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PubMed1995

Age-related changes in perimicrovascular protein distribution.

The diffusion hypothesis for physiological aging proposes that an increase in interstitial matrix fiber-to-gel ratio causes a decrease in nutrient diffusion to the cells. This hypothesis predicts a decrease in interstitial matrix protein with age. The objective was to test this hypothesis by determining age-related changes in plasma protein distribution in perimicrovascular and distal regions of rat mesentery interstitial matrix. Rats that were 77, 140, 210, 315, 455, and 630 days old were anesthetized with pentobarbital sodium, and a mesenteric loop was exteriorized. Intravital video microspectrophotometry was performed using wavelengths of 280, 320, and 700 nm. Perimicrovascular protein concentrations from the protein absorbance images were used to obtain the histogram, mean, and skewness of the proximal and distal protein concentration distributions. An exponential gradient model was also used to obtain the proximal and distal protein concentrations and gradient decay constants. Proximal protein concentration increased from 77- to 140-day-old rat and then decreased gradually through 210-, 315-, 455-, and 630-day-old rats. Distal concentration decreased gradually from 140- to 630-day-old rats. There was an increase in positive skewness of the proximal protein distributions from 140- through 630-day-old rats. We found an age-related decrease in perimicrovascular protein and propose that this is due to a decrease in protein permeability with age. The results support the diffusion theory of aging.

Aging↗
PubMed1998

Direct observation of radial intracellular PO2 gradients in a single cardiomyocyte of the rat.

The purpose of the present study was to directly visualize radial gradients of intracellular PO2 in a single individual cardiomyocyte isolated from the rat ventricle. Microspectrophotometry with the use of cytosolic myoglobin as an oxygen probe was conducted at 410 nm. When the quiescent cell was incubated with 1 microM carbonyl cyanide m-chlorophenylhydrazone to increase oxygen consumption approximately eightfold, gradual decreases in myoglobin oxygen saturation (SMb) were demonstrated toward the core of the cell, whereas these decreases disappeared when the cell was treated with 2 mM NaCN. These results highlighted the importance of diffusional oxygen transport in determining intracellular oxygenation in cardiac cells. From the measured SMb, we assessed the profile of radial changes in intracellular PO2 at the mean SMb comparable to that in vivo ( approximately 0.5). Quite steep PO2 gradients were demonstrated in the vicinity of the sarcolemma that were rapidly attenuated toward the cell core. These radial profiles of intracellular PO2 demonstrate the significance of myoglobin-facilitated diffusion of oxygen. Furthermore, the shallow gradients of PO2 near the center of the cell might arise from partial depression of oxygen consumption near the cell core.

Animals↗
PubMed1990

Sendai virus infection potentiates neurogenic inflammation in the rat trachea.

The goal of this study was to determine whether Sendai (parainfluenza type I) virus infection potentiates neurogenic inflammatory responses in the trachea. Pathogen-free F344 male rats were inoculated into each nostril with Sendai virus or with sterile culture medium. Six days after the inoculation, a threshold dose of capsaicin (75 micrograms/kg) or the vehicle used to dissolve the capsaicin was injected intravenously, and Monastral blue pigment was injected to measure the increase in vascular permeability produced by the capsaicin or vehicle (n = 6 per group). Five minutes later the tracheae were fixed by vascular perfusion, removed, and treated with a histochemical reaction for myeloperoxidase to stain the neutrophils in the mucosa, and the magnitude of the Monastral blue extravasation was determined both by stereological point counting and by microspectrophotometry. Capsaicin produced a larger number of Monastral blue-labeled blood vessels in the rats infected with Sendai virus than in the uninfected rats or in any of the rats that received only the vehicle. In addition, capsaicin further increased the already large number of neutrophils in the tracheal mucosa of the infected rats. However, in the dose used, it had no detectable effect on neutrophils in the uninfected rats. We conclude that Sendai virus infection causes neutrophil chemotaxis but does not increase vascular permeability in the trachea; it does, however, make the trachea abnormally susceptible to neurogenic inflammation, as manifested by an unusually large increase in vascular permeability and an exaggerated influx of neutrophils in response to sensory nerve stimulation by capsaicin.

Animals↗
PubMed2001

Oxygen binding by single red blood cells from the red-eared turtle Trachemys scripta.

Oxygen-binding properties of single red blood cells from the red-eared turtle Trachemys scripta were measured by microspectrophotometry to describe the variation in oxygen affinity of red blood cells and to gain insight into the distribution of functionally different hemoglobins among red blood cells. Methodologically, this study represents the first report on the cell-to-cell variation in oxygen-binding properties based on oxygen-binding curves of single vertebrate red blood cells. The cells differed significantly with respect to oxygen affinity. Mean oxygen pressure at half saturation of the cells in a blood sample was found to be 20.1 +/- 3.3 (SD) Torr. The distribution of oxygen affinities among red blood cells is unimodal, indicating that the two hemoglobins found in turtle blood are not segregated in distinct cells. Therefore, the functional interaction shown by these hemoglobins in vitro is likely to take place in vivo. The considerable variation in oxygen affinity between individual red blood cells calls for its incorporation in models of tissue oxygenation.

Adenosine Triphosphate↗
PubMed2004

Negative metabolic and coronary flow effects of decreases in cAMP and increases in cGMP in control and renal hypertensive rabbit hearts.

The interaction during stimulation of cGMP and inhibition of cAMP was investigated in control and renal hypertensive hearts. Control and hypertensive [1 kidney, 1 clip (1K1C)] rabbits were used. The anesthetized open-chest groups were vehicle, 8-bromo-cGMP (8-Br-cGMP; 10(-3)M), propranolol (Prop; 2 mg/kg), and Prop + 8-Br-cGMP. O(2) consumption levels (Vo(2)) in the subepicardium (Epi) and subendocardium (Endo) were determined from coronary flow (microspheres) and O(2) extraction (microspectrophotometry). Wall thickening and cAMP levels were also determined. In control, no significant change in Vo(2) was seen for the 8-Br-cGMP group, but Vo(2) was decreased from Epi (9.7 +/- 1.5 ml O(2) x min(-1) x 100 g(-1)) and Endo (10.5 +/- 0.4 ml O(2) x min(-1) x 100 g(-1)) to 6.8 +/- 0.6/7.8 +/- 0.5 ml O(2) x min(-1) x 100 g(-1) in the control Prop group. Control Prop + 8-Br-cGMP did not cause a further fall in Vo(2) but lowered Endo flow. In 1K1C, Vo(2) decreased from Epi/Endo (10.8 +/- 1.3/11 +/- 1.0 ml O(2).min(-1).100 g(-1)) to 7.8 +/- 1.1/8.7 +/- 0.5 ml O(2) x min(-1) x 100 g(-1) in the 1K1C 8-Br-cGMP group and to 7 +/- 0.5/8.1 +/- 0.5 ml O(2) x min(-1) x 100 g(-1) in the 1K1C Prop group. 1K1C Prop + 8-Br-cGMP did not cause a further fall in Vo(2) but lowered blood flow. No significant changes in cAMP levels were present with 8-Br-cGMP in control or 1K1C rabbits, but significant decreases were seen with Prop in both control and 1K1C rabbits. No further change was seen in Prop + 8-Br-cGMP for either control or 1K1C. Thus the negative metabolic effect of stimulating cGMP was seen only in the hypertensive rabbit heart. The negative metabolic effect of inhibiting cAMP was seen in both the control and the hypertensive rabbit heart. However, the negative metabolic effects of cGMP and cAMP were nonadditive.

Adrenergic beta-Antagonists↗
PubMed1998

Cytogenetics of Hodgkin's disease.

Cytogenetic studies over the past 35 years have made a major contribution towards the understanding of the nature of Hodgkin's disease by demonstrating unequivocally the consistent presence of a clonal population of cells that have the cardinal features of malignancy e.g. more or less gross aneuploidy, frequently with complex chromosomal changes and showing considerable variation from case to case, thus comparable to the findings in carcinomas and other solid cancers. The mode is frequently in the triploid-tetraploid region, as we found in 17 of 27 cases studied in this laboratory by Feulgen microspectrophotometry, compared to only 10 cases with neardiploid modes. It is disappointing that no specific change, such as a translocation that could give a clue to the chromosomal location of a gene or genes involved in the etiology of Hodgkin's disease, has yet been found. Nevertheless it is clear that a number of nonrandom changes, including several that are also common in other malignancies including the non-Hodgkin's lymphomas, are frequently present, e.g., deletions of 1p, 6q, and 7q. Interestingly, deletions of 4q, with loss of 4q25 --> q27, that have also been reported may show some specificity for Hodgkin's disease.

Chromosome Aberrations↗
PubMed1987

New strategies in the management of acute leukemias.

The rapidly emerging knowledge of cytogenetics, cell surface markers, cytochemistry, electron microscopy and flow microspectrophotometry makes the objective identification of subsets of acute leukemia patients possible. These new classification schemes have revealed a unique biology and response to therapy for the different subsets. New regimens result in a significant cured fraction of patients. Identification of the potentially cured subsets of patients has become possible. Molecular genetics has the potential for further improving the assessment of residual disease and indications for both intensification and continuation treatments. These advances in knowledge bring the clinical scientist closer to the potential for prescribing specific therapy for individual patients.

Acute Disease↗
PubMed1985

Hemoglobin content of single erythrocytes from fetuses with parents having heterozygous beta-thalassemia.

The hemoglobin content of single erythrocytes was determined by microspectrophotometry as total extinction (TE) at 415 nm in subjects with a wide spectrum of hemoglobin levels and erythrocyte indices. Precise measurements of the cellular area (A) and the ratio TE/A were also recorded. A significant correlation was found between TE and mean corpuscular hemoglobin (MCH), A and mean corpuscular volume, and TE/A and mean cell hemoglobin concentration (MCHC) for the cases studied. In addition, TE, A and TE/A were determined in fetal erythrocytes obtained by fetoscopy at the 20th week of gestation for prenatal diagnosis of beta-thalassemia. The mean red cell TE and TE/A of the group of fetuses diagnosed to have thalassemia major were significantly lower than those of the group of normal controls and with heterozygous beta-thalassemia. The significant differences of TE, A and TE/A between maternal and fetal blood allowed a safe distinction of the latter. The calculated MCH of the fetuses with thalassemia major was 12% less than that of the normal controls; the respective difference of the mean cell size was 5% and that of MCHC 3% lower than normal.

Erythrocyte Indices↗
PubMed1991

Flowcytometric measurement of the cell cycle of experimental tumors: some devices for accurate measurement of proliferative activity.

In this paper we present the cell cycle analysis using the latest flowcytometer, FACScan and dedicated softwares. Although the 5-bromodeoxyuridine (BrdU) and anti-BrdU method developed by Gratzner has become popular for analysis of cell kinetics, contaminating interstitial cells and cell doublets are still factors responsible for some mistakes in measuring the cell cycle accurately. These hampering cells were almost gated out on the catogram consisting of the forward scatter and DNA content (propidium iodide, PI). After that, the bivariate distribution of BrdU and PI according to the tumor growth showed reliable patterns. In LLC tumor the population, of which DNA content was compatible with S phase cells, was detected in the cytogram. Thus, the two-dimensional analysis of the cell cycle can demonstrate each population clearly. The results obtained from the BrdU method are more beneficial than autoradiography or microspectrophotometry, and reproducible data of the cell cycle parameters can be acquired with some devices described here.

Adenocarcinoma↗
PubMed2002

Imaging of oxygen transfer among microvessels of rat cremaster muscle.

BACKGROUND: The proximity of capillaries, arterioles, and venules provides complex spatial relationships that lead to oxygen transfer among microvessels. Although a conceptual image of complex oxygen transfer among microvessels has been hypothesized, in vivo mapping of oxygen saturation (SO2) levels in microvessels had never been performed. METHODS AND RESULTS: The oxygen profile of the arterioles and venules of the rat cremaster muscle during normoxia and hypoxia was visualized by preparing pseudo-color images of SO2 levels based on microspectrophotometry data obtained by using 3 different optical filters and a cooled CCD camera. The SO2 images showed lower SO2 levels in arterioles close to their walls, and the SO2 levels in the paired venules showed higher SO2 levels close to the arterioles. There were capillaries that crossed the microvessels whose SO2 levels changed as they crossed the microvessels. The SO2 levels were lower close to the vessel wall than in the centerline level of the microvessels, and the highest SO2 levels in venules paralleling arterioles were skewed toward the arterial side. The SO2 images showed that the SO2 level in arterioles decreased after crossing venules, whereas the SO2 level in venules increased after crossing arterioles. CONCLUSIONS: Visualization of intravascular SO2 levels suggested that oxygen is transferred between paired microvessels and between crossing microvessels in rat cremaster muscle. The possibility that oxygen is transported from some arterioles to venules and tissue through adjacent capillaries is proposed.

Animals↗
PubMed1988

Myocardial oxygen consumption, oxygen supply/demand heterogeneity, and microvascular patency in regionally stunned myocardium.

Although oxygen consumption closely parallels mechanical work in the normal heart, previous studies have found that stunned myocardium may have normal or even increased oxygen consumption despite depressed function. In this study we used microspectrophotometry to measure the oxygen saturations within arteries and veins of less than 100 micron diameter in quick-frozen biopsy samples from normal and regionally stunned myocardium of 10 open-chest anesthetized dogs. Regional myocardial blood flow, measured by radioactive microspheres, was similar in stunned and normal regions, as was mean arteriolar oxygen saturation. However, mean venous oxygen saturation was lower in the stunned region (epicardium 38.0% vs 43.8%, p less than .02; endocardium 36.2% vs 39.5%, p = .12), indicating increased oxygen extraction and consumption, despite a marked reduction in mean systolic segmental shortening from 14.4% to 0.5%. In addition, there was greater vein-to-vein heterogeneity of oxygen saturation in the stunned region, with an excess of veins having low saturations (statistically significant in epicardium, nonsignificant trend in endocardium). Microvascular injections studies with Microfil or drafting ink revealed filling of over 95% of arterioles and 85% of capillaries in the stunned region, similar to the findings in the normal region. Our results are consistent with an inefficient transfer of energy into myocyte contraction or an increased use of energy for noncontractile activities in stunned myocardium. In addition, the finding of increased heterogeneity of oxygen extraction suggests that the injury to stunned myocardium may not be uniform to all contractile elements, but instead may be focally and irregularly distributed.

Animals↗
PubMed1988

Increased preferential absorption in human atherosclerotic plaque with oral beta carotene. Implications for laser endarterectomy.

Patients undergoing carotid endarterectomy were pretreated with low-dose, oral beta carotene to determine whether the carotenoid content of plaque could be increased in vivo. Beta carotene-treated patients had a 50-fold increase in their plaque beta carotene level from 0.066 to 3.3 micrograms beta carotene/g plaque. Microscopy and microspectrophotometry demonstrated that plaque from beta carotene-treated patients had higher carotenoid levels and higher absorption (450-500 nm) compared with control specimens, but normal media was unaffected. This demonstration of increased preferential absorption by plaque suggests that selective ablation of atherosclerotic plaque may be enhanced by pretreating patients with oral beta carotene.

Absorption↗
PubMed1990

Hypertension-induced cardiac hypertrophy. Oxygen supply and consumption with pacing.

The purpose of this study was to determine if hypertrophied myocardium was associated with diminished cardiac function, restricted oxygen supply, or oxygen consumption during tachycardia. Myocardial oxygen supply and oxygen consumption were determined during baseline and atrial pacing conditions 30 days after New Zealand White rabbits were prepared as one-kidney, one clip Goldblatt hypertensive or uninephrectomized sham control rabbits. Coronary blood flow and cardiac output, using radioactive microspheres, and small vessel oxygen saturations, using microspectrophotometry, were measured in hypertrophied and nonhypertrophied hearts. After 30 days, baseline blood pressure was significantly higher in the Goldblatt rabbits compared with sham controls, and hypertension was maintained during pacing. The myocardium was hypertrophied in the Goldblatt hypertensive rabbits compared with sham controls. Baseline heart rates were not different between animal groups (242 +/- 32 and 244 +/- 24 beats/min, respectively). Both groups were paced 35% above baseline heart rates; during pacing, cardiac output was similar to baseline values in the sham controls (304 +/- 99 versus 321 +/- 116 ml/min, respectively) but reduced in the hypertensive rabbits (248 +/- 43 versus 325 +/- 62 ml/min). Myocardial oxygen consumption increased twofold in both nonhypertrophied and hypertrophied ventricles during tachycardia. Oxygen extraction was significantly elevated, but coronary blood flow was not altered during pacing in either animal group. Therefore, at the pacing level chosen the diminished function in cardiac hypertrophy was not associated with reduced oxygen consumption. Conversely, reduced efficiency during pacing in the hypertrophied myocardium was suggested.

Animals↗
PubMed1994

Nitric oxide synthase inhibition alters cerebral blood flow and oxygen balance in focal cerebral ischemia in rats.

BACKGROUND AND PURPOSE: This study investigated whether the nitric oxide synthase inhibitor NG-nitro-L-arginine-methyl ester (L-NAME) would alter blood flow and oxygen balance in the ischemic cerebrocortex of isoflurane-anesthetized Long-Evans rats. METHODS: Fifteen minutes after middle cerebral artery occlusion, L-NAME (1.5 mg/min per kilogram) was infused intravenously to the L-NAME group (n = 14), and normal saline was given to the control group (n = 14) for 45 minutes. In each group, regional cerebral blood flow was determined with [14C]iodoantipyrine, and arterial and venous oxygen saturations were determined by microspectrophotometry. RESULTS: In both groups regional cerebral blood flow of the ischemic cortex was significantly lower than that of the contralateral cortex ([mean +/- SD] 55 +/- 13 versus 110 +/- 29 mL/min per 100 g in the control group and 35 +/- 13 versus 90 +/- 24 mL/min per 100 g in the L-NAME group). Compared with the blood flow in the ischemic cortex of the control group, L-NAME significantly reduced ischemic blood flow by 36%. Venous oxygen saturation was significantly increased in the ischemic cortex (41 +/- 1% versus 44 +/- 3%) but decreased in the contralateral cortex (65 +/- 3% versus 61 +/- 4%) by L-NAME. Calculated ischemic cortical oxygen consumption in the L-NAME group was 39% lower than that in the corresponding control group, whereas the difference was only 11% in the contralateral sides between groups. In both groups, the ratio of oxygen supply to consumption was lower in the ischemic than in the nonischemic regions. In the ischemic cortex, this ratio was significantly lower in the control group than in the L-NAME group (1.7 +/- 0.1 versus 1.9 +/- 0.1). In contrast, the ratio tended to be decreased by L-NAME in nonischemic regions. CONCLUSIONS: These observations suggest that despite a decrease in cerebral blood flow, inhibition of nitric oxide synthesis mildly improves the oxygen supply and consumption balance in the ischemic cortex.

Amino Acid Oxidoreductases↗
PubMed2001

Functional characterization of heme proteins encapsulated in wet nanoporous silica gels.

Myoglobin and hemoglobin were encapsulated in wet, nanoporous silica gels. A rigorous evaluation of the effect of the encapsulation on protein dynamics and function was carried out by measuring the kinetics of carbon monoxide rebinding after nanosecond laser flash-photolysis with transient absorption detection, and the oxygen affinity with absorption microspectrophotometry. The time course of carbon monoxide binding to myoglobin evidenced a strongly enhanced geminate recombination and a faster bimolecular rebinding with respect to solution, whereas T and R quaternary states of hemoglobin exhibited a geminate phase and a bimolecular binding rate very similar to those observed in solution. Oxygen affinity of T-state hemoglobin was found to be close to that observed for the binding of the first oxygen to T-state hemoglobin in solution. Results indicate that some conformational transitions are kinetically restricted, allowing to isolate distinct tertiary and quaternary states. This opens the way to their detailed functional characterization and application to biodevices.

Absorption↗
PubMed1975

Determination of the glycogen content in single neutrophil leukocytes using a micromodel of leukocyte glycogen.

The kinetics of the periodic acid oxidation as part of the periodic acid-Schiff reaction was studied by combined microinterferometry and microspectrophotometry in micromodel systems of liver glycogen and leukocyte glycogen as well as in neutrophil leukocytes. The initial formation of Schiff-positive chromogens was more rapid in neutrophil leukocytes than in liver or leukocyte glycogen. The chromogen formation was, however, practically complete within 60 min in both neutrophil leukocytes and leukocyte glycogen, but this did not appear to be the case in liver glycogen. Differences in the rate of chromogen formation may depend on various factors such as differences in the source and treatment of the glycogen. The complete periodic acid-Schiff reaction appears to be a measure of the glycogen amount in neutrophil leukocytes and the microdroplet system of leukocyte glycogen is considered to be an appropriate model for the estimation of the glycogen amount in single neutrophil leukocytes. A mean value of 13.3 10-12 g glycogen per normal human neutrophil was found.

Glycogen↗
PubMed1979

Applications of microcomputer technology to cytophotometry.

Lack of appropriate software support for microprocessor program development has previously limited the applications of such technology in the field of microspectrophotometry. This paper describes our use of a Vickers M86 integrating microdensitometer coupled through a custom-designed interface circuit to a Processor Technology SOL III microcomputer. W e have developed a series of interactive, user-oriented programs for DNA-Feulgen cytophotometry with this instrument to allow automatic storage of data in files on floppy disks and instant retrieval of sets of measurements for statistical processing. The same data files can also be used to generate graphic displays in the form of bar histograms or plots of linear regressions on a video monitor and to produce hardcopy output of data files and graphic displays through the use of a high speed DIABLO printer.

Animals↗
PubMed1981

Mithramycin- and 4'-6-diamidino-2-phenylindole (DAPI)-DNA staining for fluorescence microspectrophotometric measurement of DNA in nuclei, plastids, and virus particles.

The properties of two DNA-specific fluorochromes, 4'-6-diamidino-2-phenylindole (DAPI) and mithramycin, have been analyzed as reagents to quantitate cellular DNA by fluorescence microspectrophotometry. Optimal staining conditions and concentrations, and the effects of other cellular materials to which the dyes bind, have been evaluated in measurements of the DNA of rat, chick, and yeast nuclei, Gonyostomum chloroplasts, and T4 particles. Use of either fluorochrome permits a high degree of resolution of different DNA quantities in nuclei and in cell organelles, and the DAPI-DNA complex is sufficiently fluorescent to permit quantitation of the DNA content in genomes as small as those of individual T4 bacteriophage particles. Fluorescence of mithramycin- or DAPI-stained DNA is proportional to DNA quantity when DNA of the same has composition is compared. Quantitation does not appear to be affected discernably by the state of the DNA, whether in different stages of the cell cycle, in condensed chromosomes, or in noncycling, differentiated nuclei. The use of chicken red blood cells is recommended as an internal monitor for variations in staining conditions.

Amidines↗