm (differential path length factor, DPF = 2.1) was found to be similar to the path length of NIR light of 1.5–2.2 mm (DPF = 1.8–2.6) in a water–CW phantom of density similar to chicken breast. We conclude that the NIR light path length can serve as a characteristic of muscle tissue density.

Defects in myoglobin oxygenation in KATP-deficient mouse hearts under normal and stress conditions characterized by near infrared spectroscopy and imaging

BACKGROUND Disruption of ATP-sensitive potassium (K(ATP)) channel activity results in the development of dilated cardiomyopathy in response to different forms of stress, likely due to the underlying metabolic defects. To further understand the role of Kir6.2-containing channels in the development of cardiac disease, we analysed the left ventricular (LV) wall oxygenation and the physiologic responses induced by acute stress in non-dilated Kir6.2(-/-) hearts. METHODS Control (C57BL6) and Kir6.2(-/-) mouse hearts were perfused in constant flow Langendorff mode with Krebs-Henseleit buffer. Myocardial oxygenation was evaluated using a newly developed technique, near infrared spectroscopic imaging (NIRSI) of the myoglobin (Mb) oxygen saturation parameter (OSP, ratio of oxy- to total Mb). RESULTS 2,4-dinitrophenol (DNP, 50-µM) and isoproterenol (0.1-µM) failed to produce a transient vasodilatory response and caused a significant diastolic pressure increase in Kir6.2(-/-) hearts. DNP strongly suppressed contractile function in both groups and induced severe mean OSP decreases in Kir6.2(-/-) hearts. Isoproterenol-induced decreases in OSP were similar despite the lack of contractile function stimulation in the Kir6.2(-/-) group. The index of OSP spatial heterogeneity (relative dispersion, RD) was lower by 15% in the Kir6.2(-/-) group at the baseline conditions. Recovery after stress caused reduction of RD values by 20% (DNP) and 8% (isoproterenol) in controls; however, these values did not change in the Kir6.2(-/-) group. CONCLUSIONS 1) NIRSI can be used to analyse 2-D dynamics of LV oxygenation in rodent models of cardiomyopathy; 2) Kir6.2-containing K(ATP) channels play an important role in maintaining myocardial oxygenation balance under acute stress conditions and in post-stress recovery.

NIR spectroscopic imaging to map hemoglobin + myoglobin oxygenation, their concentration and optical pathlength across a beating pig heart during surgery.

The purpose of this paper is to demonstrate that near-infrared (NIR) spectroscopic imaging can provide spatial distribution (maps) of the absolute concentration of hemoglobin + myoglobin, oxygen saturation parameter and optical pathlength, reporting on the biochemico-physiological status of a beating heart in vivo. The method is based on processing the NIR spectroscopic images employing a first-derivative approach. Blood-pressure-controlled gating compensated the effect of heart motion on the imaging. All the maps are available simultaneously and noninvasively at a spatial resolution in the submillimeter range and can be obtained in a couple of minutes. The equipment has no mechanical contact with the tissue, thereby leaving the heart unaffected during the measurement.

Circularly Polarized Luminescence (CPL) of Proteins and Protein Complexes

The review considers general theory of circularly polarized luminescence (CPL), instrumentation employed for its accurate measurement including artifacts, calibration, principle parts, and its application to proteins and protein complexes. Circularly polarized intrinsic fluorescence of proteins includes CPL of peptides, poly-α-amino acids, tyrosine and tryptophan residues in proteins, complexes of proteins with functional non-fluorescent agents, and protein conformation perturbation. CPL of fluorescent agents in protein complexes addresses to artificial probes, enzymatic cofactors, bilirubin, lanthanides and light-harvesting chlorophyll-protein complex of photosynthetic photosystem II of higher plants.

Non-invasive measurements of hemoglobin + myoglobin, their oxygenation and NIR light pathlength in heart in vivo by diffuse reflectance spectroscopy

The existing non-invasive optical methods of the hemoglobin (Hb) and myoglobin (Mb) estimation in cardiac tissues imply knowledge of the light pathlength (L) when various modifications of Lambert-Beer law for either spectrophotometry or light diffuse reflectance is applied. For Hb and/or Mb quantification in tissue, a few invasive (biochemical) approaches were applied. For L (differential pathlength factor; DPF) determination in tissue, special optical methods were used. No approaches have been proposed to simultaneously and non-invasively determine Hb/Mb and L in cardiac or other muscle tissues. In the present study, the first derivative of the NIR diffuse reflectance spectrum is shown to be effective in simultaneous determination of Hb+Mb concentration (in mM) and L (in mm) in cardiac tissue in vivo. The results showed that measured in a few minutes in a normal pig heart in vivo the total Hb+Mb concentration was 0.9-1.2 mM of heme, tissue oxygen saturation parameter (OSP) was approximately 65%, and DPF at 700-965 nm was of 2.7-2.8. At the experimental ischemia, total [Hb+Mb] decreased by 25%, OSP reduced to zero, while DPF did not change. These results correlated with the previously published. The method may be applied during open-heart surgery, heart studies ex vivo or to any muscle tissue to continuously and non-invasively monitor the [Hb+Mb] content and oxygenation as well as L, which may reflect the changes in tissue structure.