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PubMed1985

Distribution and axial diffusion of retinol in bleached rod outer segments of frogs (Rana pipiens).

Isolated retinas and rod outer segments from frogs (Rana pipiens) were exposed to light that produced axially uniform total bleaches of rhodopsin. Using fluorescence video microscopy, it was shown that the formation and equilibrium distribution of all-trans-retinol, the final chromophore product of rhodopsin bleaching is axially uniform. This result shows that the rate and amount of oxidoreductase-mediated reduction of all-trans-retinal to all-trans-retinol is not affected by the relative age of the disk membranes to which the enzymes are bound. Therefore previously reported axial differences in regeneration of rhodopsin and recovery of photocurrent after exposure to bright light probably are not due to axial differences in the formation of rhodopsin photoproducts. In addition, measurements on individual rod cells show that there is no significant redistribution of retinol for up to 2 hr following localized partial bleaches of rhodopsin. This raises the perplexing question of how retinol is shuttled between disk membranes and the pigment epithelium during visual pigment regeneration following substantial bleaches.

Animals↗
PubMed1992

Sabeluzole, a memory-enhancing molecule, increases fast axonal transport in neuronal cell cultures.

Morphological rearrangements, such as synapse number changes, have been observed in the adult mammalian brain after various experimental paradigms of learning and behavioral experience. The role of axonal transport in the physical translocation of material during this form of brain plasticity has not been fully appreciated. We show here by quantitative video microscopy that sabeluzole (R58735), a new memory-enhancing drug in humans, effectively increases fast axonal transport in rat neuronal cell cultures. Long-term incubation (24 hr) with sabeluzole in the concentration range between 0.1 and 1 microM increases both velocity and jump length of saltatory movements maximally by 20-30% in embryonic hippocampal neurons. Acute treatment only increases the velocity by 15-20%. Furthermore, the inhibition of axonal transport by 0.1 mM vanadate in N4 neuroblastoma cells is reversed by 1 microM sabeluzole. Observations on the kinesin-induced microtubule mobility in a reconstituted system show a 10% enhancement by sabeluzole at an optimal concentration of 2 microM, but no increase in kinesin ATPase activity. To our knowledge, this is the first pharmacological compound shown to increase fast axonal transport. The mechanism of fast axonal transport enhancement is discussed as a rationale for new therapeutic treatment in neuropathology.

Animals↗
PubMed1992

Regeneration in the auditory system.

The auditory organs of birds and mammals normally stop producing sensory hair cells during embryonic development, so loss of those cells later in life results in hearing deficits that have been considered irreversible. In contrast to this, the ears of some fish and amphibians produce hair cells continuously throughout life and even increase in sensitivity. The lateral line organs in the skin of fish and aquatic amphibians also contain hair cells and have long been known to be replaceable through regeneration. Recently, it was discovered that after acoustic trauma or antibiotic poisoning, injured hair cells in the mature auditory organs of birds also could be replaced through regeneration. This is especially notable because it occurs in populations of cells that are mitotically quiescent in undamaged ears. More recent investigations have focused on identifying the cells that give rise to new hair cells during regeneration. In the lateral line organs of salamanders, time-lapse video microscopy has revealed that surviving supporting cells divide to give rise to progeny that can differentiate either as hair cells or as supporting cells. Definitive identification of the progenitors of regenerated hair cells in the avian cochlea awaits further investigation, but evidence that points to two possible candidate cell types is discussed.

Animals↗
PubMed1992

Magnesium deficiency in vitro enhances free radical-induced intracellular oxidation and cytotoxicity in endothelial cells.

The effect of magnesium (Mg)-deficient culture on endothelial cell susceptibility to oxidative stress was examined. Bovine endothelial cells were cultured in either control sufficient (0.8 mM) or deficient (0.4 mM) levels of MgCl2. Oxygen radicals were produced extracellularly by the addition of dihydroxyfumarate and Fe(3+)-ADP. Isolated Mg-deficient endothelial cells produced 2- to 3-fold higher levels of thiobarbituric acid (TBA)-reactive materials when incubated with this free radical system. Additional studies were performed using digitized video microscopy and 2',7'-dichlorofluorescein diacetate (DCFDA) as an intracellular indicator for oxidative events at the single cell level. In response to the exogenous oxidative stress, endothelial cells exhibited a time-dependent increase in fluorescence, suggestive of intracellular lipid peroxidation. The increase in cellular fluorescence began within 1 min of free radical addition; the Mg-deficient cells exhibited a more rapid increase in fluorescence than that of Mg-sufficient cells. In separate experiments, cellular viability was assessed using the Trypan blue exclusion assay. Mg deficiency increased cytotoxicity of the added oxyradicals, but the loss of cellular viability began to occur only after 15 min of free radical exposure, lagging behind the detection of intracellular oxidation products. These results suggest that increased oxidative endothelial cell injury may contribute to vascular injury during Mg deficiency.

Adenosine Diphosphate↗
PubMed1993

Analysis of lymphocyte aggregation using digital image analysis.

We present the development and testing of a novel assay of lymphocyte adhesion based on time-resolved morphological measurements of intercellular aggregation. Homotypic lymphocyte aggregation is induced according to various protocols and monitored for several hours using video microscopy and time-lapse recording. Digital images of the aggregating cell population are acquired and analyzed to obtain the size distribution and the shape of cell aggregates. By following the temporal evolution of the size distribution of aggregates, the rates of aggregation events can be accurately quantified and compared. In addition, an analysis of the two- and three-dimensional structures of the aggregates using appropriately defined shape factors allows comparisons of mechanical binding strengths and cytoskeletal activity. To demonstrate the capabilities of the assay, we present results from a series of aggregation experiments with Jurkat cells treated with 33B6, 19H8, IC9, and 20E4 monoclonal antibodies. These monoclonal antibodies bind to various epitopes of known adhesion molecules and induce aggregation phenomena that proceed at different rates. Our results show that the assay has small repeatability error and is sensitive enough to compare aggregation events induced through distinct molecular epitopes. Used in conjunction with current biochemical detection assays and adhesion pathway modulation experiments, the developed assay will facilitate the study of cellular adhesion and aggregation mechanisms.

Antibodies, Monoclonal↗
PubMed1995

Cytokine-stimulated chemotaxis of human neutrophils in a 3-D conjoined fibrin gel assay.

The ability of neutrophils to migrate through three-dimensional (3-D) tissues in response to chemical stimuli is critical to their host defense function. However, studies characterizing stimulated migration in vitro have been largely limited to two-dimensional (2-D) surfaces. In this study, we have employed direct observation methods to quantify human neutrophil migration in 3-D fibrin gel using time-lapse video microscopy and automated cell tracking methods. A novel 3-D conjoined gel assay was developed to establish experimentally quantifiable and theoretically predictable diffusion gradients of chemotactic factors. This assay was used to measure objective migration parameters, namely the random motility and chemotaxis coefficients, in response to the cytokine, interleukin-8 (IL-8). The random motility coefficient, mu, showed a biphasic dependence on IL-8 concentration with a maximum of 1.1 x 10(-8) cm2/s at 5 x 10(-8) M IL-8; no significant motility was observed in the absence of IL-8. We further established the dependence of cell orientation bias, phi, on the concentration and gradient steepness (i.e., specific gradient, SG) of IL-8. Results indicate that phi increases with increasing SG, provided the concentration is maintained sufficiently low, which we conjecture to result from minimizing IL-8 receptor down-regulation. The chemotaxis coefficient, chi, was maximum at an intermediate SG for both IL-8 concentrations studied. We also examined the applicability of this assay to estimate mu and chi from indirect measurements of chemotaxis, namely the simpler measurement of cell redistribution after a prescribed incubation time, as opposed to direct cell tracking measurements. By virtue of measuring chi, this is the first quantitatively objective study of mammalian cell chemotaxis in a physiologically relevant 3-D gel and, in particular, of neutrophil chemotaxis on any substratum in response to the physiologically relevant chemotactic factor, IL-8.

Chemotactic Factors↗
PubMed1994

Increased sensitivity of diagnostic latex agglutination tests in an ultrasonic standing wave field.

A technique is described which increases the sensitivity of latex agglutination tests for soluble and particulate antigens. The levels of detection of tests for C-reactive protein and E. coli O157 respectively have been improved by x256 and x1024 compared with the standard test procedure of sample rotation on a test-card. This new method combines dilution of the test latex particles, a 2 min sample treatment in the ultrasonic standing wave field of a tubular piezo-electric transducer and subsequent examination by video-microscopy. Ultrasonic treatment is required to achieve increased localised concentrations of the latex particles in the standing wave field, and dilution of the latex is a critical requirement to allow agglutination to occur at low antigen concentrations.

Antigens, Bacterial↗
PubMed1982

Time parameters of contraction of inotropically responding cultured heart cells in relation to cellular cyclic AMP levels.

Television video microscopy combined with photoelectric recording was used to determine the influence of a number of positive inotropic agents on the amplitude (peak height) and the course of the contraction of electrically paced myocytes in 4-day monolayer cultures derived from the heart ventricles of 1 to 2-day old rats. Cyclic AMP was determined in parallel cultures of the same cell population. Reductions in time to 90% of peak height, 90% of relaxation time, and duration of contraction caused by peak height-augmenting concentrations of isoproterenol, epinephrine, dibutyryl cyclic AMP, and 1-methyl-3-isobutylxanthine, but not of theophylline, correlated with rises in cellular cyclic AMP levels. Ouabain, a rise in extracellular CaCl2, and, in some experiments, phenylephrine in the presence of propranolol increased peak height, but did not change time to 90% of peak height, 90% of relaxation time, duration of contraction, and cyclic AMP content. These responses are compared to those observed by other authors in intact cardiac muscle and are discussed in the light of evidence linking increased myocardial cyclic AMP levels with an abbreviation of systole.

1-Methyl-3-isobutylxanthine↗
PubMed1987

Anatomic and hemodynamic characteristics of the blood vessels feeding the cremaster skeletal muscle in the rat.

The anatomic arrangement, pressure distribution, and resting vascular tone of the feed arteries located upstream from the rat cremaster microcirculation were determined to characterize the sites of the vascular resistance in this macrovessel segment of the cremaster circulation. The cremaster microcirculation and its feeding arteries were studied using an intravital video microscopy system. Vascular diameters and pressures were measured with an image shearing monitor and servo-null micropipet system, respectively. The central arteriole of the cremaster muscle was found to be a distal segment of the external spermatic artery which branched from the pudic-epigastric artery that in turn arose from the common iliac artery. Together the length of these vessels, from the aorta to the cremaster muscle, was 37 mm and they accounted for 42% of the total pressure drop across the cremaster vascular network. The largest pressure drop (31 mm Hg) upstream from the cremaster occurred across the external spermatic artery which was also the longest (17.7 mm) feed vessel. Topical application of adenosine (1 X 10(-3) M) significantly dilated the pudic-epigastric artery and the external spermatic artery, indicating that these vessels had significant tone. In summary, our data indicate that the large fraction of network vascular resistance located in the feed vessels upstream from the cremaster is the result of both architectural features and vascular tone.

Adenosine↗
PubMed1987

Responses of sequentially branching macro- and microvessels during reactive hyperemia in skeletal muscle.

Small artery and microvascular responses during reactive hyperemia were compared to determine which resistance-bearing vessels played a role in controlling blood flow and resistance for the cremaster skeletal muscle. Using an intravital video microscopy system, measurements of microvessel pressure, flow velocity, and diameter were obtained from cremaster muscles in anesthetized rats. These were compared with measurements of diameter that were obtained from the small arteries feeding the cremaster muscle. After a 60-sec occlusion of the sacral aorta, total cremaster blood flow increased approximately 28% and calculated microvascular resistance for the cremaster muscle fell 50%. During the period of occlusion, diameters of small arteries (159-292 micron) decreased despite the presence of smooth muscle tone. Likewise, the diameters of large arterioles (65-117 micron) decreased whereas small arterioles (16-30 micron) dilated. The decrease in diameter of the small arteries and large arterioles was accompanied by a significant fall in intravascular pressure, suggesting that the behavior of these vessels was largely passive. Immediately following the release of occlusion, small arteries and large arterioles returned to their control diameters while small arterioles remained in a dilated state for approximately 2 min. These results indicate that for the cremaster muscle, vascular responses vary along the length of the arterial tree during reactive hyperemia, small but not large arterioles are primarily responsible for the decrease in network resistance and subsequent hyperemia following occlusion, and the small feeder arteries did not dilate during reactive hyperemia but instead acted to set a limit on the decrease in network resistance and the increase in blood flow.

Animals↗
PubMed1988

Vascular isolation of the rat cremaster muscle.

The vascular supply to the rat cremaster muscle was completely isolated to provide a microvascular preparation with a controllable blood flow. The anatomy of the cremaster vascular supply and the surgical approach to isolate the cremaster muscle on its neurovascular pedicle are described. The functional integrity of this isolated cremaster preparation was tested using intravital video microscopy to evaluate the tissue's response to vasoactive agents and to peripheral nerve stimulation. The isolated cremaster muscle was positioned in situ in a controlled tissue bath and concentration response curves to the topical application of norepinephrine (NE) and acetylcholine (Ach) were determined. Vasoconstriction elicited by the topical application of NE or by stimulation of the genitofemoral nerve trunk was similar for both the isolated and standard cremaster preparations. Application of 10(-5) M Ach caused maximal vasodilation equal to that produced by 10(-3) M papaverine in both preparations. In summary, the isolated cremaster muscle of the rat is an acceptable model of a skeletal muscle microcirculation which can be used to investigate microvascular function when precise monitoring or control of perfusion to the entire muscle is needed.

Acetylcholine↗
PubMed1988

Vasomotion patterns in skeletal muscle arterioles during changes in arterial pressure.

The effects of stepwise reductions of arterial pressure on arteriolar diameter and on vasomotion patterns in the rabbit tenuissimus muscle were investigated in eight New Zealand White rabbits (0.8-1.2 kg) anesthetized with 20% urethane. The vasomotor activity of 13 bifurcations, where first-order terminal arterioles branch from a transverse arteriole, was recorded by video microscopy and related to the pressures in the femoral artery. The arterial pressure was lowered in steps by partial occlusion of the abdominal aorta. Changes of mean diameter due to reduced perfusion pressures were most pronounced in transverse arterioles. Dilation in terminal arterioles at reduced arterial pressures did not exceed resting condition vasomotion peak diameters. Regular vasomotion in the terminal arterioles was intercalated with periods of no vasoactivity which became progressively longer when perfusion pressure was reduced. The oscillation frequency was maintained during the periods with regular vasomotion. Vasomotion ceased in transverse and terminal arterioles at arterial pressures between 50 and 30 mm Hg. During reactive hyperemia vasomotion reappeared after 0.5 to 4 min with the original fundamental frequency. We postulate that vasomotion in terminal arterioles is due to a vascular pacemaker which acts as a local oscillator that can be influenced by perfusion pressure in an on/off-type fashion. The pacemaker oscillation frequency is constant and independent of myogenic factors.

Animals↗
PubMed1990

Microvascular response to ischemia, and tissue structure, in normal and atrophied skeletal muscle.

The objective of this study was to explain why the normally observed reactive hyperemia in frog sartorius muscle following ischemia is absent when this muscle atrophies. Two possibilities were addressed: (1) absence is due to lowered O2 consumption, making the muscle more tolerant to ischemia, and (2) absence is linked to impaired vascular function in atrophy. We used 10 frogs after 2-3 months and 8 frogs after 7-14 months of laboratory captivity. Animals in the latter group had a significantly lower sartorius muscle weight, i.e., 85 +/- 33 vs 24 +/- 11 SD mg. Using intravital video microscopy, we measured red cell velocity in capillaries at the muscle surface, and densities of capillaries with moving (NCPER) and stationary red cells (NCSTAT) before and after 30 min ischemia. Ischemia induced a significant temporary increase in overall velocity (from 0.10 to 0.27 mm/sec) in normal muscles, but no increase in atrophied muscles. It resulted in no difference in NCPER between the two groups (preischemic levels in both groups: 15.0 cap/mm of test line), but in a significant difference in NCSTAT (3.8 vs 11.5 cap/mm in atrophy). Using light and electron microscopy, we also measured structural and ultrastructural parameters in both groups. In atrophied muscles the mean fiber cross-sectional area was lower (568 vs 1935 microns 2) and anatomical capillary density higher (892 vs 282 cap/mm2) than in normal muscles. Mitochondrial volume density was not statistically different from the 1.5% level in the normal muscle, while the lipid droplet volume density was larger (2.33 vs 0.58%). The percentage of capillaries with damaged endothelium was larger (33.5 vs 12.6%). Using histology, the white cell volume density per capillary volume was also found to be larger in atrophy (1.96 vs 0.83%). From the discrepancy between the lack of intergroup difference in preischemic NCPER and the 3.2-fold difference in anatomical capillary density we estimate that about 60% of capillaries were perfused with red cells in atrophied muscles. Although the preischemic rate of perfusion in these capillaries was comparable between the two groups, the postischemic response was not: reactive hyperemia was absent in atrophy. Our mitochondrial and lipid volume density data do not support the possibility that this absence was due to lowered O2 consumption, as these densities did not decrease with atrophy.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Fluorescent imaging in vivo of developing blood vessels on the optic tectum of Xenopus laevis.

The growth and development of individual living capillaries, venules, and endothelial sprouts on the pial surface of the brain were examined with video microscopy and intravascular FITC-dextran in anesthetized tadpoles of pigment-deficient Xenopus laevis, stages 42-50. The fluorescent tracer, injected intracardially through glass micropipets, was well tolerated by the tadpoles and improved the visibility of vessels compared to transmitted light. Case histories of vascular development on the optic tectum confirmed the sprouting of new capillaries during angiogenesis. The caudal tectum and its vascular domains grew faster than the rostral, but the densities of caudal surface vessels were at least as high as rostral densities, indicating that angiogenesis was well matched to neural development. Internal capillary branches were further elaborated and pial venules increased in diameter in premetamorphic tadpoles and in Xenopus frogs.

Animals↗
PubMed1991

Neutrophil-derived oxidants promote leukocyte adherence in postcapillary venules.

The objective of this study was to determine whether hydrogen peroxide (H2O2), hypochlorous acid (HOCl), and monochloramine (NH2Cl), at concentrations produced by activated neutrophils, promote leukocyte adherence to microvascular endothelium in post-capillary venules. Cat mesenteric venules (30-45 microns diameter) were examined using intravital video microscopy. Red blood cell velocity (VRBC), venular diameter (DV), and the number of adherent leukocytes (NWBC) were measured in postcapillary venules. Venular blood flow and wall shear rate (tau) were calculated from the measured values of VRBC and DV. Different concentrations (0.01-1.0 mM) of H2O2, HOCl, or NH2Cl were superfused on the mesentery. In some experiments, the contributions of the leukocyte adhesive glycoprotein CD11/CD18 and platelet-activating factor (PAF) in the oxidant-induced leukocyte adherence were determined using a CD18-specific antibody (IB4) and a PAF-receptor antagonist (WEB 2086), respectively. The results of our in vivo experiments indicate that H2O2 and NH2Cl, but not HOCl, promote leukocyte adhesion to venular endothelium. Incubation of isolated cat neutrophils with either NH2Cl or H2O2 resulted in activation of CD11/CD18, as assessed by flow cytometry. Although the leukocyte adhesion induced by both H2O2 and NH2Cl was associated with a reduction in venular wall shear rate, corresponding decrements in shear rate induced by partial occlusion of the mesenteric artery did not lead to similar levels of leukocyte adherence. The leukocyte adherence induced by H2O2 and NH2Cl was largely prevented by monoclonal antibody IB4, indicating that both oxidants promote leukocyte adherence via activation of CD11/CD18. The H2O2-induced, CD18-mediated leukocyte adherence appears to be elicited by PAF and by a direct effect of the oxidant on CD11/CD18 expression. The mechanism underlying the NH2Cl-induced leukocyte adherence remain unclear.

Animals↗
PubMed1992

Capillary as a communicating medium in the microvasculature.

The preceding study (Dietrich and Tyml, 1992. Microvasc. Res. 43) demonstrated that a local application of norepinephrine (NE) on a capillary in a skeletal muscle produces a temporary reduction in blood flow within this capillary. The reduction is mediated via constriction of the supplying arteriole. The objective of the present study was to address the mechanism by which the local NE stimulus is propagated from the capillary to the arteriole. Using intravital video microscopy we measured red blood cell velocity in capillaries, and diameter of supplying arterioles, in the sartorius muscle in anesthetized frogs. Velocity responses were measured following iontophoretic application of NE (3 mM in the pipette) on the capillary, with or without pretreatment with 0.9 mM tetrodotoxin (nerve-specific sodium channel blocker), 30 mM lidocaine (nonspecific sodium channel blocker), and 30 mM yohimbine (alpha 2-receptor blocker). Diameter responses were measured before and after capillary damage introduced by microcautery. Tetrodotoxin did not block the NE-induced velocity reduction (i.e., from 0.2 to 0.07 mm/sec), while lidocaine attenuated it. Yohimbine blocked it only when applied on the same site as NE. Capillary damage abolished the NE-induced arteriolar constriction (i.e., from 27.8 to 21.5 microns). We conclude that the observed responses were not due to (1) direct diffusion of NE from the capillary to the arteriole, (2) conduction along adrenergic nerves, or (3) venous-arteriolar diffusional cross-talk. We interpret our data to indicate that the capillary itself could function as a communicating medium.

Animals↗
PubMed1992

Acute endothelial cell contraction in vitro: a comparison with vascular smooth muscle cells and fibroblasts.

The contractile responses of cultured rat and calf endothelial cells (EC), vascular smooth muscle cells (VSMC), and fibroblasts (FB) to vasoactive mediators (thrombin, serotonin, bradykinin, and histamine), forskolin, and cytochalasin B were compared. Cells were grown on a pliable silicone membrane, and contraction was assessed, using time-lapse video microscopy, by recording changes in the wrinkling of the silicone as the cells exerted tension on the surface. We found that all cells contracted in the presence of serum or thrombin and that VSMC and FB also contracted with serotonin stimulation. Bradykinin and histamine were not contractants in this system. Discrepancies between these results and reports of changes in permeability of endothelial layers in vitro and in vivo may be due to (1) the vascular segment from which EC were studied or (2) the possibility that certain mediators may provoke a noncontractile response that results in gap formation. Thus changes in vascular permeability, which occur during inflammation, may have both contractile and noncontractile components. Forskolin, known to indirectly inhibit myosin light-chain kinase activity, and cytochalasin B were potent relaxants, suggesting a similar smooth muscle-like contractile mechanism for all three cell types.

Actin Cytoskeleton↗
PubMed1992

Application of image analysis for evaluation of red blood cell dynamics in capillaries.

We have devised a method to display and directly evaluate red blood cell (rbc) dynamics in capillaries using the same dual camera intravital video microscopy system employed to determine rbc oxygen saturation (Ellis et al., 1990). Capillary images are recorded on videotape and an interactive graphics system is used for analysis. Data are sampled once a frame for 60 sec using a window (one pixel wide (0.93 micron) and 100 pixels high) positioned along the axis of a capillary. The resulting data are displayed as sequential space-time images 100 pixels high by 300 pixels wide (10 sec). The space-time images thus created represent the dynamics of the rbc's in a single comprehensive static image in which the rbc's appear as dark, diagonal bands separated by light bands representing plasma gaps. From these images one can obtain information on velocity of individual rbc's (micron/sec), lineal density of rbc's (rbc/mm), and rbc supply rate (rbc/sec). This information can be used to delineate the temporal and spatial heterogeneity of hemodynamics in capillary networks. These data can then be combined with coincident data on red blood cell oxygenation to provide a complete picture of oxygen transport in capillaries or it can be used alone as a tool for the evaluation of basic in vivo and in vitro rheological questions.

Animals↗