NIR spectroscopic detection of breast cancer.

Near infrared spectroscopy (NIRS) utilizes intrinsic optical absorption signals of blood, water, and lipid concentration available in the NIR window (600-1000 nm) as well as a developing array of extrinsic organic compounds to detect and localize cancer. This paper reviews optical cancer detection made possible through high tumor-tissue signal-to-noise ratio (SNR) and providing biochemical and physiological data in addition to those obtained via other methods. NIRS detects cancers in vivo through a combination of blood volume and oxygenation from measurements of oxy- and deoxy-hemoglobin giving signals of tumor angiogenesis and hypermetabolism. The Chance lab tends towards CW breast cancer systems using manually scannable detectors with calibrated low pressure tissue contact. These systems calculate angiogenesis and hypermetabolism by using a pair of wavelengths and referencing the mirror image position of the contralateral breast to achieve high ROC/AUC. Time domain and frequency domain spectroscopy were also used to study similar intrinsic breast tumor characteristics such as high blood volume. Other NIRS metrics are water-fat ratio and the optical scattering coefficient. An extrinsic FDA approved dye, ICG, has been used to measure blood pooling with extravasation, similar to Gadolinium in MRI. A key future development in NIRS will be new Molecular Beacons targeting cancers and fluorescing in the NIR window to enhance in vivo tumor-tissue ratios and to afford biochemical specificity with the potential for effective photodynamic anti-cancer therapies.

Oxygenation and blood volume changes in flaps according to near-infrared spectrophotometry.

OBJECTIVE: To test the ability of near-infrared spectrophotometry (NIRS) to predict vascular compromise in flaps postoperatively. DESIGN: Pilot study. SUBJECTS: Eleven denervated latissimus dorsi flaps were assessed in 8 pigs. INTERVENTIONS: Flaps were isolated on their vascular pedicle. We used NIRS to demonstrate tissue oxygen saturation and quantities of deoxygenated hemoglobin and oxygenated hemoglobin when flaps underwent venous or arterial occlusions. Oxygen saturation (percentage of oxygenated hemoglobin) was calculated as the difference between the 2 light intensities (860-750 nm) with the use of 2 time periods: preoperative (80%) oxygen saturation and during arterial occlusion (0%) oxygen saturation with NIRS. Blood volume changes within the flap were also measured. RESULTS: Arterial occlusion resulted in significant decreases in oxygen saturation and in blood volume with immediate recovery. Venous occlusion resulted in an initial rapid increase in blood volume with no appreciable early deoxygenation. CONCLUSIONS: Near-infrared spectrophotometry appears promising as a noninvasive, low-cost, portable bedside monitor that can demonstrate in real time changes in blood volume and oxygen saturation within a flap at a variety of tissue depths.

Effects of hypercapnia on ECoG and oxidative metabolism in neonatal dog brain.

The effects of hypercapnia on cerebral electrical activity and mitochondrial oxidative phosphorylation were studied in the anesthetized neonatal dog by using the electrocorticogram (ECoG) and 31P-magnetic resonance spectroscopy. Three levels of hypercapnia with arterial PCO2 values of approximately 70, 100, and 140 Torr reduced the intracellular pH of the brain from 7.11 to 6.99, 6.87, and 6.76, respectively. These levels of hypercapnia also reduced ADP concentration ([ADP]) from 21.5 to 18.1, 14.8, and 12.9 microM as well as the average ECoG power output by 20, 30, and 40%. A Michaelis-Menten relationship for the mitochondrial respiratory enzymes was fitted with [ADP] and the change in the average ECoG. The result suggests that mitochondrial respiration is regulated by [ADP] and that the in vivo Michaelis-Menten constant for ADP was 21 microM, a value close to the in vitro value. The mitochondrial maximal reaction velocity was reduced by only 10% during hypercapnia and showed no relationship with the degree of acidosis, suggesting that mitochondrial respiratory enzymes are not responsible for the inhibition of the brain electrical activity.

Bilateral carotid artery occlusion causes periventricular leukomalacia in neonatal dogs.

At 14 days of age, seven mongrel puppies were anesthetized and bilateral carotid arteries, not only the common but also the external and internal carotid arteries, were ligated with sutures. Seven sham-operated littermates served as controls. They were sacrificed at 3 months of age, and their brains were examined macro- and microscopically. Neuropathological examination revealed dilated posterior communicating and basilar arteries in bilateral carotid artery occlusion (BCAO) animals. Six out of 7 BCAO brains had uni- or multiloculated cysts in the periventricular white matter which were surrounded by a band of GFAP-positive glial cells. Scattered small areas of gliosis were found in all experimental animals. Four BCAO brains also showed ventricular dilatation. Although the cerebral cortex seemed to be intact, the periventricular white matter and the corpus callosum were reduced in width in experimental animals. Myelination in the white matter was significantly reduced in BCAO animals compared with the controls. This study directly demonstrates that cerebral hypoperfusion alone can produce periventricular leukomalacia in neonatal dogs.

Optical imaging of human breast cancer.

Since an increasing number of breast cancers have been reported in recent years, there is a need for improving techniques for early detection of the breast cancer. Here we tested a time gated optical imaging technique as a tool for imaging human breast. Pulsed laser light at wavelengths of 780 and 830 nm are transmitted through human breast tissues and time spectra of the diffused light through the tissue are recorded over nanoseconds. Data from different locations are acquired and used to reconstruct a two dimensional image as a set of spectra in pixel form. The imaging consists of absorption and scattering coefficients, and the absorption coefficients at the two wavelengths are related to oxygen concentration and blood volume. The analysis of these coefficients is based upon the early arrival photons, therefore allowing construction of a better image than those from the current diaphanography. We demonstrate images of breast cancer, cysts created after lumpectomy, and consequences of radiation therapy. Results show that time gated optical imaging can image oxygen concentration in the cancerous and fibrotic breasts. Resolution of the imaging for smaller tumor size needs to be improved.

Consequences of reduced cerebral blood flow in brain development. II. Retardation of neurological outcome and phosphorus metabolism.

We investigated the temporal relationship of the emergence of biochemical abnormalities to the development of behavioral dysfunction to identify the central factors of ischemic neurological disorders in developing brains. To induce early ischemia, bilateral carotid artery occlusion (BCAO) was surgically performed on 21 cats at the second week of age. BCAO produces histopathological damage, including neuronal loss and thinning of white matter. 31P magnetic resonance spectroscopy was used to monitor brain oxidative metabolism, neuronal membrane growth, and myelination of the prefrontal cortex in the first 3 months. Neurological development was monitored by conducting 25 tests of reflex, motor, sensory, and integrated behavioral function. At 1 month, phosphodiester (PDE) levels, a component of membranes and myelin, were low in animals showing complete ligation. At 2 months, the growth of PDE was low (1/4 to 1/2 of normal) in BCAO animals, whereas normal animals demonstrated a 23% increase. Phosphocreatine (PCr) levels, indicated by PCr/ATP and PCr/inorganic phosphate ratios, were retarded at 2 months in completely ligated animals (1/4 of normal). Neurologically, the completely ligated animals showed retardation of general development. The retardation was most pronounced for integrative functions, including visual function, and became more pronounced later in development. The time course of emergence of the retardation generally coincided with emergence of abnormalities in phosphorous compounds. The simultaneous occurrence of several biochemical and functional abnormalities in development following early ischemic insult suggests a causal relationship between membrane and mitochondrial development and neurological function.

31P-NMR spectroscopy of isolated perfused rat lung.

We obtained 202.5-MHz 31P-nuclear magnetic resonance (NMR) spectra of isolated perfused rat lungs, degassed and inflated, and of lung extract. The spectra included those of ATP, ADP, phosphocreatine (PCr), inorganic phosphate (Pi), phosphomonoesters, phosphodiesters, and a broad component due to the membrane phospholipids. The line width at one-half peak height for beta-ATP was 1.0 ppm for the degassed lung and 1.2 ppm for the inflated lung. This suggests that the air-water interfaces in inflated lung, which produce proton NMR line broadening, do not act prominently in 31P-NMR spectroscopy. In a degassed lung, when perfusion was stopped for up to 30 min, PCr and ATP peaks decreased progressively with time while Pi and phosphomonoester peaks increased. On return of flow, these changes reversed. The intracellular pH values calculated from the difference in magnetic field between PCr and Pi peaks of inflated and degassed lungs were 7.16 +/- 0.10 (SD; n = 4) and 6.99 +/- 0.10 (n = 4), respectively. The change of intracellular pH caused by 30 min of ischemia was -0.2 pH units. Our findings indicate that air-water interfaces should not broaden lung 31P peaks in vivo.

Multiparametric evaluation of brain functions in the Mongolian gerbil in vivo.

We have developed the multiprobe assembly (MPA) by which metabolic, ionic and electrical activities can be monitored from the surface of the brain. In the present study we included optical fibers for the monitoring of intracapillary hemoglobin oxygenation by use of the Erlangen Microlight Guide Spectrophotometer (EMPHO-I) from the surface of the gerbil brain. The newly developed MPA provides simultaneous information about oxygen delivery (oxydeoxy Hb), tissue pO2 level, as well as the intracellular oxygen balance (intramitochondrial redox state). The ionic homeostasis was evaluated by monitoring extracellular K+ and Ca2+ activities reflecting the permeability changes of cation channels as well as the activities of Na+,K(+)-ATPase and other ion linked transport processes. The electrical activities were monitored by a bipolar electrocortical surface probe and DC steady potential. The subjects of the present study were Mongolian gerbils (Meriones unguiculatus) anesthetized and operated according to our routine techniques. After 30 min of recovery from the operation each gerbil was exposed to a short anoxia, graded hypoxia, ischemia as well as spreading depression. The results can be summarized as follows: 1. A clear correlation was recorded between the changes in oxydeoxy Hb spectra, tissue pO2 level and oxidation-reduction state of intramitochondrial NADH under oxygen deficiency situations (hypoxia, ischemia). 2. Blood volume changes under various perturbations monitored by various probes (366 reflectance and EMPHO-I) correlated very well with each other. 3. The degree of inhibition of Na+,K(+)-ATPase induced by oxygen deficiency could be interpreted by changes in extracellular levels of K+ measured by the surface mini-electrode. 4. Brain stimulation induced by spreading depression mechanism led to transient changes in ionic homeostasis and increase in energy requirements. The major HbO2 response was an increase in oxygenation due to the large CBF increase as monitored by the laser Doppler flowmeter. 5. Changes in oxy-deoxy Hb under fast scanning of 500-600 nm during 2-3 seconds of bilateral carotid arterial occlusion provided an indirect index for tissue O2 consumption.

Substrate regulation of mitochondrial oxidative phosphorylation in hypercapnic rabbit muscle.

Endurance muscle performance is highly dependent on ATP production from mitochondrial oxidative phosphorylation. To study the role of the mitochondrial oxidative enzymes in muscle fatigue, we analyzed the relationship between the concentrations of substrates associated with ATP synthesis and the muscle performance of electrically stimulated rabbit muscle under CO2-induced acidosis. Two different conditions of pacing-induced muscle performance were produced in the gastrocnemius and soleus muscle groups in anesthetized rabbits by stimulating the sciatic nerve submaximally at two frequencies. Phosphorus nuclear magnetic resonance was used to measure ATP, phosphocreatine, and Pi and to provide data for a calculation of intracellular pH and free ADP. To induce acidosis, the animal was ventilated with 20% CO2. The administration of CO2 effectively reduced the intracellular pH from 6.9 to 6.7 and reduced the isometric tension-time integral (TTI) to below half the value measured in normocapnia at the low pacing frequency. A twofold increase in the pacing frequency resulted in a doubling of the TTI in normocapnia and a tripling of TTI in hypercapnia. The increases in TTI corresponded with increases in free ADP and Pi concentrations. Under the various conditions, all free ADP values were near the in vitro Michaelis-Menten constant (Km) of ADP. The Michaelis-Menten relationship of the oxidative phosphorylative enzymes was applied to the change in substrate concentrations with respect to TTI. From this relationship we observed that the in vivo Km of free ADP was 26 microM, which is close to the in nitro Km, and that Km and maximal reaction velocity did not change under hypercapnia and increased pacing frequency.(ABSTRACT TRUNCATED AT 250 WORDS)

31P NMR and enzymatic analysis of cytosolic phosphocreatine, ATP, Pi and intracellular pH in the isolated working perfused rat heart.

Hearts from fed male Wistar rats (200-350 g) were perfused at low and high workloads with Pi-free Krebs-Henseleit medium containing either 10 mM glucose or 10 mM glucose plus 15 mU/mL insulin. The intracellular pH by 31P NMR ranged between 6.99 and 7.02 and agreed to within 0.1 pH unit of estimates calculated using enzymatically determined total tissue HCO3-/CO2 contents. At high work, where the tissue contents of phosphocreatine (PCr) and ATP were determined on the same heart as NMR areas (n = 16), the proportionality factors, defined as the 31P NMR area units divided by the total enzymatically determined tissue content (area units/mumol/g dry wt), were 112 +/- 8 for PCr, 99 +/- 4 for gamma-ATP, 138 +/- 9 for alpha-ATP and 100 +/- 4 for beta-ATP. These values were normalized by taking beta-ATP as 100 area units/mumol/g dry wt. Since the proportionality factor for PCr and gamma- and beta-ATP were not statistically different (p less than 0.05), it was concluded that each was equally visible by 31P NMR and that no significant breakdown of PCr occurred during freezing or tissue acid extraction procedures. The cytosolic Pi estimated from NMR in glucose plus insulin perfused hearts at low and high work was 4.92 +/- 0.67 and 6.33 +/- 0.42 mumol/g dry wt. Using the near-equilibrium expression of KCK/KG + G and the metabolite levels in heart extracts, the calculated cytosolic Pi was 13.08 +/- 1.83 and 16.17 +/- 3.08 mumol/g dry wt, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Age dependence of steady state mitochondrial oxidative metabolism in the in vivo hypoxic dog brain.

Mitochondrial bioenergetics were investigated in newborn, neonatal and adult dog brains during normoxia and hypoxia. The ratio of the rate of ATP synthesis to the maximum synthesis rate (V/Vmax), phosphorylation potential, [ADP] and PCr/Pi, were used to evaluate age related mitochondrial hypoxic tolerance. These indicators were calculated from the phosphorus compounds measured by in vivo 31P MRS quantitatively using ATP as an internal reference. Indicators and substrates of mitochondrial function, V/Vmax, ADP, and Pi reached a peak value during the neonatal (3-21 days) period of development, suggesting that the oxidative metabolism of the neonate is more vulnerable to stress when compared to newborns and adults. Distinction among newborns and neonates became apparent during hypoxia. Newborns (0-2 days old) showed substantial tolerance by maintaining V/Vmax until exposure to severe hypoxia. Older neonates (3-21 days old) showed increases in V/Vmax, [Pi] and [ADP] under less than severe conditions of hypoxia. Adults exhibited low V/Vmax values even during exposure to severe hypoxia, further indicating that mitochondrial oxidative processes are more stable in adults than in newborns and neonates. This study provides evidence that newborns and adults are more capable of maintaining mitochondrial function under conditions of minimal to moderate hypoxia than 3-21 day old neonates.

Quantitation of high energy phosphate compounds and metabolic significance in the developing dog brain.

The phosphate metabolites, PCr, ATP, ADP and inorganic phosphate (Pi), were quantitated in the brain of the newborn, neonatal, juvenile and adult dog to investigate the potential control mechanisms responsible for increased ATP demands during development. The concentrations of PCr and Pi were measured in vivo by MRS using the enzymatic-measured ATP as the internal standard. Phosphocreatine values increased during development from 2.08 mmol/kg wet weight in the 0-2 day newborn to 5.11 mmol/kg wet weight in the adult brain and paralleled the increases in the total creatine pool (PCr + Cr) from 4.12 to 10.05 mmol/kg wet weight. Brain ATP concentrations increased approximately 40% during postnatal development; however, when expressed as intracellular concentration, no increase in ATP was apparent due to the age-dependent decrease in extracellular space. The Pi concentration, estimated by MRS, increased significantly during postnatal development with a range of 1.78 to 2.52 mmol/kg wet wt, then decreased to 1.97 mmol/kg wet weight at adulthood. In those developmental stages where total Pi was measured enzymatically on freeze-clamped tissue, the NMR visible Pi comprised about 48 to 93% of the total, with the highest percentage being visible in the newborn brain. The intracellular pH decreased from 7.21 in the newborn to 7.10 in the adult. With development, the free ADP concentration, calculated from the components of the creatine kinase equilibrium, ranged from 27 to 34 microM. These values are close to the apparent in vitro Km of ADP for oxidative phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)

The detection of cytochrome oxidase heme iron and copper absorption in the blood-perfused and blood-free brain in normoxia and hypoxia.

The resolution of cytochrome and hemoglobin changes in in vivo rat and cat brains has required studies over wide wavelength ranges (580-1100 nm) with a novel spectroscopic technique using blood-free and blood-perfused brains. Tissue oxygen was varied from physiological levels to 0 and hematocrits were varied from normal to less than 1%. The experimental results were subjected to a multicomponent analysis using the Beer-Lambert law. At normal hematocrits, the oxygen saturation of hemoglobin in the brain was found to be 30-50% in rats and cats, indicating that the optical method responded primarily to the saturation of the venous ends of the capillary beds. With low hematocrits, both brains showed the absorption band of reduced cytochrome c, the iron component of cytochrome aa3, plus the absorption band of the oxidized copper component. In cat brains, the background absorption changed at all wavelengths. Thus, no isosbestic points were observed in the spectra. In rat brains, however, they were readily observed. The "overtones" of water absorption in the NIR region were found to be significant in the difference spectra of the cat brain, but not in the rat brain. Parallel absorbance changes in the heme and copper components of cytochrome aa3 were obtained in rat and cat brains during the normoxic-hypoxic transition. The ratio of the iron absorbance at 605 nm to the copper absorbance at 830 nm is much smaller in both brains than the in vitro value due to the shorter path length of photon migration at the shorter wavelengths.(ABSTRACT TRUNCATED AT 250 WORDS)

Experimental brain ischaemia: assessment of injury by magnetic resonance spectroscopy and histology.

Phosphorus magnetic resonance spectroscopy (31P-MRS) was made to measure changes in brain high energy phosphate compounds, adenosine triphosphate (ATP) and phosphocreatine (PCr), inorganic phosphorus (Pi) and intracellular pH (pHi) during a prolonged period of incomplete brain ischaemia produced, in anaesthetized dogs, by bilateral carotid occlusion together with haemorrhagic hypotension for intervals of up to 300 min. Mean arterial blood pressure (MABP) was lowered in a stepwise fashion, until signs of metabolic decompensation (as estimated by MRS) occurred. At that point MABP was varied against further evidence of metabolic decompensation in an attempt to maintain a more constant degree of insult. At the end of the ischaemic period MABP was restored and the animals observed during a 3 h recovery period. At the end of the recovery period the brains were perfusion-fixed for histological examination. A semi-quantitative method of histological evaluation was used to determine the degree of histological damage. This permitted assignment of an 'ischaemic score' to the tissue sampled from each animal. Comparisons were then made between the magnitude of this 'ischaemic score' and the changes in metabolic and physiological variables (ATP, PCr, pHi and MABP) as well as an estimator of phosphorylation potential (PCr/Pi), which were all measured during the ischaemic insult. Histological examination showed a wide variety of neuronal alterations, including dark and pale type injury, which correlated directly with the metabolic derangements brought about by ischaemia. The degree of damage determined from this histological assessment correlated best with the duration and degree of change in PCr/Pi, supporting the use of this ratio as a critical index of cellular energy state. In particular there was a strong linear relationship between the degree of leucocyte recruitment and changes in PCr/Pi. To summarize, metabolic changes, determined by MRS, correlate with the degree of histological damage, and in turn, the classical descriptions of acute ischaemic neuronal injury appear to be validated by MRS determinations of metabolic changes during ischaemia.

Oxidative phosphorylation system during steady-state hypoxia in the dog brain.

The relationship between biochemical and physiological responses and tissue O2 during hypoxia was investigated in vivo in the dog brain by 31P nuclear magnetic resonance (NMR) spectroscopy. Our findings demonstrate how ATP synthesis in the brain can be maintained during hypoxia because of compensatory changes in NADH, ADP, and Pi. Eleven beagle dogs were anesthetized and mechanically ventilated, and a steady-state graded hypoxia was induced by decreasing the fraction of inspired O2 (FIO2) stepwise at 20-min intervals. Biochemical metabolites were measured using 31P-NMR and fluorescence spectroscopy. When sagittal sinus O2 partial pressure (PVO2) had decreased to 15 Torr, NADH increased by 30%, Pi increased by 50%, and phosphocreatine (PCr) decreased by 20%. In contrast, ATP remained constant. There was a 10% increase in ADP in dogs that maintained a steady temperature, but ADP decreased by as much as 30% in dogs in which body temperature decreased with the falling PVO2. PCr/Pi was logarithmically related to the phosphorylation potential during steady-state hypoxia. Compensation for the O2 lack is attributed to increases in ADP, Pi, and NADH as a result of the reciprocal relationship of the Michaelis-Menten equation. If the Michaelis-Menten constants (Km) of ADP, Pi, and O2 are the same as determined in vitro in mitochondria, the minimum brain cytosolic O2 capable of maintaining a steady-state ATP is near its Km (0.1 Torr) at a PVO2 of 7.5 Torr. At this critical O2 level, PCr/Pi is 0.9, intracellular pH is 6.75, phosphorylation potential is 38.5 mM-1, and the calculated maximum velocity of ATP formation by oxidative phosphorylation is 55% of normal.

Activation of the Na+, K(+)-ATPase in Narcine brasiliensis.

The in vivo activation and turnover rates of the sodium pump (Na+, K(+)-ATPase) were investigated in the electrocytes of the electric organ of the elasmobranch Narcine brasiliensis. The Narcine electric organ appears to be an excellent model for the study of sodium pump activation in an excitable tissue. The sodium transmembrane gradient and high-energy phosphagens were concurrently measured by 23Na and 31P NMR spectroscopy. The resting electric organ, which depends primarily on anaerobic metabolism, displays a high concentration of phosphocreatine (PCr). It has an intracellular sodium concentration ([Na+]i) of 20 +/- 10 milliequivalents/liter as estimated by NMR. Electrical stimulation of the nerves innervating the electric organ results in an increase in [Na+]i in the electrolyte and rapid depletion of PCr. Ouabain causes an 85% decrease in utilization of high-energy phosphagens, indicating that rapid PCr turnover in this tissue is mainly due to Na+, K(+)-ATPase activity. From these data we can determine that the rate of sodium pump turnover increases by greater than 3 orders of magnitude within several hundred milliseconds. In excised unstimulated electric organ slices, changes in [Na+]i equivalent to those occurring with stimulation, but induced by hyperosmolar conditions, do not result in increased PCr hydrolysis. We conclude that cholinergic stimulation of the electric organ causes a rapid and extremely large increase in sodium pump turnover, which is regulated predominantly by factors other than [Na+]i.

Brain oxidative metabolism of the newborn dog: correlation between 31P NMR spectroscopy and pyridine nucleotide redox state.

The effects of both anoxia and short- and long-term hypoxia on brain oxidative metabolism were studied in newborn dogs. Oxidative metabolism was evaluated by two independent measures: in vivo continuous monitoring of mitochondrial NADH redox state and energy stores as calculated from the phosphocreatine (PCr)/Pi levels measured by 31P nuclear magnetic resonance (NMR) spectroscopy. The hemodynamic response to low oxygen supply was further evaluated by measuring the changes in the reflected light intensity at 366 nm (the excitation wavelength for NADH). The animal underwent surgery and was prepared for monitoring of the two signals (NADH and PCr/Pi). It was then placed inside a Phosphoenergetics 260-80 NMR spectrometer magnet with a 31-cm bore. Each animal (1-21 days old) was exposed to short-term anoxia or hypoxia as well as to long-term hypoxia (1-2 h). The results can be summarized as follow: (a) In the normoxic brain, the ratio between PCr and Pi was greater than 1 (1.2-1.4), while under hypoxia or asphyxia a significant decrease that was correlated to the FiO2 levels was recorded. (b) A clear correlation was found between the decrease in PCr/Pi values and the increased NADH redox state developed under decreased O2 supply to the brain. (c) Exposing the animal to moderately long-term hypoxia led to a stabilized low-energy state of the brain with a good recovery after rebreathing normal air. (d) Under long-term and severe hypoxia, the microcirculatory autoregulatory mechanism was damaged and massive vasoconstriction was optically recorded simultaneously with a significant decrease in PCr/Pi values.(ABSTRACT TRUNCATED AT 250 WORDS)