Biomedical subjects

Dino J Ravnic

Publications and source records attributed to Dino J Ravnic.

8 recordsLinked to original sources

PubMed2007

The murine bronchopulmonary microcirculation in hapten-induced inflammation.

OBJECTIVE: The clinical observation of central bronchial artery hypertrophy in chronic lung inflammation suggests the possibility that the bronchial circulation may also participate in adaptive responses in peripheral lung inflammation. METHODS: To investigate the potential role of the bronchial microcirculation in peripheral lung inflammation, we developed a murine model of lung inflammation using the intratracheal instillation of the peptide-hapten trinitrophenol in presensitized mice. RESULTS: Clinical parameters indicated a peak inflammatory response at 96 hours. Similarly, gross and microscopic evidence of inflammation was observed 96 hours after antigen instillation. Using a forced oscillation technique to probe peripheral lung mechanics at 96 hours, we detected no change in central airway resistance (P > .05), but a significant increase in peripheral tissue resistance (P < .05). The structure of the bronchial circulation was investigated by microsphere occlusion of the pulmonary circulation and corrosion casting of the bronchial circulation. SEM of the bronchial artery casts demonstrated (1) the presence of the peripheral bronchial circulation in mice, (2) interconnections of the two systems in the distal bronchial arteries and at the level of alveolar capillaries, and (3) functional evidence of increased bronchial perfusion of alveolar capillaries during mononuclear inflammation. CONCLUSION: These results suggest an important adaptive role of the bronchial circulation in pulmonary inflammation.

Animals↗

PubMed2006

Structural adaptations in the murine colon microcirculation associated with hapten-induced inflammation.

BACKGROUND: Blood flowing across the vascular endothelium creates wall shear stress, dependent on velocity of flow and vessel geometry, that tends to disrupt lymphocyte-endothelial cell adhesion. OBJECTIVE: The microcirculation in a murine model of acute colitis was investigated to identify structural adaptations during acute colitis that may facilitate transmigration. METHODS: In 2,4,6-trinitrobenzenesulphonic acid-induced acute colitis, the infiltrating cells and colonic microcirculation was investigated by cellular topographic mapping, corrosion casting and three-dimensional scanning electron microscopy (SEM). Colonic blood velocimetry was performed using intravital microscopy. RESULTS: Clinical and histological parameters suggested a peak inflammatory response at 96 h (p<0.001). The infiltrating cells were spatially related to the mucosal capillary plexus by three-dimensional topographic mapping (p<0.001). In normal mice, corrosion casting and three-dimensional SEM showed a polygonal mucosal plexus supplied by ascending arteries and descending veins. After 2,4,6-trinitrobenzenesulphonic acid stimulation, three-dimensional SEM showed preserved branch angles (p = 0.52) and nominal vessel lengths (p = 0.93), but a significantly dilated mucosal capillary plexus (p<0.001). Intravital microscopy of the mucosal plexus showed a greater than twofold decrease in the velocity of flow (p<0.001). CONCLUSIONS: The demonstrable slowing of the velocity of flow despite an increase in volumetric flow suggests that these microvascular adaptations create conditions suitable for leucocyte adhesion and transmigration.

Acute Disease↗

PubMed2006

Multi-image particle tracking velocimetry of the microcirculation using fluorescent nanoparticles.

Particle tracking velocimetry provides a Lagrangian description of flow properties in the microcirculation. To determine the utility of fluorescent nanoparticles to provide Lagrangian coordinates, we tracked these particles both in vitro and in vivo. The particles had a neutral charge and fluorescence intensity greater than 1,000 times the PKH26-labeled red blood cells. At image acquisition rates of 60 frames per second, particles were tracked at velocities up to 4,000 microm/s. Morphometric changes reflecting streaking artifact were significant at velocities of 4,000 microm/s (P < 0.05), but not at lower velocities (P > 0.05). Intravital microscopy monitoring after intravenous injection of the particles demonstrated a circulation half-life that was inversely related to particle size: 500 nm nanoparticles demonstrated a smaller change in plasma concentration than larger particles. Regardless of the size of the particles, more than 50% of the recovered fluorescence was located in the liver. These results suggest that fluorescent nanoparticles provide a convenient and practical Lagrangian description of flow velocity in the microcirculation.

Animals↗

PubMed2006

Structural mapping of immunoglobulin subclasses using multiplexed bead flow cytometry.

Monoclonal hybridomas secrete immunoglobulin molecules with a single specificity and distinct class/subclass structure. The determination of immunoglobulin structure can be used to facilitate hybridoma colony management and predict monoclonality. In this report, we used multiplexed bead flow cytometry to define hybridoma class/subclass. The assay was sufficiently sensitive to detect 50 ng/mL of antibody. The multiplexed bead assay efficiently defined traditional class/subclass determinants as well as more subtle patterns of crossreactivity. Further, the assay was combined with Poisson statistics to provide a numerical estimate of hybridoma monoclonality. The sensitivity and flexibility of this approach suggests the utility of multiplexed bead flow cytometry in the early management of immunoglobulin-secreting hybridomas.

Animals↗

PubMed2006

Inflammation-responsive focal constrictors in the mouse ear microcirculation.

In many capillary exchange beds, blood flow is locally regulated by precapillary sphincter-like activity. In this study, we used intravascular tracers and scanning electron microscopy to investigate precapillary blood flow regulation in the mouse ear. Gelatin ink injections of the normal mouse ear demonstrated 6.8 +/- 2.3 axial vessels with a cutoff of detectable tracer in the early branches: 19 +/- 11 focal constrictions were observed along the 1st to 5th order branches of the axial vessels. A perfusion tracer consisting of biotinylated anti-endothelial lectins (Ricinus Communis Agglutin, Lycopersicon Esculentum and Griffonia Simplicifolia) was circulated for 30 min under physiological conditions. Subsequent enzyme histochemistry demonstrated no significant change in distal perfusion or in the number of focal constrictions (P > 0.05). Furthermore, the focal constrictions were unresponsive to vasodilators such as organic nitrates and prostaglandin E1. By contrast, the presence of oxazolone-induced inflammation resulted in significant and sustained vasodilatation for more than 96 h (P > 0.001). Scanning electron microscopy demonstrated discrete constricting bands morphologically distinct from known precapillary sphincters. These results suggest that these previously unappreciated inflammation-responsive precapillary constrictors regulate capillary recruitment in the mouse ear microcirculation.

Animals↗

PubMed2006

Multiframe particle tracking in intravital imaging: defining Lagrangian coordinates in the microcirculation.

The cellular composition of the microcirculation creates blood flow that can be unsteady and nonuniform. To obtain information about nonuniform cellular trajectories, we describe in vivo imaging techniques that provide both detailed tracking of individual particles as well as an approach to simultaneous multicolor particle tracking. Particularly relevant to biologic systems, Lagrangian methods provide information about the fate of individual particles and flow in the system.

Animals↗

PubMed2005

Vessel painting of the microcirculation using fluorescent lipophilic tracers.

Flexible approaches to defining microvessel morphometry are useful in the study of both acute and chronic structural changes of the microcirculation. In this report, we examined the utility of the intravascular infusion of lipophilic carbocyanine tracers in the structural assessment of the retina, skin, lung, and colon microcirculation. The microvessel labeling technique, here termed fluorescent vessel painting, involved the intravascular injection of sulfonated lipophilic carbocyanine tracers. The utility of vessel painting in morphometry was assessed using morphometric comparisons with corrosion casting and 2-dimensional and 3-dimensional scanning electron microscopy. The comparisons demonstrated that fluorescent vessel painting modestly overestimated the interbranch angles, interbranch distances, and vessel diameters of the 2D mucosal plexus of the colon. These differences were narrowed with the application of confocal microscopy. The advantages of fluorescence vessel painting included (1) the filling of all tissues including the relatively high resistance microvessels of the mouse skin, (2) the ability to use tissue counterstains such as DAPI, and (3) the prolonged stability of the lipophilic tracer after aldehyde fixation. These studies suggest the utility of fluorescent vessel painting as a complementary technique to corrosion casting in the morphometric study of the microcirculation.

Animals↗

PubMedDate not supplied

Melittin-induced membrane permeability: a nonosmotic mechanism of cell death.

Derived from honeybees, melittin is a 26-amino acid, alpha-helical, membrane-attack protein that efficiently kills mammalian cells. To investigate the contribution of colloid-osmotic effects to the mechanism of cell death, we studied the effect of melittin on lymphocyte membrane permeability and cell volumes. Melittin concentrations of 0.5 to 2.0 microM induced release of membrane permeability markers without total disruption of the cell membrane. At these melittin concentrations, electrical-impedance cytometry demonstrated melittin-induced changes in red blood cell volumes (P<0.01), but no change in lymphocyte cell volumes (P>0.05). Streaming video microscopy, obtaining images of melittin-treated lymphocytes at 80-ms intervals, demonstrated a loss of optical density (P<0.001) suggesting a flattening of the cell but no significant increase in cell perimeter (P>0.05). Real-time multiparameter flow cytometry of melittin-treated lymphocytes confirmed simultaneous loss of the cytoplasmic marker, calcein, and uptake of the DNA dye, ethidium homodimer, but demonstrated no increase in forward light scatter. Transmission-electron microscopy of melittin-treated lymphocytes showed normal cell volumes but discontinuities in the cell membrane suggesting direct membrane toxicity. We conclude that melittin causes lymphocyte death by a "leaky patch" mechanism that is independent of colloid-osmotic effects.

Animals↗