Fluorescence Video Microscopy Research Articles

Role of 11beta-hydroxysteroid dehydrogenase in nongenomic aldosterone effects in human arteries.

The aim of the present study was to demonstrate rapid effects of aldosterone on the Na(+)-H(+) exchanger in strips of human vascular vessels and to determine whether 11beta-hydroxysteroid dehydrogenase enzyme (11beta-HSD) could play a protective role in this response, such as that described for the classic type I mineralocorticoid receptor (MR). The activity of 11beta-HSD isoforms 1 and 2 were measured in fetal and adult arteries. Both isoforms are present in adult and fetal vessels. However, a significant difference in the proportion of each isoform was found. Isoform 1 activity (in pmol x min(-1) x 100 mg(-1) protein) was 42+/-5 in fetal vessels and 29+/-2 in adult arteries, and isoform 2 activity was 78+/-7 in fetal and 12+/-2 in adult tissue. The nongenomic effect of aldosterone on Na(+)-H(+) exchanger activity was measured in strips of chorionic and radial uterine arteries loaded with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein. Recordings of intracellular pH (pH(i)) were made by videofluorescence microscopy. Aldosterone (0.5 nmol/L) rapidly increased pH(i), with a half-maximal effect between 2 and 3 nmol/L in both fetal and adult vessels. Ethylisopropylamiloride, a specific inhibitor of the Na(+)-H(+) exchanger, inhibited this effect. The hormone-mediated increase in pH(i) was unaffected by spironolactone, a classic antagonist of MR, but was completely blocked by RU28318. Cortisol (up to 1 micromol/L) had no effect on pH(i), but when applied in the presence of carbenoxolone, a dramatic increase in Na(+)-H(+) exchanger activity was evident. The increments on pH(i) for each cortisol concentration were similar to those observed for aldosterone. These findings suggest that vascular 11beta-HSD plays an active role in maintaining the specificity of the rapid effects of aldosterone.

Formation of three-dimensional thyroid follicle-like structures by polarized FRT cells made communication competent by transfection and stable expression of the connexin-32 gene.

Pig thyrocytes, either in the intact gland or cultured under conditions leading to thyroid follicle reconstitution, coexpress two gap junction proteins, connexin-32 (Cx32) and connexin-43 (Cx43). As thyrocytes cultured in the form of a monolayer only express Cx43, we hypothesized that Cx32 could play a role in thyroid folliculogenesis. In the present work, we analyzed the ability of polarized FRT cells (that are gap junction deficient) to form follicle-like structures after stable transfection with either Cx32 or Cx43 genes. Wild-type and transfected FRT cells, while growing, showed the capacity to form three-dimensional structures corresponding to domes that result from the accumulation of fluid underneath limited areas of the cell layer. The number of domes formed by FRT cells expressing Cx32 (FRT-Cx32) was 2- to 3-fold higher than that obtained with either wild-type or Cx43-transfected FRT cells (FRT-Cx43). Domes generated by FRT-Cx32 cells were stable (beyond 3 weeks of culture), whereas those formed from wild-type or FRT-Cx43 cells were transient, disappearing when cells reached confluence. Inspection of the cell organization within domes formed from FRT-Cx32 cells by phase contrast and confocal microscopy revealed a progressive transition from domes toward closed structures with a lumen. The tightness of the lumen was demonstrated by the retention of a fluorescent probe, lucifer yellow, introduced by microinjection. Electron microscope examinations showed that the neoformed follicle-like structures had an inside-out polarity. Analyses of cell motion and division with time, by fluorescence video microscopy, indicated that the transformation of domes into inside-out follicles brings into play the migration of cells and, to a lesser extent, cell multiplication underneath the domes. In conclusion, FRT cells forced to express Cx32 give rise to domes that transform into closed inside-out follicles. This gain of function appears Cx specific, as FRT-Cx43 cells did not form similar structures. Our data suggest that the formation and/or functioning of Cx32 gap junctions might represent a key event in thyroid epithelium morphogenesis, i.e. formation of a lumen from a tight epithelial cell layer.

Measuring VEGF-Flk-1 Activity and Consequences of VEGF-Flk-1 Targeting In Vivo Using Intravital Microscopy: Clinical Applications

Vascular endothelial growth factor (VEGF)-Flk-1/KDR tyrosine kinase signaling pathway plays a pivotal role in tumor angiogenesis. Targeting this angiogenic signaling pathway presents a promising alternative for the treatment of neoplasms. However, recent experimental and clinical studies have suggested that VEGF-Flk-1/KDR activity is unevenly distributed throughout the tumor microvasculature. To further evaluate this phenomenon, the regional differences in VEGF-Flk-1/KDR signaling activities in vivo were studied using intravital fluorescence videomicroscopy in an experimental murine brain tumor model. Regional VEGF-Flk-1/KDR was assessed using the small molecule inhibitor SU5416, which selectively inhibits the tyrosine kinase receptor Flk-1. C6 glioblastoma cells were implanted into the dorsal skinfold chamber preparation of nude mice. The process of tumor vascularization was repeatedly assessed over 22 days. SU5416 treatment resulted in a significant reduction in tumor vascular density (p < 0.05). Regional microvascular evaluation indicated that the magnitude of this antiangiogenic effect was pronounced in the more angiogenic and better vascularized peritumoral areas than in the intratumoral areas of the tumor microvasculature. These results demonstrate regional differences in Flk-1 activity in vivo that may have significant impact on the susceptibility of tumors to compounds that target VEGF-Flk-1/KDR. This finding should be considered in upcoming clinical trials targeting individual signal transduction systems in cancer patients.

Measuring VEGF-Flk-1 Activity and Consequences of VEGF-Flk-1 Targeting In Vivo Using Intravital Microscopy: Clinical Applications

Vascular endothelial growth factor (VEGF)-Flk-1/KDR tyrosine kinase signaling pathway plays a pivotal role in tumor angiogenesis. Targeting this angiogenic signaling pathway presents a promising alternative for the treatment of neoplasms. However, recent experimental and clinical studies have suggested that VEGF-Flk-1/KDR activity is unevenly distributed throughout the tumor microvasculature. To further evaluate this phenomenon, the regional differences in VEGF-Flk-1/KDR signaling activities in vivo were studied using intravital fluorescence videomicroscopy in an experimental murine brain tumor model. Regional VEGF-Flk-1/KDR was assessed using the small molecule inhibitor SU5416, which selectively inhibits the tyrosine kinase receptor Flk-1. C(6) glioblastoma cells were implanted into the dorsal skinfold chamber preparation of nude mice. The process of tumor vascularization was repeatedly assessed over 22 days. SU5416 treatment resulted in a significant reduction in tumor vascular density (p<0.05). Regional microvascular evaluation indicated that the magnitude of this antiangiogenic effect was pronounced in the more angiogenic and better vascularized peritumoral areas than in the intratumoral areas of the tumor microvasculature. These results demonstrate regional differences in Flk-1 activity in vivo that may have significant impact on the susceptibility of tumors to compounds that target VEGF-Flk-1/KDR. This finding should be considered in upcoming clinical trials targeting individual signal transduction systems in cancer patients.

Investigation of the hydrolysis of single DNA molecules using fluorescence video microscopy.

We developed a method of studying the hydrolysis of single DNA molecules by exonuclease (exo) III using fluorescence video microscopy. A single DNA molecule immobilized at one end on a coverslip and labeled with a fluorescent bead at the other end confined the motion of a bead to a two-dimensional projected circular area determined by the contour length of DNA. The radius of this area decreased with time after the addition of exo III, Mg2+, or a single-stranded DNA-binding protein (SSB), which caused the single-stranded (ss) DNA to twist around itself. However, the radius was relatively constant over time in the absence of Mg2+, which is a cofactor in exo III activity, even when exo III and SSB were both present. These observations indicated that the decrease in the radius was due to hydrolysis of DNA by exo III. We then evaluated the rate of exo III hydrolysis of single DNA molecules by monitoring the decrease in the radius.

The effects of various organ preservation solutions on hepatocyte membrane potentials, intracellular calcium concentrations, and outcome following liver transplantation

Background: Hepatocyte membrane potential differences (PDs) may be altered by the preservation solutions used in liver transplantation. Such alterations could impact on the survival of the donor liver, extent of biochemical injury, and flux of important ionic compounds. The purpose of the present study was to document these outcomes in the presence of four different preservation solutions. Methods: Livers of adult male Sprague-Dawley rats (N = 3 to 4 per group) were impaled with intracellular microelectrodes prior to and at various time periods for 6 hours following complete hepatic resection. Just prior to resection, each liver was perfused with preservation solutions associated with high (normal saline [NS]), moderate (Euro-Collins [EC]), and low (University of Wisconsin solution [UW]) risks of reperfusion injury. Results: Baseline (in situ) PDs were similar in all groups (−37 ± 4 mV, mean ± SD). Ten minutes postresection, hepatic PDs were as follows: NS, −23.8 ± 3.5 mV; EC, −11.4 ± 0.4 mV; and UW, −8.7 ± 0.3 mV ( P <0.01 for all groups). Maximum depolarization occurred at 6 hours postresection (NS, −8.1 ± 1.1 mV; EC, −7.7 ± 1.3 mV; and UW, −8.6 ± 1.0 mV). To determine whether these changes are of pathophysiologic importance, the NS solution was modified (addition of 0.1% ethanol) to achieve similar PD changes as those observed with UW. Liver transplants were then performed where the donor livers had been perfused and preserved for 6 hours with either NS or the modified NS (MNS) solution. Posttransplant (10 day) survival was 1 of 6 (17%) in the NS group and 4 of 6 (67%) in the MNS group ( P <0.05). Regarding the effects of PD changes on ionic flux, intracellular calcium levels were documented for up to 4 hours by fluorescence video microscopy using Fura-2 in isolated hepatocytes exposed to NS, UW, and MNS solutions. Intracellular calcium levels were similar in all solutions at each time point studied. Conclusions: The results of this study indicate that hepatocytes undergo prompt and marked depolarization following hepatic resection, and the extent of the depolarization correlates with survival following transplantation.

Mechanical activation of dorsal root ganglion cells in vitro: comparison with capsaicin and modulation by κ-opioids

The aim of this study was to characterize plasma membrane pathways involved in the intracellular calcium ([Ca 2+] i) response of small DRG neurons to mechanical stimulation and the modulation of these pathways by κ-opioids. [Ca 2+] i responses were measured by fluorescence video microscopy of Fura-2 labeled lumbosacral DRG neurons obtained from adult rats in short-term primary culture. Transient focal mechanical stimulation of the soma, or brief superfusion with 300 nM capsaicin, resulted to [Ca 2+] i increases which were abolished in Ca 2+-free solution, but unaffected by lanthanum (25 μM) or tetrodotoxin (10 −6 M). 156 out of 465 neurons tested (34%) showed mechanosensitivity while 55 out of 118 neurons (47%) were capsaicin-sensitive. Ninty percent of capsaicin-sensitive neurons were mechanosensitive. Gadolinium (Gd 3+; 250 μM) and amiloride (100 μM) abolished the [Ca 2+] i transient in response to mechanical stimulation, but had no effect on capsaicin-induced [Ca 2+] i transients. The κ-opioid agonists U50,488 and fedotozine showed a dose-dependent inhibition of mechanically stimulated [Ca 2+] i transients but had little effect on capsaicin-induced [Ca 2+] i transients. The inhibitory effect of U50,488 was abolished by the κ-opioid antagonist nor-Binaltorphimine dihydrochloride (nor-BNI; 100 nM), and by high concentrations of naloxone (30–100 nM), but not by low concentrations of naloxone (3 nM). We conclude that mechanically induced [Ca 2+] i transients in small diameter DRG somas are mediated by influx of Ca 2+ through a Gd 3+- and amiloride-sensitive plasma membrane pathway that is co-expressed with capsaicin-sensitive channels. Mechanical-, but not capsaicin-mediated, Ca 2+ transients are sensitive to κ-opioid agonists.

7-Nitroindazole impedes erythrocyte flow response to isovolemic hemodilution in the cerebral capillary circulation.

The role of nitric oxide (NO) in the mechanism of hemodilution-induced cerebral hyperemia is unclear. Based on findings in hypoxemia, the authors hypothesize that NO of neuronal origin contributes to an increase in velocity of erythrocytes in the cerebral microcirculation during anemia produced by isovolemic hemodilution. The change in erythrocyte velocity in cerebrocortical capillaries was assessed by intravital fluorescence video microscopy. A closed cranial window was implanted over the frontoparietal cortex of barbiturate-anesthetized, ventilated adult rats. Erythrocytes were labeled in vitro with fluorescein isothiocyanate and infused intravenously, and their velocity in subsurface capillaries was measured by frame-to-frame image tracking. Arterial blood was withdrawn in increments of 2 mL and replaced by serum albumin; arterial blood pressure was maintained at control level with an infusion of methoxamine. Erythrocyte velocity increased progressively, reaching 215% of baseline, as arterial hematocrit was reduced from 45% to 17%. Pretreatment of a separate group of rats with 7-nitroindazole (20 mg/kg intraperitoneally), a relatively selective inhibitor of neuronal NO synthase, abolished the increase in velocity at hematocrits greater than 20%, but the maximum velocity attained at the lowest hematocrit was similar to that in the control group. The results suggest that NO from neuronal source may contribute to the increase in capillary erythrocyte flow during moderate isovolemic hemodilution.

Role of the Cytoplasmic Tail of Ecotropic Moloney Murine Leukemia Virus Env Protein in Fusion Pore Formation

Fusion between cells expressing envelope protein (Env) of Moloney murine leukemia virus and target cells were studied by use of video fluorescence microscopy and electrical capacitance measurements. When the full-length 632-amino-acid residue Env was expressed, fusion did not occur at all for 3T3 cells as target and only somewhat for XC6 cells. Expression of Env 616*-a construct of Env with the last 16 amino acid residues (617 to 632; the R peptide) deleted from its C terminus to match the proteolytically cleaved Env produced during viral budding-resulted in high levels of fusion. Env 601*, lacking the entire cytoplasmic tail (CT) (identified by hydrophobicity), also led to fusion. Truncation of an additional six residues (Env 595*) abolished fusion. The kinetics of forming fusion pores did not depend on whether cells were first prebound at 4 degrees C and the time until fusion measured after the temperature was raised to 37 degrees C or whether cells were first brought into contact at 37 degrees C and the time until fusion immediately measured. This similarity in kinetics indicates that binding is accomplished quickly compared to subsequent steps in fusion. The fusion pores formed by Env 601* and Env 616* had the same initial size and enlarged in similar manners. Thus, once the R peptide is removed, the CT is not needed for fusion and does not affect formed pores. However, residues 595 to 601 are required for fusion. It is suggested here that the ectodomain and membrane-spanning domain of Env are directly responsible for fusion and that the R peptide affects their configurations at some point during the fusion process, thereby indirectly controlling fusion.

Targeting angiogenesis inhibits tumor infiltration and expression of the pro-invasive protein SPARC.

The solid growth of high-grade glioma appears to be critically dependent on tumor angiogenesis. It remains unknown, however, whether the diffuse infiltration of glioma cells into healthy adjacent tissue is also dependent on the formation of new tumor vessels. Here, we analyze the relationship between tumor angiogenesis and tumor cell infiltration in an experimental glioma model. C6 cells were implanted into the dorsal skinfold chamber of nude mice, and tumor angiogenesis was monitored by intravital fluorescence videomicroscopy. Glioma infiltration was assessed by the extent of tumor cell invasion into the adjacent chamber tissue and by expression of SPARC, a cellular marker of glioma invasiveness. To test the hypothesis that glioma angiogenesis and glioma infiltration are codependent, we assessed tumor infiltration in both the presence and the absence of the angiogenesis inhibitor SU5416. SU5416 is a selective inhibitor of the VEGF/Flk-1 signal-transduction pathway, a critical pathway implicated in angiogenesis. Control tumors demonstrated both high angiogenic activity and tumor cell invasion accompanied by strong expression of SPARC in invading tumor cells at the tumor-host tissue border. SU5416-treated tumors demonstrated reduced vascular density and vascular surface in the tumor periphery accompanied by marked inhibition of glioma invasion and decreased SPARC expression. A direct effect of SU5416 on glioma cell motility and invasiveness was excluded by in vitro migration and invasion assays. These results suggest a crucial role for glioma-induced angiogenesis as a prerequisite for diffuse tumor invasion and a possible therapeutic role for anti-angiogenic compounds as inhibitors of both solid and diffuse infiltrative tumor growth.

Glycerol attenuates the adherence of leukocytes in rat pial venules after transient middle cerebral artery occlusion

Intravenous infusion of glycerol has been used in patients with a cerebral infarction, expecting improvement in brain edema and cerebral blood flow (CBF). However, the mechanism of the improvement of CBF has not been clearly demonstrated. The aim of this study in the rat pial microvasculature after transient middle cerebral artery occlusion (MCAO) is to examine the effects of glycerol on leukocyte- endothelium interaction, which plays a critical role in the pathogenesis of brain injury by ischemia/ reperfusion and concerns induction of secondary brain damage. Rhodamine 6G-labeled leukocytes at the brain surface were visualized with intra-vital fluorescence videomicroscopy through a closed cranial window and an analysis was made of the number of adherent leukocytes and the centerline leukocyte velocity in the venule before MCAO, after reperfusion of MCAO and after infusion of glycerol (Croup 1) or saline (Croup 2). The number of adherent leukocytes decreased and the centerline leukocyte velocity increased statistically significantly immediately after the infusion of glycerol in Croup I, but there was no significant change in Croup 2. The infusion of glycerol washes away the adherent leukocytes and prevents them from interfering with the blood cell and plasma flow. Furthermore, secondary brain damage may be relieved by decreasing the adherence of leukocytes. In conclusion, modulating the adherence of leukocytes is one of the important factors in the neuroprotective effect of glycerol. [Neurol Res 1999; 21: 785-790]

Visualization of defective mitochondrial function in skeletal muscle fibers of patients with sporadic amyotrophic lateral sclerosis.

The mitochondrial function in skeletal muscle was investigated in skeletal muscle biopsies of 26 patients with sporadic amyotrophic lateral sclerosis (ALS) and compared with investigations of 28 age-matched control muscle samples and biopsies of 6 patients with spinal muscular atrophy (SMA) and two patients with Tay-Sachs disease. In comparison to the control, SMA and Tay-Sachs biopsies, we observed in the ALS samples a significant about two-fold lower activity of complex I of mitochondrial respiratory chain. To visualise the distribution of the mitochondrial defect in skeletal muscle fibers we applied confocal laser-scanning microscopy and video fluorescence microscopy of NAD(P)H and fluorescent flavoproteins. The redox change of mitochondrial NAD(P)H and flavoproteins on addition of mitochondrial substrates, ADP, or cyanide were determined by measurement of fluorescence intensities with dual-photon UV-excitation and single-photon blue excitation. In skeletal muscle fibers of ALS patients with abnormalities of mitochondrial DNA (multiple deletions, n=1, or lower mtDNA levels, n=14) we observed a heterogeneous distribution of the mitochondrial defects among individual fibers and even within single fibers. In some patients (n=3) a mitochondrial defect was also detectable in cultivated skin fibroblasts. These findings support the viewpoint that the observed impairment of mitochondrial function in muscle of certain ALS patients is caused by an intrinsic mitochondrial defect which may be of pathophysiological significance in the etiology of this neurodegenerative disease.

Regional pleural filtration and absorption measured by fluorescent tracers in rabbits.

In prone anesthetized rabbits, we used Evans blue-dyed albumin (EBA) to study regional pleural filtration and FITC dextran to study regional pleural absorption. Evans blue was injected intravenously, and the animals were ventilated for 6 h at either of two levels of ventilation. Postmortem the right rib cage was frozen and thawed before study. EBA fluorescence emitted from the rib cage surface was measured along the cranial-caudal axis near the mid chest with fluorescence videomicroscopy. Fluorescent light intensity increased from the third to the eighth rib in a cyclic fashion, with peaks at the ribs and troughs at the intercostal spaces. This increase was greater at the higher ventilation. Fluorescent images of cross sections of a frozen rib cage verified a cranial-caudal gradient in filtration. Fluorescent images of FITC dextran absorbed from the pleural space into the rib cage surface indicated major areas of absorption at the ventral, caudal, and cranial regions adjacent to the lung margins and areas of absorption scattered in the intercostal spaces. Simultaneous measurements of EBA filtration and FITC absorption showed sites of maximal filtration that were different from sites of maximal absorption. Pleural uptake of fluorescent microspheres (2-microm diameter) located lymphatic stomata distributed randomly within clusters in the intercostal spaces and channels of lymphatic lacunae parallel to blood vessels. Diaphragmatic uptake of microspheres was confined mainly to the ventral surface of the central tendon. Visceral pleural absorption was minimal.

Role of nitric oxide in intrarenal hemodynamics in experimental diabetes mellitus in rats.

The role of nitric oxide (NO) in the regulation of the intrarenal microcirculation in streptozotocin (STZ)-induced diabetes mellitus in rats is not clear. We examined renal cortical and papillary hemodynamics in STZ rats and determined the effects of systemic inhibition and stimulation of NO synthesis. Renal blood flow in cortical (QCC), and inner medullary ascending (QAV) and descending (QDV) vasa recta capillaries was measured by fluorescence videomicroscopy in STZ Munich-Wistar rats and nondiabetic control rats. Ten days after STZ injection (80 mg/kg ip), basal QCC and QDV were significantly greater in STZ rats (n = 16) compared with control rats (n = 15). Infusion of N(G)-monomethyl-L-arginine (L-NMMA, 15 mg/kg bolus, 500 microg. min(-1). kg(-1) iv) decreased Q(CC) (-41%), QAV (-38%), and QDV (-37%) in control rats (n = 6) and to a significantly greater magnitude than in STZ rats (n = 7), Q(CC) (-14%), QAV (-20%), and QDV (-25%). Coinfusion of L-arginine (L-Arg, 1 mg. kg(-1). min(-1) iv) with L-NMMA increased Q(CC) to a significantly greater extent (P < 0.01) in control rats compared with STZ rats. In subsequent studies, infusion of L-Arg alone increased QCC (+50%), QAV (+16%), and QDV (+11%) in control rats (n = 5) but had no effect in STZ rats (n = 5). These results show that the response of renal cortical and papillary capillary blood flow to both inhibition and stimulation of NO synthesis is attenuated in the early onset of STZ-diabetes mellitus rats compared with control rats.

Selectins and beta 2-integrins mediate post-ischaemic venular adhesion of polymorphonuclear leukocytes, but not capillary plugging, in isolated hearts.

Leukocytes adhering to venular endothelium and emigrating into the tissue contribute to myocardial reperfusion injury. The aim of the present study was to characterize the contribution of two different families of adhesion molecules, selectins and integrins, to post-ischaemic capillary plugging and venular adhesion of leukocytes in an isolated heart model. Guinea-pig hearts were perfused using the Langendorff technique. After 20 min stabilization global ischaemia was induced for 15 min at 37 degrees C. With the onset of reperfusion 10(7) isolated polymorphonuclear leukocytes (PMN), prelabelled with rhodamine 6G, were infused within 1 min. Perfusion was continued for 2 min to wash out all cells not firmly adhering to the vascular endothelium. Hearts were then arrested, mounted on a microscope stage and perfused with a cardioplegic solution containing 0.01% fluorescein isothiocyanate (FITC)-dextran (MW 150,000). In situ videofluorescence microscopy was used to quantify PMN plugging and adherent PMN. Four groups were studied: control (no treatment or ischaemia, n = 6); ischaemia (no treatment and 15 min ischaemia, n = 5); fucoidin (pretreatment of hearts and PMN with 0.3 mg/ml selectin inhibitor fucoidin and 15 min ischaemia, n = 5) and CD18 (pretreatment of PMN with 0.1 mg monoclonal antibody against CD18 and 15 min ischaemia, n = 5). Capillary plugging by PMN was 25 +/- 5 PMN/mm2 epicardial surface area and increased moderately to 55 +/- 6 PMN/mm2 in reperfused hearts. This increase was not affected by fucoidin or CD18 antibody. In contrast, post-ischaemic adhesion of PMN in small venules increased ninefold from 21 +/- 5 to 196 +/- 23 PMN/mm2 endothelial surface area. The increase in PMN adhesion to venular endothelium was blocked completely by pretreatment with fucoidin (19 +/- 5 PMN/mm-2) or CD18 antibody (7 +/- 2 PMN/mm-2). We conclude that selectin interaction alone is not sufficient to account for post-ischaemic PMN adhesion in the small venules of the coronary vasculature, because blocking the integrin subunit CD18 also inhibited PMN adhesion completely. On the other hand, neither integrins nor selectins seem to be involved in post-ischaemic capillary plugging by PMN in our perfused heart model.

Leukocyte Sequestration in Pulmonary Microvessels and Lung Injury following Systemic Complement Activation in Rabbits

Inflammatory reactions are associated with sequestration of leukocytes in the lung. Complement activation leads to accumulation of leukocytes in alveolar septa and alveoli, to lung edema and hemorrhage. Although in organs other than the lung leukocytes interact with the vascular endothelium only in postcapillary venules, alveolar capillaries are considered to be the site of leukocyte sequestration in the lung. However, pulmonary venules and arterioles have not been investigated systematically after complement activation so far. A closed thoracic window was implanted in anesthetized rabbits; leukocytes and red blood cells were stained, and the movement of these cells was measured in superficial pulmonary arterioles, venules and alveolar capillaries using fluorescence video microscopy before and 30 and 60 min after infusion of cobra venom factor (CVF). Erythrocyte velocity and macrohemodynamic conditions did not change after CVF infusion and were not different from the sham-treated controls. The number of sticking leukocytes increased significantly compared to baseline and control: by 150% in arterioles and in venules and by 740% in alveolar capillaries within 60 min after CVF infusion. The width of alveolar septa in vivo was significantly enlarged after CVF infusion, indicating interstitial pulmonary edema. At the end of the experiments, myeloperoxidase activity was higher in the CVF group, showing leukocyte sequestration in the whole organ. It is concluded that complement activation by CVF induces leukocyte sequestration in lung arterioles, venules and alveolar capillaries and leads to mild lung injury.

Directly observed membrane fusion between oppositely charged phospholipid bilayers.

A novel method was developed for the direct examination of pairwise encounters between positively and negatively charged phospholipid bilayer vesicles. Giant bilayer vesicles (unilamellar, 4-20 micron in diameter) prepared from 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, a new cationic phospholipid derivative, were electrophoretically maneuvered into contact with individual anionic phospholipid vesicles. Fluorescence video microscopy revealed that such vesicles commonly underwent fusion within milliseconds (1 video field) after contact, without leakage. Fusion occurred at constant volume and, since flaccid vesicles were rare, the excess membrane was not available after fusion. Hemifusion (the outer monolayers of each vesicle fused while the inner monolayers remained intact) was inferred from membrane-bound dye transfer and a change in the contact area. Hemifusion was observed as a final stable state and as an intermediate to fusion of vesicles composed of charged phospholipids plus zwitterionic phospholipids. Hemifusion occurred in one of three ways following adhesion: either delayed with an abrupt increase in area of contact, immediately with a gradual increase in area of contact, or with retraction during which adherent vesicles dissociated from a flat contact to a point contact. Phosphatidylethanolamine strongly promoted immediate hemifusion; the resultant hemifused state was stable and seldom underwent complete fusion. Although sometimes single contacts between vesicles led to rupture of both, in other cases, a single vesicle underwent multiple fusion events. Direct observation has unequivocally demonstrated the fusion of two, isolated bilayer-bounded bodies to yield a stable, non-leaky product, as occurs in cells, in the absence of proteins.

Intracellular calcium oscillations induced by ATP in airway epithelial cells.

In airway epithelial cells, extracellular ATP (ATP(o)) stimulates an initial transient increase in intracellular Ca(2+) concentration that is followed by periodic increases in intracellular Ca(2+) concentration (Ca(2+) oscillations). The characteristics and mechanism of these ATP-induced Ca(2+) responses were studied in primary cultures of rabbit tracheal cells with digital video fluorescence microscopy and the Ca(2+)-indicator dye fura 2. The continual presence of ATP(o) at concentrations of 0.1-100 microM stimulated Ca(2+) oscillations that persisted for 20 min. The frequency of the Ca(2+) oscillations was found to be dependent on both ATP(o) concentration and intrinsic sensitivity of each cell to ATP(o). Cells exhibited similar Ca(2+) oscillations to extracellular UTP (UTP(o)), but the oscillations typically occurred at lower UTP(o) concentrations. The ATP-induced Ca(2+) oscillations were abolished by the phospholipase C inhibitor U-73122 and by the endoplasmic reticulum Ca(2+)-pump inhibitor thapsigargin but were maintained in Ca(2+)-free medium. These results are consistent with the hypothesis that in airway epithelial cells ATP(o) and UTP(o) act via P2U purinoceptors to stimulate Ca(2+) oscillations by the continuous production of inositol 1,4,5-trisphosphate and the oscillatory release of Ca(2+) from internal stores. ATP-induced Ca(2+) oscillations of adjacent individual cells occurred independently of each other. By contrast, a mechanically induced intercellular Ca(2+) wave propagated through a field of Ca(2+)-oscillating cells. Thus Ca(2+) oscillations and propagating Ca(2+) waves are two fundamental modes of Ca(2+) signaling that exist and operate simultaneously in airway epithelial cells.

Comparative Conformational Dynamics of Supercoiled Plasmids and Linear DNA during Capillary Electrophoresis

The electrophoretic migration of 40 and 90 kbp supercoiled (sc) double-stranded (ds) plasmid DNA in dilute and entangled solutions of linear hydroxyethyl cellulose (HEC, Mn = 438 800) was visualized by video fluorescence microscopy at 60−222 frames/s. The sc-DNA molecules migrated as elastic rods with stretch−contraction cycles directed along the electric field axis. The elastic rod electrophoretic behavior of sc-DNA is completely different from the familiar random coil electrophoretic dynamics of linear ds-DNA. Extension−contraction cycles were more periodic for sc-DNA than for linear ds-DNA. The consequences of the different sc-DNA and linear ds-DNA electrophoretic dynamics on mobility dispersion are discussed.

Nitric oxide synthase inhibitor augments post-ischemic leukocyte adhesion in the cerebral microcirculation in vivo

The objective was to examine the effect of the nitric oxide synthase inhibitor, N^-nitro-L-arginine methyl ester (L-NAME) on leukocyte adhesion in the cerebral microcirculation during reperfusion following partial forebrain ischemia in the rat. intravital fluorescence video-microscopy through a closed cranial window was used to visualize leukocyte-endothelium interaction in small pial veins of 15-100 /im diameter. Forebrain ischemia was produced by the ligation of both common carotid arteries plus elevation of the intracranial pressure to 20 mmHg for 60 min. The number of leukocytes adhering to the endothelium for longer than 3 sec was determined during ischemia (5 min and 60 min) and during reperfusion (5 min and 60 min). Two experimental groups were treated with either L-NAME or its inactive enantiomer D-NAME (20 mg kg~1 i.v.) 30 min prior to reperfusion. In a third group, also treated with D-NAME, post-ischemic hyperemia was prevented by lowering the ICP without removing the occlusion of common carotid arteries (partial reperfusion). The velocity of flow adjacent to the endothelial surface of pial veins was measured by tracking the movement of fluorescently labeled red blood cells as flow markers before and after ischemia. During ischemia, the number of adhering leukocytes increased approximately two-fold at 5 min, and threefold at 60 min. In the D-NAME-treated group with complete reperfusion, leukocyte adhesion returned to the baseline level by 60 min of reperfusion. However, in the L-NAME-treated group, leukocyte adhesion remained elevated at 60 min of reperfusion. Post-ischemic flow velocity was significantly decreased (-66%) from control after L-NAME treatment whereas it was increased (+53%) in the D-NAME-treated group. In the partial reperfusion group, leukocyte adhesion continued to increase after the first hour of ischemia and reached a level 2.7-fold over baseline at 60 min reperfusion. Flow velocity remained below control (-26%) at 60 min reperfusion. Leukocyte adhesion was absent in pial arteries and no plugging by leukocytes was observed in cortical capillaries. The results suggest that leukocyte adhesion in small pial veins increases during I h forebrain ischemia and continues to increase during reperfusion if the velocity of flow or shear rate is low. The increase in leukocyte adhesion is reversible if flow velocity is elevated during reperfusion. L-NAME prevents post-ischemic hyperemia and augments leukocyte adhesion principally via a ,decrease in velocity or shear rate. [Neurol Res 1999; 21: 378-384]