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PubMed1990

Toxic injury from mercuric chloride in rat hepatocytes.

The relationship between cytosolic free Ca2+, mitochondrial membrane potential, ATP depletion, pyridine nucleotide fluorescence, cell surface blebbing, and cell death was evaluated in rat hepatocytes exposed to HgCl2. In cell suspensions, 50 microM HgCl2 oxidized pyridine nucleotides between 1/2 and 2 min, caused ATP depletion between 2 and 5 min, and produced an 89% loss of cell viability after 20 min. Rates of cell killing were identical in high (1.2 mM) and low (2.6 microM) Ca2+ buffers. Cytosolic free Ca2+ was determined in 1-day cultured hepatocytes by ratio imaging of Fura-2 employing multiparameter digitized video microscopy. In high Ca2+ medium, HgCl2 caused a 3-4-fold increase of free Ca2+ beginning after 6-7 min, but free Ca2+ did not change in low Ca2+ medium. Bleb formation occurred after about 4-5 min in both buffers prior to any increase of free Ca2+. Subsequently, in high Ca2+ medium, blebs became hot spots of free Ca2+ (greater than 600 nM). After about 2 min of exposure to HgCl2, rhodamine 123 fluorescence redistributed from mitochondrial to cytosolic compartments signifying collapse of the mitochondrial membrane potential. The results taken together demonstrate that bleb formation, ATP depletion, and the onset of cell death are not dependent on an increase of cytosolic free Ca2+. HgCl2 toxicity appears to be a consequence of inhibition of oxidative phosphorylation leading to ATP depletion and cell death.

Adenosine Triphosphate↗
PubMed1990

Ethanol inhibition of thrombus formation on collagen-coated glass.

Epi-fluorescent video microscopy was used to evaluate the effect of ethanol on platelet mural thrombus formation. Whole blood, treated with ethanol, was perfused over collagen coated glass in a parallel-plate flow chamber at a shear rate of 1,000/s. Digital image processing and photodiode measurements were used to analyze the dynamics of thrombus growth on this surface. Ethanol concentrations as low as 0.02% v/v were found to inhibit 45 +/- 23% (+/- S.D.) of normal platelet accumulation on the slide while 0.2% v/v ethanol effected an 82 +/- 15% inhibition of mural thrombus formation. While platelet adhesion to the collagen surface appeared unaffected by ethanol concentrations up to 0.1% v/v, 0.2% v/v ethanol had an effect on adhesion as well as aggregation. These results imply that low ethanol concentrations inhibit the formation of mural thrombi in a model of a damaged blood vessel at physiological shear rates. This inhibition would not be detected in systems which measure bulk aggregation, e. g. in aggregometric determinations.

Collagen↗
PubMed2004

On the acoustic vaporization of micrometer-sized droplets.

This paper examines the vaporization of individual dodecafluoropentane droplets by the application of single ultrasonic tone bursts. High speed video microscopy was used to monitor droplets in a flow tube, while a focused, single element transducer operating at 3, 4, or 10 MHz was aimed at the intersection of the acoustical and optical beams. A highly dilute droplet emulsion was injected, and individual droplets were positioned in the two foci. Phase transitions of droplets were produced by rarefactional pressures as low as 4 MPa at 3 MHz using single, 3.25 micros tone bursts. During acoustic irradiation, droplets showed dipole-type oscillations along the acoustic axis (average amplitude 1.3 microm, independent of droplet diameter which ranged from 5 to 27 microm). The onset of vaporization was monitored as either spot-like, within the droplet, or homogeneous, throughout the droplet's imaged cross section. Spot-like centers of nucleation were observed solely along the axis lying parallel to the direction of oscillation and centered on the droplet. Smaller droplets required more acoustic intensity for vaporization than larger droplets, which is consistent with other experiments on emulsions.

Acoustics↗
PubMed1989

Localized heterogeneity of red cell velocity in skeletal muscle at rest and after contraction.

Using intravital video microscopy, the present study focussed on a detailed analysis of Vrbc heterogeneity in a 2.4 x 1.8 x 0.15 surface volume of a frog sartorius muscle, before and after supramaximal contraction. Heterogeneity of Vrbc was evaluated (1) for an entire population of capillaries seen in this volume, (2) for a series of optical cross-sections, (3) along a series of longitudinal muscle strips, and (4) in terms of an asymmetry ratio for pairs of concurrent capillaries surrounding a muscle fibre. All four types of analysis showed an increased Vrbc homogeneity after contraction. Velocities became more homogeneous along rather than across muscle fibres. The mean asymmetry ratio became significantly larger during post-contraction hyperemia suggesting that each fibre receives a more uniform blood supply that will contribute to an improved exchange of materials across the capillary wall. The analysis of localized Vrbc heterogeneity provides new means of pinpointing the sources of perfusion heterogeneity. It enables, therefore, a specific experimental intervention that is aimed at an improved perfusion under both normal and abnormal conditions.

Animals↗
PubMed1990

Rapid changes in the distribution of GAP-43 correlate with the expression of neuronal polarity during normal development and under experimental conditions.

Hippocampal neurons growing in culture initially extend several, short minor processes that have the potential to become either axons or dendrites. The first expression of polarity occurs when one of these minor processes begins to elongate rapidly, becoming the axon. Before axonal outgrowth, the growth-associated protein GAP-43 is distributed equally among the growth cones of the minor processes; it is preferentially concentrated in the axonal growth cone once polarity has been established (Goslin, K., D. Schreyer, J. Skene, and G. Banker. 1990. J. Neurosci. 10:588-602). To determine when the selective segregation of GAP-43 begins, we followed individual cells by video microscopy, fixed them as soon as the axon could be distinguished, and localized GAP-43 by immunofluorescence microscopy. Individual minor processes acquired axonal growth characteristics within a period of 30-60 min, and GAP-43 became selectively concentrated to the growth cones of these processes with an equally rapid time course. We also examined changes in the distribution of GAP-43 after transection of the axon. After an axonal transection that is distant from the soma, neuronal polarity is maintained, and the original axon begins to regrow almost immediately. In such cases, GAP-43 became selectively concentrated in the new axonal growth cone within 12-30 min. In contrast, when the axon is transected close to the soma, polarity is lost; the original axon rarely regrows, and there is a significant delay before a new axon emerges. Under these circumstances, GAP-43 accumulated in the new growth cone much more slowly, suggesting that its ongoing selective routing to the axon had been disrupted by the transection. These results demonstrate that the selective segregation of GAP-43 to the growth cone of a single process is closely correlated with the acquisition of axonal growth characteristics and, hence, with the expression of polarity.

Animals↗
PubMed1996

Synchronous oscillations in cerebrocortical capillary red blood cell velocity after nitric oxide synthase inhibition.

Low-frequency (4-12 cpm) spontaneous oscillations in cerebral blood flow are well known and their augmentation after nitric oxide synthase inhibition has recently been described. However, the presence of these oscillations in blood flow velocity in the capillary network of the brain has not been demonstrated. In this paper, low-frequency oscillations in red blood cell flow velocity in cortical capillaries using intravital video microscopy were studied before and after infusion of the nitric oxide synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME). Fluorescein isothiocyanate-labeled red blood cells were injected intravenously and served as markers of capillary flow. Red cell velocity was measured by off-line image tracking. After infusion of L-NAME (30 mg/kg), red cell velocity decreased from an average of 1.0 +/- 0.1 mm/sec to 0.53 +/- 0.1 mm/sec. Simultaneously, low-frequency oscillations in velocity emerged as indicated by an 81 +/- 17% increase in standard deviation of the 4- to 8-cpm frequency components. There was a significant temporal correlation (r = 0.58, P < 0.01) in red cell velocity between neighboring capillaries after L-NAME. Principal component analysis suggested that the high temporal correlation was a consequence of low-frequency oscillations rather than phase. These results are consistent with a model in which low-frequency spontaneous oscillations in flow velocity are brought about by precapillary vasomotion. This study provides for the first time direct evidence for low-frequency synchronous oscillations of red cell flow velocity in the cerebral capillary network.

Animals↗
PubMed1994

Borrelia burgdorferi swims with a planar waveform similar to that of eukaryotic flagella.

Borrelia burgdorferi is a motile spirochete with multiple internal periplasmic flagella (PFs) attached near each end of the cell cylinder; these PFs overlap in the cell center. We analyzed the shape and motion of wild type and PF-deficient mutants using both photomicrography and video microscopy. We found that swimming cells resembled the dynamic movements of eukaryotic flagella. In contrast to helically shaped spirochetes, which propagate spiral waves, translating B. burgdorferi swam with a planar waveform with occasional axial twists; waves had a peak-to-peak amplitude of 0.85 micron and a wavelength of 3.19 microns. Planar waves began full-sized at the anterior end and propagated toward the back end of the cell. Concomitantly, these waves gyrated counter-clockwise as viewed from the posterior end along the cell axis. In nontranslating cells, wave propagation ceased. Either the waveform of nontranslating cells resembled the translating form, or the cells became markedly contorted. Cells of the PF-deficient mutant isolated by Sadziene et al. [Sadziene, A., Thomas, D. D., Bundoc, V. G., Holt, S. C. & Barbour, A. G. (1991) J. Clin. Invest. 88, 82-92] were found to be relatively straight. The results suggest that the shape of B. burgdorferi is dictated by interactions between the cell body and the PFs. In addition, the PFs from opposite ends of the cell are believed to interact with one another so that during the markedly distorted nontranslational form, the PFs from opposite ends rotate in opposing directions around one another, causing the cell to bend.

Borrelia burgdorferi Group↗
PubMed1994

Cell movement and shape are non-random and determined by intracellular, oscillatory rotating waves in Dictyostelium amoebae.

We present evidence for a mechanism of eukaryotic cell movement. The pseudopodial dynamics and shape of Dictyostelium discoideum amoebae were investigated using computer-supported video microscopy. An examination of the cell periphery by the novel method of serial circular maps revealed explicit, classical wave patterns, which indicate the existence of intrinsic intracellular oscillations. The patterns are generated by the transit of self-organized, super-positioned, harmonic modes of rotating oscillatory waves (ROWS). These waves are probably associated with the dynamics of intracellular actin polymerisation and depolymerisation. A Karhunen-Loève expansion was conducted on one cell during 10 min of locomotion using points each 10 degrees around the cell's boundary. The results show that only 2-3 modes are necessary to describe the most essential features of cell movement and shape. Based on this analysis, a wave model was developed, which accurately simulates the dynamics of cell movement and shape during this time. The model was tested by reconstructing the cell's dynamical form by means of the Karhunen-Loève transform. No difference was detected between this reconstruction and the actual cell outline. Although cell movement and shape have hitherto been viewed as random, our results demonstrate that ROWS determine the spatio-temporal expression of pseudopodia, and consequently govern cell shape and movement, non-randomly.

Animals↗
PubMed1992

Intracellular cyclic AMP produces effects opposite to those of cyclic GMP and calcium on shape and motility of neuroblastoma cells.

We have directly evaluated the effects of various intracellular second messengers including cyclic nucleotides, calcium ion, and inositol polyphosphates on shape and motility of differentiating mouse neuroblastoma cells. The messengers were microinjected into cells and the responses of the soma, neurite, and growth cone were monitored using time-lapse video microscopy. Each messenger altered cell shape and motility in a characteristic manner. Cyclic AMP promoted lamellipodial expansion, neurite outgrowth, and motility. The other injected messengers opposed motility. Cyclic GMP caused motile structures to freeze and to retract permanently, while the inhibitory effects of calcium injection were concentration-dependent. Small calcium injections affected specifically actin-containing motile structures which froze and retracted temporarily. Intermediate calcium injections caused a strong contraction at the site of injection in all cells. With large injections, cells retracted long neurites, rounded up, and frequently began vigorous blebbing that continued to cell death. Injections of the inositol polyphosphates IP3(1,4,5) and IP4(1,4,5,6) mimicked the effects of small calcium injections, as did electrical stimulation that elicited action potentials. The results suggest that in mouse neuroblastoma cells, intracellular cAMP elevation increases cytoskeletal organization and promotes neurite extension perhaps through an enhancement of cell-substratum adhesion. On the other hand, a rise of intracellular cGMP or intracellular calcium interferes directly with the function and organization of the actin-microfilament system. The integrated action of these second messenger systems may, therefore, operate in vivo to allow substances released from neighboring cells to regulate neuronal architecture.

Bucladesine↗
PubMed1988

Three-dimensional localization and redistribution of F-actin in higher plant mitosis and cell plate formation.

The distribution of F-actin cables in dividing endosperm cells of a higher plant, Haemanthus, was visualized with the immunogold-silver-enhanced method and compared with the arrangement of immunogold-stained microtubules in the same cells. The three-dimensional distribution of F-actin cables and microtubules during mitosis and cell plate formation was analyzed using ultrathin optical sectioning of whole mounts in polarized light video microscopy. F-actin cables form a loose irregular network in the interphase cytoplasm. Much of this network remains outside of the spindle during mitosis. A few F-actin cables were detected within the spindle. Their pronounced rearrangement during mitosis appears to be related to the presence and growth of microtubule arrays. During prometaphase, actin cables located on the spindle surface and those present within the spindle tend to arrange parallel to the long axis of the spindle. Cables outside the spindle do not reorient, except those at the polar region, where they appear to be compressed by the elongating spindle. Beginning with mid-anaphase, shorter actin cables oriented in various directions accumulate at the equator. Some of them are incorporated into the phragmoplast and cell plate and are gradually fragmented as the cell plate is formed and ages. Actin cables adjacent to microtubule arrays often show a regular punctate staining pattern. Such a pattern is seldom observed in the peripheral cytoplasm, which contains few microtubules. The rearrangement of F-actin cables mimicks the behavior of spindle inclusions, such as starch grains, mitochondria, etc., implying that F-actin is redistributed passively by microtubule growth or microtubule-related transport. Thus F-actin or actomyosin-based motility does not appear to be directly involved in mitosis and cytokinesis in higher plants.

Actins↗
PubMed1988

Flagellar quiescence and transience of inactivation induced by rapid pH drop.

The effects of rapid pH drop on the flagellar movement of reactivated sea urchin sperm were studied by video microscopy and by a newly developed pH jump method. Triton-demembranated sperm were reactivated in a thin layer of the reactivation medium containing ATP and potassium acetate and supported by a ring-shaped Millipore filter stuck to the lower surface of a supported coverslip. The pH of the medium was lowered rapidly by dissolving acetic acid vapor abruptly introduced into a gap between the cover and slide. Flagellar beating ceased immediately when the pH of the reactivation medium was lowered. At least two types of cessation were distinguished: 1) "instantaneous" cessation in a bent form closely resembling those characteristic of steady-state beating before pH drop (waveform freeze), and 2) flagellar quiescence in a cane-shaped form resembling those characteristic of Ca-induced quiescence (cane-shaped quiescence). The flagellum again began beating if the pH was raised to normal but eventually was disintegrated by tubule sliding if the pH was left lowered. Field-by-field analysis of the transient movement of flagella becoming quiescent upon pH drop demonstrated that the proximal bend of the cane-shaped form corresponded to the principal bend of the steady-state beating in some flagella, but in others, to the reverse bend. These observations indicate that low pHs affect flagellar beating by interfering with sliding-bending conversion by a mechanism different from that previously reported.

Animals↗
PubMed1989

Growth cone-growth cone interactions in cultures of rat sympathetic neurons.

Growth cones of sympathetic neurons from the superior cervical ganglia of neonatal rats were studied using video-microscopy to determine events following contact between growth cones and other cell surfaces, including other growth cones and neurites. A variety of behaviors were observed to occur upon contact between growth cones. Most commonly, one growth cone would collapse and subsequently retract upon establishing filopodial contact with the growth cone of another sympathetic neuron. Contacts resulting in collapse and retraction were often accompanied by a rapid and transient burst of lamellipodial activity along the neurite 30-50 microns proximal to the retracting growth cone. In no instances did interactions between growth cones and either fibroblasts or red blood cells result in the growth cone collapsing, suggesting that a specific recognition event was involved. On several occasions, growth cones were seen to track other growth cones, although fasciculation was rare. In some cases, there was no obvious response between contacting growth cones. Growth cone-growth cone contact was almost four times more likely to result in collapse and retraction than was growth cone-neurite contact (45% vs 12%, respectively). These observations suggest that the superior cervical ganglion may be composed of neurons with different cell surface determinants and that these determinants are more concentrated on the surface of growth cones than on neurites. These results further suggest that contact-mediated inhibition of growth cone locomotion may play an important role in growth cone guidance.

Animals↗
PubMed1992

Secretion from rat neurohypophysial nerve terminals (neurosecretosomes) rapidly inactivates despite continued elevation of intracellular Ca2+.

Cytoplasmic calcium concentration was measured in neurosecretory nerve terminals (neurosecretosomes) isolated from rat neurohypophyses by fura-2 fluorescence measurements and digital video microscopy. Hormone release and cytoplasmic calcium concentration were measured during depolarizations induced by elevated extracellular potassium concentration. During prolonged depolarizations with 55 mM [K+]o, the cytoplasmic calcium concentration remained elevated as long as depolarization persisted, while secretion inactivated after the initial sharp rise. The amplitude and duration of the increase in [Ca2+]i was dependent on the degree of depolarization such that upon low levels of depolarizations (12.5 mM or 25 mM [K+]o), the calcium responses were smaller and relatively transient, and with higher levels of depolarization (55 mM [K+]o) the responses were sustained and were higher in amplitude. Responses to low levels of depolarization were less sensitive to the dihydropyridine calcium channel blocker, nimodipine, while the increase in [Ca2+]i induced by 55 mM [K+]o became transient, and was significantly smaller. These observations suggest that these peptidergic nerve terminals possess at least two different types of voltage-gated calcium channels. Removal of extracellular sodium resulted in a significant increase in [Ca2+]i and secretion in the absence of depolarizing stimulus, suggesting that sodium-calcium exchange mechanism is operative in these nerve terminals. Although the [Ca2+]i increase was of similar magnitude to the depolarization-induced changes, the resultant secretion was 10-fold lower, but the rate of inactivation of secretion, however, was comparable.

Animals↗
PubMed1989

Single-cell analysis of Ca++ changes in human lung mast cells: graded vs. all-or-nothing elevations after IgE-mediated stimulation.

Human lung mast cells were examined by digital video microscopy for changes in cytosolic free ionized calcium [( Ca++]i) after stimulation with anti-IgE antibody or specific antigens. These studies sought to determine whether the mast cell response resembled a graded or an all-or-nothing process. Preliminary experiments indicated that labeling mast cells with fura-2 did not alter their response to IgE-mediated stimulation. Subsequent experiments established that an IgE-mediated stimulus evoked an elevation of [Ca++]i from a baseline value of 85 nM to an average of 190 nM (range 60-450 nM, n = 23), with an average histamine release of 26%. There was a good correlation (Rs = 0.67) between the average net [Ca++]i change and the subsequent histamine release (regression equation: %HR = 0.189[net(Ca)-52]). [Ca++]i elevations were found to precede histamine release (t1/2 for [Ca++]i of 35 s vs. t1/2 for histamine release of 110 s). Single-cell analysis found that even for very low values of histamine release, nearly all cells demonstrated a [Ca++]i response. However, this response was markedly heterogeneous, ranging from no response to responses two to three times the mean. Comparative studies of mast cells stimulated under optimal and suboptimal conditions established that there was a graded [Ca++]i response dependent on the strength of the stimulus. An all-or-nothing reaction for the [Ca++]i response was ruled out.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1993

Spatial relationship between leukocyte accumulation and microvascular injury during reperfusion following hepatic ischemia.

In order to further elucidate the possible contribution of leukocytes to microvascular injury during reperfusion following total hepatic ischemia, we studied the spatial relationship between areas of white cell accumulation and areas of microvascular damage in the rat liver in vivo. No-flow hepatic ischemia was produced for 90 min in vivo and during the ensuing reperfusion phase (I/R) leukocyte accumulation, absolute number of perfused sinusoids per unit area, and red blood cell velocity were quantitated using in vivo epi-fluorescence video microscopy. The total number of stationary leukocytes in the liver during reperfusion was found to be significantly elevated following ischemia compared to time-matched sham-operated controls. In addition, by 2 hr of reperfusion, approximately 80% of the leukocytes in the I/R group were extravascular compared to only about 50% in the controls. When leukocyte accumulation and microhemodynamics were expressed on the basis of whole liver, the increased accumulation of leukocytes was associated with decreased microvascular perfusion as indicated by decreased number of sinusoids perfused and decreased red blood cell velocity. However, when the data were analyzed on the basis of .05mm2 microscopic fields on the surface of the liver, there was no difference in leukocyte accumulation in areas with sinusoidal blood flow compared to areas that were devoid of perfused sinusoids. Moreover, in a correlation analysis of number of adherent leukocytes/microscopic field vs red blood cell velocity in perfused sinusoids in that field, only a very small negative correlation between leukocytes/field and red blood cell velocity was found (r = -.23, p < .05). These results demonstrate that at the whole organ level leukocyte accumulation appears to correlate well with microvascular damage; however, this increase in whole liver accumulation of leukocytes does not necessarily reflect accumulation at sites of overt microvascular damage. Thus, leukocyte-independent factors are likely to be of considerable quantitative importance in microvascular injury during reperfusion following hepatic ischemia.

Animals↗
PubMed1991

Heterogeneity of microvascular flow in rat skeletal muscle is reduced by contraction and by hemodilution.

The objective was to test the hypothesis that heterogeneity of microvascular perfusion in a mammalian muscle will decrease with increased flow. We used the extensor digitorum longus muscle in rats to quantify, via intravital video microscopy, (1) the spatial distribution of red cell velocity, VRBC, and (2) density of capillaries with moving and stationary red cells (CDPER, CDSTAT) within a large capillary bed (1.05 x 0.78 mm) at the muscle surface. From the VRBC distribution, the coefficient of variation (CV = SD/mean) was used as an index of VRBC heterogeneity. The flow was increased by two procedures: (1) 1 min supramaximal muscle contraction, and (2) hematocrit reduction from 49 to 21% by isovolemic hemodilution with a 6% PVP-40 (polyvinylpyrrolidone) solution. Among 5 rats studied, the post-contraction and post-hemodilution mean VRBC's were significantly larger than the resting VRBC (1.08 and 0.17 vs. 0.11 mm/s). The corresponding CV's were significantly lower (33 and 49 vs. 60%). The percentages of capillaries with low velocity (0-0.1 mm/s) were also significantly lower (0 and 27 vs. 52%). CDPER values did not differ from the resting level (30.8 cap/mm of test line) but CDSTAT was significantly smaller after contraction (0.8 vs. 4.9 cap/mm). The present data demonstrate that heterogeneity of VRBC in rat skeletal muscle decreases after contraction and after hemodilution. The study shows that hemodilution has a major effect on improving flow in a population of poorly perfused capillaries. Since heterogeneity also decreased after contraction in an amphibian muscle (Tyml, Microvasc. Res. 32: 84-98, 1986), the study supports the view that heterogeneity, in general, is a microcirculatory parameter that reflects the vascular network response to a given tissue stimulus.

Animals↗
PubMed1991

Inhibition of leukocyte rolling in venules by protamine and sulfated polysaccharides.

Intravital video microscopy was used to investigate leukocyte margination in 80 mesenteric venules (19 to 54 microns) of 50 anesthetized rabbits. After intravenous (IV) bolus injection, sulfated polysaccharides reduced in a reversible and dose-dependent way the number of leukocytes rolling slowly along the venular wall. The presence of sulfate groups is essential because other negatively charged or neutral polysaccharides had no effect. It was not caused by an increase in RBC velocity or chelation of divalent cations. Inhibition by sulfated dextrans (n = 7) was independent of molecular weight (mol wt 13,000 to 500,000) but was influenced by the average number of sulfate groups per monosaccharide. With substitution 0.13, the 90%-inhibition dose was 104 mg/kg, with 0.7 it was 56 mg/kg, and between substitution 1 and 2 it ranged from 20 to 23 mg/kg. At 100 mg/kg, plasma concentration was 0.6 to 0.7 mg/mL. Xylan sulfate (mol wt 6,000, substitution 1.8) gave 90% inhibition at 11 mg/kg, and heparin gave 90% inhibition at 97 mg/kg. Duration of inhibition (0.5 to 2 hours) depended on mol wt and appeared to be related to plasma clearance. Because protamine also inhibited rolling (12 mg/kg; less than 10 minutes), we propose that repetitive formation and breakup of ionic bonds between sulfate groups and positively charged amino acids is involved in leukocyte rolling. During inhibition of rolling, systemic lymphocyte/monocyte levels appeared to increase. Granulocyte counts did not change, indicating that rolling is not the main mechanism responsible for the marginal granulocyte pool.

Animals↗
PubMed2003

Non-invasive imaging of blood cell concentration and blood distribution in zebrafish Danio rerio incubated in hypoxic conditions in vivo.

This is the first study to use a combination of digital imaging techniques and vital video microscopy to study hypoxia-induced changes in blood cell concentration, angiogenesis and blood redistribution in entire animals. Zebrafish Danio rerio, which are known to be independent of convective oxygen transport until about 2 weeks post-fertilization, were raised under chronic hypoxia (P(O(2))=8.7 kPa) starting at 1 day after fertilization (d.p.f.) until 15 d.p.f. In control animals, the concentration of red cells (i.e. the number of red cells per nl blood) remained constant until 7 d.p.f., and than decreased by approximately 70% until 15 d.p.f. In hypoxic animals, however, the concentration of red cells remained significantly elevated compared to control animals at 12 and 15 d.p.f. Assuming that the hemoglobin content of the red cells is similar, hypoxic animals have a higher oxygen carrying capacity in their blood. Red cell distribution within the various parts of the circulatory system, taken as an indicator for blood distribution, revealed a significant modification in the number of blood cells perfusing the organs in hypoxic animals. At 12 d.p.f., gut perfusion was reduced by almost 50% in hypoxic animals, while perfusion of the segmental muscle tissue was increased to 350% of control values. No significant changes in brain perfusion were observed under these conditions. At 15 d.p.f., the reduction in gut perfusion was abolished, although muscle perfusion was still significantly elevated. At this time, growth of hypoxic animals was less compared to control animals, revealing that hypoxia had become deleterious for further development. The vascular bed of various organs was not obviously different in hypoxic animals compared to normoxic animals.

Analysis of Variance↗