RESUMEN
Research into the hunting behavior in members of the Cricetidae family offers an opportunity to reveal what changes in the predatory behavioral sequences occur when a rodent species shifts from an omnivorous to a predatory lifestyle. The study tests the following hypotheses: are there phylogenetic differences in the divergence of species' predatory lifestyles in hamsters or do ecological factors lead to shaping their hunting behavior? We applied the data compression approach for performing comparative analysis of hunting patterns as biological "texts." The study presents a comparative analysis of hunting behaviors in five Cricetinae species, focusing on the new data obtained for the desert hamster Phodopus roborovskii whose behavior has never been studied before. The hunting behavior of P. roborovskii appeared to be the most variable one. In contrast, behavioral sequences in P. campbelli and Allocricetulus curtatus display more significant order and predictability of behavior during hunting. Optional hunting behavior in the most ancient species P. roborovskii displayed similarities with obligate patterns in "young" Allocricetulus species. It thus turned out to be the most advanced hunter among members of the Phodopus genus. Differences in hunting sequences among Phodopus representatives suggest that the hunting behavior of these species, despite its optional mode, was subject to selection during species splitting within the genus. These results did not reveal the role played by phylogenetic differences in the divergence of species' predatory lifestyles. They suggested that ecological conditions are the main factors in speciation of the hunting behavior in hamsters.
Asunto(s)
Antígenos del Núcleo de la Hepatitis B/química , Virus de la Hepatitis B/química , Complejos Multiproteicos/química , Nanopartículas/química , Péptidos/química , Escherichia coli , Antígenos del Núcleo de la Hepatitis B/genética , Antígenos del Núcleo de la Hepatitis B/metabolismo , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/metabolismo , Virus de la Hepatitis B/ultraestructura , Humanos , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructura , Nanopartículas/ultraestructura , Péptidos/genética , Péptidos/metabolismo , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
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 T(1)-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 MnCl(2) (70 micromol/kg/14 min). In all pigs, 5 × 10(6) 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.
Asunto(s)
Tejido Adiposo/citología , Congelación , Imagen por Resonancia Magnética/métodos , Infarto del Miocardio/terapia , Pericardio/patología , Trasplante de Células Madre , Células Madre/citología , Animales , Progresión de la Enfermedad , Gadolinio DTPA , Geles , Ventrículos Cardíacos/patología , Ventrículos Cardíacos/fisiopatología , Humanos , Cinética , Infarto del Miocardio/sangre , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Miocardio/patología , Cambios Post Mortem , Sefarosa , Sus scrofa , Factores de TiempoRESUMEN
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 T(1) -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 5 x 10(6) 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.
Asunto(s)
Frío/efectos adversos , Sistemas de Liberación de Medicamentos/métodos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Imagen por Resonancia Magnética/métodos , Infarto del Miocardio/patología , Pericardio/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/farmacología , Alginatos/química , Animales , Factor 2 de Crecimiento de Fibroblastos/administración & dosificación , Factor 2 de Crecimiento de Fibroblastos/uso terapéutico , Fluorescencia , Gadolinio , Gadolinio DTPA/administración & dosificación , Gadolinio DTPA/farmacología , Ácido Glucurónico/química , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/patología , Ácidos Hexurónicos/química , Humanos , Implantes Experimentales , Cinética , Microesferas , Infarto del Miocardio/sangre , Infarto del Miocardio/tratamiento farmacológico , Perfusión , Pericardio/patología , Cambios Post Mortem , Procesamiento de Señales Asistido por Computador , Sus scrofa , Sales de Tetrazolio/metabolismo , Factores de Tiempo , Factor A de Crecimiento Endotelial Vascular/administración & dosificación , Factor A de Crecimiento Endotelial Vascular/uso terapéuticoRESUMEN
To assess infarction development in pig hearts, Mn-enhanced and Gd-enhanced MRI were used. In domestic pigs (25-35 kg, n = 37), the first and second diagonal branches of the left anterior descending coronary artery were ligated to induce acute ischemia and infarction (ischemia+reperfusion) or chronic infarction of increasing duration (3- 28 days). Ex vivo experiments were performed on hearts perfused in the Langendorff mode with a 50:50 mixture of blood and Krebs-Henseleit buffer using a spin-echo sequence on a 7 T Bruker imaging system. Signal acquisition from the heart and two reference test tubes (H(2)O and H(2)O + 10 mM CuSO(4)) was gated by the left ventricular pressure wave. T(1)-weighted images of six 8 mm short-axis slices (2 mm interslice gaps) were obtained before and after the addition of 0.2 mM MnCl(2) every 5 min over a 30-45 min period. Signal intensities were normalized to those of the H(2)O reference and fitted by a monoexponential function. The rates of intensity increase and maximal increases were significantly lower in the ischemic/infarcted areas and showed a trend to rise on infarction progression. In vivo Gd-enhanced MRI (3 T Siemens scanner) and in vivo/ex vivo near-infrared imaging confirmed major Mn-enhanced MRI findings. Triphenyltetrazolium chloride staining revealed necrotic areas in all chronic infarctions and no necrosis after acute ischemia. We conclude that MnCl(2) highlights ischemic areas because of the low collateral flow characteristic of pig hearts, whereas in the infarcted areas with substantial perfusion, scar tissue components are important for contrast distribution.
Asunto(s)
Aumento de la Imagen/métodos , Manganeso/farmacocinética , Modelos Cardiovasculares , Daño por Reperfusión Miocárdica/diagnóstico , Daño por Reperfusión Miocárdica/metabolismo , Aturdimiento Miocárdico/diagnóstico , Aturdimiento Miocárdico/metabolismo , Animales , Enfermedad Crónica , Simulación por Computador , Medios de Contraste/farmacocinética , Interpretación de Imagen Asistida por Computador/métodos , Técnicas In Vitro , Cinética , Tasa de Depuración Metabólica , PorcinosRESUMEN
It was previously shown that the adrenergic agonist dobutamine stimulates Rb(+) uptake in isolated pig hearts. In the present work we examined whether dobutamine can increase Rb(+) uptake rate in vivo. Open-chest domestic pigs (N = 14) were used under general anesthesia. The surface coil was placed against the anterior left ventricular wall to obtain (87)Rb spectra using a spectrometer interfaced with the 7T, 30-cm horizontal bore magnet. RbCl was infused at the rate of 1.35 +/- 0.14 mmol/kg/hr without or with dobutamine (0.6 mg/kg/hr) over a 60-min period, and then the infusions were terminated. The rate constant (k x 10(3), from 13 +/- 2.4 to 36 +/- 12 min(-1)) and Rb(+) influx rate (from 2.5% to 4.8%/min) were increased by dobutamine despite lower plasma [Rb(+)] (0.59 +/- 0.16 vs. 0.84 +/- 0.24 mM in control), while the tissue/plasma Rb ratios were identical (38 +/- 9). Heart rate (HR) and systolic blood pressure (SBP) were increased from 106 +/- 9 to 161 +/- 15 bpm (+52%) and from 78 +/- 7 to 93 +/- 11 mmHg (+19%), respectively. The stimulation of Rb(+) uptake by dobutamine is consistent with the activation of Na(+)/K(+) ATPase previously observed in isolated hearts. However, the 50% increase in HR and the double coronary flow caused by the SBP increase and vasodilatation may also contribute to this effect.
Asunto(s)
Dobutamina/farmacología , Espectroscopía de Resonancia Magnética/métodos , Miocardio/metabolismo , Radioisótopos de Rubidio/metabolismo , Animales , Frecuencia Cardíaca , Hemodinámica , PorcinosRESUMEN
87Rb-MRI was used to measure Rb(+) uptake in blood-perfused pig hearts during complete occlusion (120 and 70 min) of the left anterior descending artery (LAD) and subsequent reperfusion (120 and 170 min). The Rb(+) uptake rate and maximal Rb image intensity during 120-min occlusion were significantly lower in the ischemic anterior left ventricular (LV) wall (0.35 +/- 0.14%/min and 26 +/- 4.7%) relative to those in the remote posterior LV wall (2.43 +/- 0.33%/min and 98 +/- 10%). Reperfusion after 120 and 70 min of occlusion resulted in formation of damaged areas, which had 40 +/- 4 and 73 +/- 10% of the (87)Rb image intensity observed in the remote posterior wall. The infarct sizes determined histologically by triphenyltetrazolium chloride staining were 9.6 +/- 3.4 and 5.6 +/- 4.6% of the total ventricular mass (LV + RV) in the 120- and 70-min occlusion groups, respectively. The sizes determined by MRI were 13.1 +/- 2 and 2.8 +/- 4.3% of the total number of pixels, respectively. The Rb(+) uptake in the anterior wall during 120-min occlusion was somewhat lower than that previously observed in crystalloid-perfused hearts. It is concluded that blood does not interfere with the ability of (87)Rb MRI to detect ischemic and infarct areas.
Asunto(s)
Imagen por Resonancia Magnética , Infarto del Miocardio/diagnóstico , Isquemia Miocárdica/diagnóstico , Radioisótopos de Rubidio , Animales , Colorantes , Técnicas In Vitro , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patología , Reperfusión Miocárdica , Miocardio/metabolismo , Miocardio/patología , Compuestos de Potasio , PorcinosRESUMEN
The role of adrenergic stimulation in Rb(+) uptake in normal and ischemic areas of pig hearts was investigated using an agonist (dobutamine) and an antagonist (S-propranolol). The left anterior descending artery (LAD) in isolated pig hearts was cannulated to maintain adequate perfusion of the LAD bed (2.2-2.8 ml/min/g). Rb(+) loading was initiated by switching perfusion to Rb(+)-containing Krebs-Henseleit (KH) buffer in the presence of 0.1 microM dobutamine, 0.5 microM propranolol, or no drug (control), and the LAD flow was reduced to 10% of the baseline for 120 min. The flow through the LAD was then restored to normal and the drugs were removed. In all groups the rate of Rb(+) uptake obtained from serial (87)Rb images was severely depressed in the ischemic anterior wall relative to that in the normal posterior wall. Dobutamine increased the rate of Rb(+) uptake in the posterior wall (6.3% +/- 1.4% vs. 2.5% +/- 0.25% per minute in control) and did not affect the rate in the anterior wall (0.86% +/- 0.40% vs. 1.14% +/- 0.19% per minute in control). Propranolol did not affect Rb(+) uptake in either of these areas. We conclude that the stimulation of Rb(+) uptake by dobutamine is related to activation of Na(+)/K(+) ATPase and passive Na(+) influx in normal tissue. The lack of any effect of propranolol implies a low level of endogenous norepinephrine during low-flow (LF) ischemia.
Asunto(s)
Epinefrina/fisiología , Isquemia Miocárdica/metabolismo , Radioisótopos de Rubidio/metabolismo , Agonistas Adrenérgicos beta/farmacología , Antagonistas Adrenérgicos beta/farmacología , Animales , Dobutamina/farmacología , Técnicas In Vitro , Propranolol/farmacología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , PorcinosRESUMEN
The study compared the effects of regional hypoxia and acidosis on Rb(+) uptake and energetics in isolated pig hearts perfused by the Langendorff method. The left anterior descending artery (LAD) was cannulated and the LAD bed was perfused with the same specific flow as the whole heart. Following equilibration with normal Krebs-Henseleit buffer (KHB, pO(2) 568 mm Hg, pH 7.42) the perfusate was switched to one that contained Rb(+) (Rb-KHB). Simultaneously, perfusion through the LAD was carried out with hypoxic (pO(2)=31 mm Hg), an acidemic (pH 7.12) or normal (pO(2)=550 mm Hg) Rb-KHB for 120 min. (87)Rb images of the entire heart or localized (31)P spectra from the left ventricular anterior wall were acquired. Hypoxia decreased the maximal (87)Rb image intensity and Rb(+) flux in the anterior wall to 79+/-9% and 85+/-7%, respectively, of that in the posterior wall. Extracellular acidosis did not affect (87)Rb image intensity and reduced Rb(+) flux (83+/-10%). During hypoxia phosphocreatine and ATP decreased to 36+/-10 and 50+/-15% of baseline, respectively and intracellular pH (pHi) decreased to 6.90+/-0.05. Extracellular acidosis did not affect the phosphocreatine or ATP levels but reduced pHi (7.06+/-0.18 vs. 7.26+/-0.06 in control). We suggest that intracellular acidosis plays a role in the inhibition of Rb(+) uptake during hypoxia.
Asunto(s)
Acidosis/fisiopatología , Hipoxia de la Célula/fisiología , Corazón/fisiopatología , Rubidio/metabolismo , Acidosis/metabolismo , Animales , Metabolismo Energético , Concentración de Iones de Hidrógeno , Imagen por Resonancia Magnética , Espectroscopía de Resonancia Magnética , Miocardio/metabolismo , Miocardio/patología , Perfusión , Radioisótopos de Rubidio , PorcinosRESUMEN
In this work the roles of coronary flow (CF) and metabolism in Rb(+) (K(+) congener) uptake were studied. In isolated pig hearts the left anterior descending artery (LAD) was cannulated to maintain adequate perfusion of the LAD bed. Rb(+) loading was initiated and the LAD flow was either completely stopped (no flow (NF)) or reduced to 12% (low flow (LF)), or buffer was deoxygenated without change in flow (hypoxic flow (HYP)) for 2 h. CF through the LAD was then restored to normal, or perfusion was switched to oxygenated buffer. Serial (87)Rb MR images or localized (31)P spectra were acquired to compare the Rb(+) uptake and energetics in the left ventricular (LV) anterior (ischemic/hypoxic) and posterior (normal) walls. End-ischemic/hypoxic (87)Rb signal intensities in the anterior wall were higher and the fluxes were greater in the HYP and LF groups than in the NF group. Phosphocreatine and ATP decreased less significantly and recovered better in the HYP and LF groups. Upon reperfusion/reoxygenation, the HYP and LF groups showed higher (87)Rb signal intensities and smaller or no infarctions in the anterior wall compared to those in the NF group. Ischemia reduces Rb(+) uptake due to both flow limitations and metabolic inhibition of cellular transport. (87)Rb MRI has a potential for distinguishing necrotic and reversibly damaged tissue.
Asunto(s)
Circulación Coronaria , Metabolismo Energético , Imagen por Resonancia Magnética , Espectroscopía de Resonancia Magnética , Daño por Reperfusión Miocárdica/metabolismo , Miocardio/metabolismo , Animales , Daño por Reperfusión Miocárdica/fisiopatología , Rubidio , Radioisótopos de Rubidio , Porcinos , Función Ventricular IzquierdaRESUMEN
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 (23)Na and (87)Rb MR imaging to evaluate Na(+)/K(+) balance, (31)P MR spectroscopy to measure energetics, and optical spectroscopy to assess oxymyoglobin (MbO(2)). 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 (23)Na (37 +/- 18% of the posterior wall) and decrease in (87)Rb (55 +/- 15%) image intensities, partial recovery of PCr, ATP, the total phosphates, and MbO(2) 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.
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Isquemia Miocárdica/diagnóstico , Radioisótopos de Rubidio , Análisis Espectral/métodos , Animales , Modelos Animales de Enfermedad , Técnicas In Vitro , Espectroscopía de Resonancia Magnética/estadística & datos numéricos , Isquemia Miocárdica/metabolismo , Miocardio/metabolismo , Consumo de Oxígeno , Perfusión/métodos , Isótopos de Fósforo , Isótopos de Sodio , Análisis Espectral/estadística & datos numéricos , PorcinosRESUMEN
87Rb MR imaging and spectroscopy were used to study the effects of ischemia on the properties of K(+) in cardiac tissue. Isolated pig and rat hearts perfused by the Langendorff method with Krebs-Henseleit buffer were preloaded with Rb(+). Ischemia (Isc) was induced by 120-min occlusion of the left anterior descending artery in the pig hearts or by stopping perfusion for 33 min in the rat hearts. Serial (87)Rb MR images or spectra from the anterior (An) LV wall of pig hearts were acquired continuously. The intensities of the Rb images of the An and posterior (Pos) walls were similar and stable during the first 45 min of ischemia. The intensity of signal from the An wall (Isc) then gradually increased by 60 +/- 8% relative to the preischemic value (vs. 31 +/- 5% increase in Pos wall) and necrosis (19 +/- 5% of the LV wall mass) developed upon reperfusion. The Rb(+) content was lower in the ischemic (An) than in the normal (Pos) area (22.3 +/- 3 vs. 28.4 +/- 1.3 mmol/g wet wt). A similar pattern was observed in the peak heights of (87)Rb spectra from the An wall, which increased by 40 +/- 16% (vs. 21 +/- 11% in control) due to a 12% decrease in the apparent Rb linewidth (LW) and a 24 +/- 14% increase in the peak area. The Rb peak comprised narrow (297 +/- 21 Hz) and broad (1098 +/- 40 Hz, 59 +/- 3% of total area) Lorentzian components. The LW of the broad component decreased by 14%, while the narrow component did not change markedly. In the rat hearts ischemia caused a 33 +/- 4% increase in the (87)Rb peak height as a result of peak narrowing (13 +/- 1%), and an increase in peak area (17 +/- 5%). The decreases in LW and increases in Rb(+) visibility can be explained by an increase in Rb(+) mobility caused by displacement of Rb(+) from anionic binding sites by H(+) (ischemic acidosis) and changes in intracellular compartmentalization of Rb(+). Magn Reson Med 44:193-200, 2000.
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Isquemia Miocárdica/metabolismo , Potasio/metabolismo , Rubidio/metabolismo , Análisis de Varianza , Animales , Isótopos , Masculino , Ratas , Ratas Sprague-Dawley , PorcinosRESUMEN
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 (87)Rb 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 (31)P NMR spectroscopy in the infarcted area, decreased to 59 +/- 17, 32 +/- 6%, and 6.7 +/- 0.36 (from 7.05 +/- 0.13), respectively. Serial (87)Rb images were acquired according to both protocols. Rate constants (k x 10(3), 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.
Asunto(s)
Imagen por Resonancia Magnética , Espectroscopía de Resonancia Magnética , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patología , Miocardio/metabolismo , Potasio/metabolismo , Análisis de Varianza , Animales , Procesamiento de Imagen Asistido por Computador , Necrosis , Isótopos de Potasio , Radioisótopos de Rubidio/farmacocinética , Porcinos , Supervivencia Tisular/fisiologíaRESUMEN
A number of medical applications of near-infrared spectroscopy are growing closer to clinical acceptance, and new techniques involving both spectroscopy and imaging are evolving rapidly. In vivo spectroscopy and, more recently, imaging techniques are largely based upon optical electronic transitions involving the metal centers of hemoglobin (blood), myoglobin (muscle) and cytochrome aa3 (mitochondria). The wide variety of near-IR based applications includes heart and stroke research, monitoring cerebral oxygenation of premature babies, and 'functional activation' (response of brain to mental tasks). All of these applications are founded upon changes in hemoglobin O2 saturation; these changes are monitored by following trends in the near-infrared absorptions of deoxyhemoglobin (760 nm) and oxyhemoglobin (920 nm). The same absorptions provide a basis for imaging regional variations in blood oxygenation. This report presents and discusses examples, both from the literature and from our recent work, of near-infrared spectroscopy and imaging in medical applications.
Asunto(s)
Metaloproteínas/análisis , Animales , Complejo IV de Transporte de Electrones/análisis , Complejo IV de Transporte de Electrones/química , Hemoglobinas/análisis , Hemoglobinas/química , Humanos , Metaloproteínas/química , Mioglobina/análisis , Mioglobina/química , Oxihemoglobinas/análisis , Oxihemoglobinas/química , Espectrofotometría/métodos , Espectrofotometría Infrarroja/métodosRESUMEN
This study investigates the effects of intracellular (pH(i)) and extracellular pH (pH(e)) on the efflux of Rb(+) and Li(+) in isolated rat hearts. (87)Rb and (7)Li NMR were used to measure Rb(+) and Li(+) content, respectively, of hearts, and (31)P NMR was used to monitor pH(i), pH(e), and phosphate levels. After 30-min equilibration with Rb(+) or Li(+), effluxes were initiated by switching perfusion to a Rb(+)- or Li(+)-free, high-K(+) (20.7 mM) Krebs-Henseleit buffer with 15 microM bumetanide. Monensin (2 microM) increased pH(i) from 7.10 +/- 0.05 to 7.32 +/- 0.07 and resulted in activation of Rb(+) efflux; the first-order rate constant (k x 10(3), in min(-1)) increased from 42 +/- 2 to 116 +/- 16. Glibenclamide (4 microM) did not inhibit monensin-activated Rb(+) efflux (k = 110 +/- 17), whereas quinine (0.2 mM) slightly inhibited it by 19 +/- 9%. Infusion of 15 mM NH(4)Cl during Rb(+) washout increased k for Rb(+) efflux by 93% (81 +/- 8), which was glibenclamide and quinine insensitive, and caused a transient increase in pH(i) to 7.25 +/- 0.08. Intracellular Li(+) inhibited NH(4)Cl-stimulated Rb(+) efflux by 55%. Monensin and NH(4)Cl stimulated Li(+) efflux by 40%, increasing k from 29 +/- 3 to 43 +/- 7 and 41 +/- 3, respectively. The stimulation was not sensitive to 10 microM dimethylamiloride. Intracellular acidosis that resulted from the washout of NH(4)Cl (pH 6.86 +/- 0.2) slightly inhibited Rb(+) efflux (k = 36 +/- 5), whereas NH(4)Cl itself in the absence of pH(i) changes did not markedly affect Rb(+) efflux. A moderate increase in pH(i) (7.17 +/- 0.06) produced by washout of 15 mM 2, 2-dimethylpropionate (DMP)-Tris from hearts preequilibrated with DMP did not markedly affect Rb(+) efflux. Neither global alkalosis (pH(i) 7.4, pH(e) 7.55) nor acidosis (pH(i) approximately pH(e) 6.8) produced by 3 mM Tris base or 5 mM MES, respectively, affected Rb(+) efflux. We suggest that intracellular alkalosis stimulates Rb(+) (K(+)) and Li(+) effluxes by activating a nonselective sarcolemmal K(+) (Li(+))/cation exchanger or a K(+) (Li(+))-anion symporter.
Asunto(s)
Miocardio/citología , Miocardio/metabolismo , Potasio/metabolismo , Acidosis/metabolismo , Alcalosis/metabolismo , Cloruro de Amonio/farmacología , Animales , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Litio , Espectroscopía de Resonancia Magnética , Masculino , Monensina/farmacología , Fósforo , Ratas , Ratas Sprague-Dawley , Radioisótopos de RubidioRESUMEN
This study investigated whether Na+/K+ ATPase is inhibited and KATP channels activated during low flow ischemia (LFI) by monitoring Rb+ uptake and efflux from rat hearts using 87Rb NMR. In the uptake experiments, isolated Langendorff perfused hearts were exposed to Rb+-containing Krebs-Henseleit buffer (2.14 m m+3.76 m m K+) for 60 min. When Rb+ uptake started the flow of perfusate was decreased from 10 to 1 ml/min/g wet weight for 44 min and then returned to normal. The rate of Rb+ uptake and its equilibrium level decreased to 40 and 65% of the control (no ischemia) levels, respectively. Phosphocreatine and cytoplasmic [ATP]/[ADP] measured by 31P NMR decreased by half, intracellular pH (pHi) decreased to 6.8+/-0.1, and Pi increased two-fold. In wash-out experiments the hearts were pre-loaded with Rb+ for 30 min following which Rb+ wash-out was initiated. Four minutes later, flow was either decreased in the absence or presence of 10 microm 2,4-dinitrophenol (DNP), or 0.1 m m DNP was infused at normal flow for 20 min. LFI resulted in biphasic Rb+ efflux; during the initial phase, which lasted 8 min, the rate constant (kx10(3)/min) did not differ from control (43+/-3). The efflux was slightly inhibited by 5 microm glibenclamide (36+/-6) or 100 microm 5-hydroxydecanoic acid (32+/-4). In the second phase k decreased to half its initial value (18+/-2). More significant changes in energy state caused by LFI+10 microm DNP had no effect on the efflux kinetics. Similar changes in energy state induced by 0.1 m m DNP at normal flow were associated with activation of Rb+ efflux (71+/-5). DNP-stimulated Rb+ efflux was inhibited by acidosis (pHi approximately pHe = 6.7) produced with 5 m m morpholinoethane sulphonic acid (53+/-5) and by 100 microm adenosine (58+/-7). We suggest that accumulation of ischemic products such as H+and adenosine decreases activation of KATP channels in rat hearts.
Asunto(s)
Isquemia Miocárdica/metabolismo , Potasio/metabolismo , 2,4-Dinitrofenol/farmacología , Adenosina Trifosfato/metabolismo , Animales , Circulación Coronaria , Metabolismo Energético , Hemodinámica , Técnicas In Vitro , Transporte Iónico/efectos de los fármacos , Espectroscopía de Resonancia Magnética , Masculino , Isquemia Miocárdica/fisiopatología , Perfusión , Fosfocreatina/metabolismo , Canales de Potasio/metabolismo , Ratas , Ratas Sprague-Dawley , Radioisótopos de Rubidio , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
The rubidium efflux from hypothermic rat hearts perfused by the Langendorff method at 20 degreesC was studied. At this temperature 87Rb-NMR efflux experiments showed the existence of two 87Rb pools: cytoplasmic and mitochondrial. Rat heart mitochondria showed a very slow exchange of mitochondrial Rb+ for cytoplasmic K+. After washout of cytosolic Rb+, mitochondria kept a stable Rb+ level for >30 min. Rb+ efflux from mitochondria was stimulated with 0.1 mM 2, 4-dinitrophenol (DNP), by sarcolemmal permeabilization and concomitant cellular energy depletion by saponin (0.01 mg/ml for 4 min) in the presence of a perfusate mimicking intracellular conditions, or by ATP-sensitive K (KATP) channel openers. DNP, a mitochondrial uncoupler, caused the onset of mitochondrial Rb+ exchange; however, the washout was not complete (80 vs. 56% in control). Energy deprivation by saponin, which permeabilizes the sarcolemma, resulted in a rapid and complete Rb+ efflux. The mitochondrial Rb+ efflux rate constant (k) decreased in the presence of glibenclamide, a KATP channel inhibitor (5 microM; k = 0.204 +/- 0.065 min-1; n = 8), or in the presence of ATP plus phosphocreatine (1.0 and 5.0 mM, respectively; k = 0.134 +/- 0.021 min-1; n = 4) in the saponin experiments (saponin only; k = 0.321 +/- 0.079 min-1; n = 3), indicating the inhibition of mitochondrial KATP channels. Thus hypothermia in combination with 87Rb-NMR allowed the probing of the mitochondrial K+ pool in whole hearts without mitochondrial isolation.
Asunto(s)
Mitocondrias Cardíacas/metabolismo , Potasio/metabolismo , 2,4-Dinitrofenol/farmacología , Animales , Citosol/metabolismo , Corazón/efectos de los fármacos , Corazón/fisiología , Concentración de Iones de Hidrógeno , Hipotermia/metabolismo , Técnicas In Vitro , Intercambio Iónico , Espectroscopía de Resonancia Magnética , Masculino , Mitocondrias Cardíacas/efectos de los fármacos , Fosfatos/metabolismo , Ratas , Ratas Sprague-Dawley , Rubidio/metabolismo , Radioisótopos de Rubidio , Saponinas/farmacología , Desacopladores/farmacologíaRESUMEN
Ion flux studies were performed on Langendorff-perfused rat hearts using 87Rb, 7Li and 23Na NMR at 36, 20 and 10 degreesC, and at constant extracellular pH (7.40). Using 31P NMR, the intracellular pH was estimated and the high energy phosphate content monitored. Compared to 36 degreesC (k=0.044+/-0.015 min-1), our measurements showed incomplete Rb+ efflux with a dramatically (5-fold) increased rate constant, k, at 20 degreesC, k=0.238+/-0.080 min-1. 5 microM glibenclamide, a KATP-channel inhibitor, completely depressed the hypothermia-activated Rb+ efflux at this temperature (k=0.052+/-0. 018 min-1). 7Li NMR efflux studies on KCl-arrested hearts at 20 degreesC also showed an increase (3-fold) in efflux rate constant: k=0.090+/-0.003 min-1 relative to its value at 36 degreesC. At 10 degreesC, both Rb+ and Li+ showed efflux rate constants similar to those observed at 36 degreesC, k=0.071+/-0.016 min-1 and k=0.050+/-0. 005 min-1, respectively, and the washout was complete. 31P NMR at 36, 20 and 10 degreesC indicated cytosolic alkalinization at pH values of 7.05, 7.21 and 7.40, respectively. The ion transport data could be interpreted in terms of a myocyte model allowing for temperature-dependent changes in transport coefficients. The incomplete efflux of Rb+ at 20 degreesC may indicate the existence of a mitochondrial Rb+-pool with a very low Rb+ permeability for efflux. These findings correlate with previously observed membrane phase transitions in these systems.
Asunto(s)
Litio/metabolismo , Miocardio/metabolismo , Rubidio/metabolismo , Sodio/metabolismo , Animales , Cationes Monovalentes , Permeabilidad de la Membrana Celular , Técnicas In Vitro , Transporte Iónico , Cinética , Espectroscopía de Resonancia Magnética , Masculino , Ratas , Ratas Sprague-Dawley , TemperaturaRESUMEN
The aim of this work was to investigate whether 1) Rb uptake is reduced in ischemic myocardium, and 2) 87Rb three-dimensional (3D) imaging can detect the ischemic area. Hearts of domestic pigs (n = 8, 20-30 kg) were perfused retrogradely with Krebs-Henseleit buffer in a 7-T, 40-cm horizontal bore magnet interfaced with Bruker MSLX spectrometer. Control (C) and ischemic (I) (45 min ligation of the left anterior descending coronary artery (LAD)) hearts were loaded with Rb+ by perfusion with a Rb(+)-containing solution (2-4.7 mM, 30-100% of K+ substitution) for 35 min and 87Rb (C) or 31P (ischemic area in I) spectra were acquired. After mechanical arrest with 0.6-0.9 mM lidocaine, 87Rb images (7 min each, 1 cm3 resolution) were acquired (30-40 min) in the presence of Rb(+)-containing perfusate. Subsequently, the hearts were stained with Evans blue (EB) and samples taken for measurements of Rb+ content. In the Group C, distribution of Rb+ in the left ventricle and the intensities of the 3D 87Rb images were uniform. In the ischemic area (Group I), verified by the lack of EB staining and changes in 31P spectra, the images showed a reduced intensity, which corresponded to decreased Rb+ content (33 +/- 11% of the normal). Thus, 87Rb imaging reveals damaged cells detecting reduced Rb+ content in the ischemic area.
Asunto(s)
Procesamiento de Imagen Asistido por Computador , Imagen por Resonancia Magnética , Espectroscopía de Resonancia Magnética , Isquemia Miocárdica/diagnóstico , Radioisótopos de Rubidio , Animales , Isquemia Miocárdica/patología , Necrosis , Fosfatos/metabolismo , Potasio/metabolismo , Radioisótopos de Rubidio/farmacocinética , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Porcinos , Supervivencia Tisular/fisiologíaRESUMEN
An experimental model was developed to evaluate the effects of activators and inhibitors of K(ATP) channels on unidirectional K+ fluxes in the whole heart. Isolated rat hearts perfused in the Langendorff mode were equilibrated with Pi-free Krebs-Henseleit buffer (KH buffer) containing 0.94-2.14 mM RbCl and 3.76 mM KCl (20-36% of K+ substituted by Rb+). Rb+ efflux was initiated by removing Rb+ from the perfusate and 87Rb spectra were acquired continuously with a 1-2 min time resolution. In hearts with normal energetics, the efflux of Rb+ fit a monoexponential function, and the rate constant did not depend on intracellular [Rb+]. Agents depressing excitability and heart rate (HR), such as 0.6 mM lidocaine (Lido), 10 microM carbachol (carb) and 20 mM MgSO4, inhibited Rb+ efflux such that the rate constant, k (10(3)/min), decreased from 50+/-1.2 in the beating heart to 26+/-1, 40+/-1.1 and 19+/-1.2, respectively. In contrast, high [K+] (21 mM) did not affect the k value (50+/-4.5), independently of the presence or absence of bumetanide (Bum, 30 microM) and glibenclamide (Glib, 5 microM). Dinitrophenol (DNP, 0.2 mM) added in the presence of high [K+] + Bum increased k three-fold, to 160+/-5. This effect was associated with a significant decrease in phosphocreatine (PCr, <10% of initial) and ATP ( 15%) levels, and a 7-fold increase in the Pi level, assessed by 31P-NMR spectroscopy. Glib completely reversed the effect of DNP. Pinacidil (Pin, 20-80 microM) did not affect the k value either in beating control hearts or in the presence of Carb or KCl + Bum. Moreover, under conditions of moderate metabolic stress induced by 0.05 mM DNP (PCr, 35%; ATP, 65%), where half-maximal activation of K(ATP) channels occurred, Pin did not further activate Rb+ efflux. We conclude that:(1) heart rate-independent Rb+ efflux accounts for 40-80% of the total Rb+ efflux in beating (300 bpm) rat hearts;(2) DNP-activated Rb+ efflux is a good model for testing inhibitors of KATP channels in whole hearts; and (3) Pin is not an effective K(ATP) channel opener in the rat heart model.