Silicon-intensified Target Research Articles

Abundant bioluminescent sources of low-light intensity in the deep Mediterranean Sea and North Atlantic Ocean

Light plays a critical role in the functioning of the marine environment. In the dark ocean, bioluminescent organisms are the only visually relevant sources of light. Cameras of different sensitivities were used to compare the density of pelagic bioluminescent sources (BL) of different light intensities at a regional scale: the image-intensified charge-coupled device for deep-sea research (ICDeep), an image-intensified silicon intensifier target (ISIT) camera and a silicon intensifier target (SIT) camera. Pelagic ICDeep values were higher than ISIT measurements by a mean factor of 7.6 in the Mediterranean Sea and 3.5 in the Atlantic Ocean. Atlantic ISIT values were higher than SIT values by a mean factor of 4.5. Standardising bioluminescence measurements to the near-seafloor (0–400 m above bottom) layer, BLNSF, a logarithmic decrease with depth was observed from three independent datasets (slopes not significantly different): ISIT (Atlantic, Mediterranean), ICDeep (Mediterranean). Intercepts from ICDeep measurements were higher than ISIT measurements by a factor of 4.4. From these trends, a conversion factor to calculate benthopelagic plankton biomass from near-seafloor BLNSF density was derived. Calibration of the ICDeep enabled calculation of the minimum intensity of source visible to that camera. BLNSF sources of low-light intensity (≥1.4 × 10−7 W m−2) outnumber fourfold sources of greater intensity (>ca. 10−6 W m−2 (λpeak = 470 nm). This reveals a high abundance of low-light bioluminescent sources in the marine environment, with mean pelagic densities of 33.15 sources m−3 (Atlantic) and 6.79 sources m−3 (Mediterranean) between 500 and 1500 m depth.

Factors influencing the abundance of deep pelagic bioluminescent zooplankton in the Mediterranean Sea

Measurements of the density of deep pelagic bioluminescent zooplankton (BL) were made with the Intensified Silicon Intensifier Target (ISIT) profiler in the Ligurian, Tyrrhenian, Adriatic, Ionian Seas and the Strait of Sicily from ∼300 m to near seafloor. Mean BL densities ranged from 2.61 m −3 at 500–1000 m depth in the Adriatic Sea to 0.01 m −3 at 4000–5000 m depth in the E Ionian Sea. We investigated drivers of spatial variation of deep pelagic BL density. Linear regression was applied between surface chlorophyll a (Chl a) concentration and underlying BL density. Chl a values were determined from satellite derived 100 km radius composites (six 10-day means per ISIT deployment, over preceding 60 days). At 500–1000 m depth we found a significant positive relationship between mean BL density and mean Chl a in the period prior to 0–10 days (at 1% level) and prior to 10–40 days (at 5% level). Beyond 40 days no relationship between BL density and Chl a was found at this depth. At depths 1000–1500 m BL density values were low and no significant relationship with Chl a was detected. Generalised additive modelling (GAM) was used to assess the influence of benthic hotspots (seamount; cold water coral mound; mud volcano) on overlying BL density. A reduction in BL density was found downstream of the Palinuro seamount from 300 to 600 m. No effect on BL density in the overlying water column was detected from the presence of cold water corals. Higher BL densities were detected over the W Madonna dello Ionio mud volcano than at other sites sampled in the NW Ionian Sea. We find surface Chl a to be a good predictor of BL density in the mesopelagic zone; below this depth we hypothesise that processes affecting the efficiency of particle export to deep water may exert greater influence on BL density.

Initial site of bile regurgitation following extrahepatic biliary obstruction in living rats

The precise mechanism of bile regurgitation from the biliary system to the blood stream still remains to be elucidated. The aim of this study was to examine the initial site of bile regurgitation in vivo after common bile duct (CBD) obstruction by digitally enhanced fluorescence microscopy. The fluorescence excreted into bile canaliculi after the administration of sodium fluorescein was continuously observed in CBD obstruction, using video-enhanced contrast (VEC) microscopy equipped with a silicon intensified target (SIT) camera. The liver histology and the localization of Mg(2+)-ATPase were examined by light and electron microscopy. By the continuous recording of canalicular fluorescence, the sequential regurgitation of the fluorescence from the canaliculi to the hepatocyte cytoplasm to the sinusoids was distinctively recognized after CBD obstruction. Bile canalicular fluorescence was enhanced, and then the fluorescence of the hepatocyte cytoplasm increased in intensity, followed by regurgitation of the fluorescence to the sinusoids. These in vivo sequences closely correlated with changes in CBD pressure. In zone 1, canalicular fluorescence focally burst into hepatocyte cytoplasm, thus resulting in the formation of fluorescent cells. By light and electron microscopy, the fluorescent cells were found to correspond to the liver cell injury. The reaction products of Mg(2+)-ATPase were incorporated into vesicles with a decreased canalicular activity, and then were transported to the sinusoidal surface after CBD obstruction. The initial site of bile regurgitation may be transcellular, and partly involves liver cell injury in zone 1 in extrahepatic biliary obstruction, associated with increased pressure of the biliary system.

Benthic bioluminescence in the bathyal North East Atlantic: luminescent responses of Vargula norvegica (Ostracoda: Myodocopida) to predation by the deep-water eel (Synaphobranchus kaupii)

Using two autonomous lander systems, one equipped with a high sensitivity intensified silicon intensifier target (ISIT) video camera and a second with a 4.1-megapixel digital stills camera, observations were made of bioluminescent emissions and fauna attracted to artificial food falls at ca. 1,000 m depth in the carbonate mound provinces of the Porcupine Seabight and Rockall Bank, North East Atlantic. On the Galway coral mound (Belgica Mound Province), seven bioluminescent events per hour were observed, whereas on an area of sediment at the base of the Kiel Mount on the Rockall Bank 133 events per hour were observed. This increase in bioluminescent activity was associated with the presence of the deep water eels Synaphobranchus kaupii, and the ostracods, Vargula norvegica. Captured ostracods luminesced readily in response to mechanical stimulation and were also observed emitting a luminous secretion. We hypothesise that V. norvegica, attracted to bait, luminesce as a defence response against the predatory activity of S. kaupii that compete for bait but also feed on the ostracods. Benthic bioluminescence in the carbonate mound provinces was not directly linked to the presence of corals.

Bioluminescence in the deep sea: Free-fall lander observations in the Atlantic Ocean off Cape Verde

A novel autonomous free-fall lander vehicle, with a capability down to 6000 m, was deployed off Cape Verde for studies on bioluminescence in the deep sea. The system was equipped with a high-sensitivity Intensified Silicon Intensified Target (ISIT) video camera, a programmable control-recording unit and an acoustic current meter with depth and temperature sensors. The ISIT lander was used in three modes: (1) free falling at 34 m min −1, with the camera looking downwards at a mesh screen, recording impacts of luminescent organisms to obtain a vertical profile down to the abyssal sea floor, sampling at >100 l s −1; (2) rotating, with the lander on the sea floor and the camera orienting to the bottom current using a servo-controlled turntable, impacts of luminescent organisms carried by the bottom current onto a mesh screen mounted 0.5 m in front of the camera were recorded to estimate abundance in the benthic boundary layer; (3) baited, with the camera focused on a bait placed on the sea floor. Profiles recorded abundance of luminescent organisms as 26.7 m −3 at 500–999 m depth, decreasing to 1.6 m −3 at 2000–2499 m and 0.5 m −3 between 2500 m and the sea floor at 4046 m, with no further detectable significant change with depth. Rotator measurements at a 0.5 m height above the sea floor gave a mean abundance of 0.47 m −3 in the benthic boundary layer at 4046 m and of 2.04 m −3 at 3200 m. Thirty five minutes after the bait was placed on the sea floor at 3200 m, bioluminescent fauna apparently arrived at the bait and produced luminescent displays at a rate of 2 min −1. Moving, flashing light sources were observed and luminescent material was released into the bottom current.

Effective permeability of hydrophilic substances through walls of lymph vessels: roles of endothelial barrier

The wall effective permeability of hydrophilic substances labeled with fluorescent dyes was evaluated in an isolated cannulated rat single lymph vessel through a videomicroscope system. Sodium fluorescein (NaFl; 332 mol wt) and FITC-dextrans (4,400, 12,000, and 71,200 mol wt) were administered into the intraluminal space of the lymph vessels and then excited by a Xenon lamp. Changes in the fluorescence intensity of the dyes were continuously measured by a silicon-intensified target camera through appropriate filters. The net flux of each dye in the wall of the lymph vessels was calculated by the relationship between the fluorescence intensity and the concentration of the dyes. NaFl and FITC-dextran 4,400 in the intraluminal space of isolated rat lymph vessels significantly penetrated the wall of the lymph vessels. FITC-dextran 12,000 in the intraluminal space of isolated rat lymph vessels slightly passed through the lymphatic wall, whereas FITC-dextran 71,200 did not penetrate the wall. Intraluminal pressures ranging from 4 to 8 cm H(2)O did not significantly affect the net flux of dyes used in the present study. After administration of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate into the lymph vessels, the net flux of FITC-dextran 4,400 and 12,000 but not 71,200 was augmented significantly. These results suggest that small molecular hydrophilic substances (< or =4,400) are permeable from the intraluminal to extraluminal space of isolated lymph vessels and that the endothelial cell surface structure may play a barrier role in the effective permeability of large molecular hydrophilic substances (4,400 to 12,000) through the wall of the lymph vessels.

The Drosophila Homologue of the Hereditary Spastic Paraplegia Protein, Spastin, Severs and Disassembles Microtubules

Hereditary spastic paraplegias (HSPs), a group of neurodegenerative disorders characterized by lower-extremity spasticity and weakness, are most commonly caused by mutations in the spastin gene, which encodes a AAA+ ATPase related to the microtubule-severing protein katanin. A Drosophila homolog of spastin (D-spastin) was identified recently, and D-spastin RNAi-treated or genetic null flies show neurological defects, and protein overexpression decreases the density of cellular microtubules. Elucidating spastin's function and disease mechanism will require a more detailed understanding of its structure and biochemical mechanism. Here, we have investigated the effects of D-spastin, individual D-spastin domains, and D-spastin proteins bearing disease mutations on microtubules in cellular and in vitro assays. We show that D-spastin, like katanin, displays ATPase activity and uses energy from ATP hydrolysis to sever and disassemble microtubules; disease mutations abolish or partially interfere with these activities.

Spectroscopic measurements of plasma plume induced during the laser deposition of the hydroxyapatite

Plasma plume induced by ArF exeimer laser ablation of a Ca10(PO4)6(OH)2 hydroxyapatite target during deposition process has been studied in different ambient conditions, i.e. in air or water vapour. ArF laser operated at the wavelength of 193 nm with the pulse energy of 300 mJ and 20 ns pulse duration. Spectroscopic measurements of the emission spectra of plasma plume have been made with the use of a fast gate, lens coupled micro-channel plate (MCP) image intensifier placed between a spectrograph and a 1254 silicon intensified target (SIT) detector connected to an optical multichannel analyser. The electron densities of 1022 ÷ 1023m−3 have been determined from the Stark broadening of Ca I lines as a function of the distance from the target. The expansion of the plasma plume has been studied using the time of flight method. The time-dependent radiation of the 422.673 nm Ca I and 393.366 nm Ca II lines has been, registered with the use of a monochromator and photomultiplier at various distances from the target. Velocities between 104 ÷ 103 m/s have been found. The velocity in air is several times higher than in the case with water vapour. The plasma plume dynamics is also different in both cases. In the presence of water vapour the spliting of the plasma plume appears.

Fast separation of oligonucleotide and triplet repeat DNA on a microfabricated capillary electrophoresis device and capillary electrophoresis.

A laser-induced fluorescence detection system coupled with a highly sensitive silicon-intensified target (SIT) camera is successfully applied to the imaging of a band for DNA fragment labeling by fluorescence dye in a microchannel, and to the visualizing of the separation process on a microfabricated chip. We demonstrated that an only 6 mm separation channel is sufficient for the separation of triplet repeat DNA fragment and DNA molecular marker within only 12 s. The separation using the microfabricated capillary electrophoresis device is confirmed to be at least 18 times faster than the same separation carried out by conventional capillary electrophoresis with 24.5 cm effective length. The use of a short capillary with 8.5 cm effective length is also efficient for fast separation of DNA; however, the microchip technology is even faster than capillary electrophoresis using a short capillary.

Single-fluorophore imaging with an unmodified epifluorescence microscope and conventional video camera

Single fluorophores in aqueous solution were imaged in real time with a conventional silicon-intensified target video camera connected to an unmodified commercial microscope (IX70, Olympus) with epifluorescence excitation with a high-pressure mercury lamp. Neither a powerful laser nor an extremely sensitive video camera was required. Three experimental systems were used to demonstrate quantitatively that individual, moving or stationary Cy3 fluorophores could be imaged with the microscope: Cy3-gelsolin attached to an actin filament sliding over heavy meromyosin, sliding actin filaments sparsely labelled with Cy3, and heavy meromyosin labelled with one or two Cy3 fluorophores. The results should encourage many laboratories to attempt 'single-molecule physiology' in which the functions and mechanisms of molecular machines are studied at the single-molecule level in an environment where the biological machines are fully active.

In vivo rat closed spinal window for spinal microcirculation: observation of pial vessels, leukocyte adhesion, and red blood cell velocity.

An in vivo closed spinal window technique in rats was designed for observing the spinal microcirculation, such as the change of vessel diameter, leukocyte adhesion, and red blood cell (RBC) velocity, which has been very rarely examined in vivo in the spinal cord. We made a very precise closed spinal window with a laminectomy at the C5 level using a dental acrylic resin and a cover glass (7 mm in diameter). Through this closed window, the dorsal surface of the rat cervical cord was observed with a video microscope, and the fluorescent images of rhodamine 6G-labeled leukocytes and fluorescein isothiocyanate-labeled RBCs were recorded and analyzed with a silicon-intensified target tube camera (30 frames/s) and an image-intensified high-speed video camera system (1000 frames/s). During CO2 inhalation, the pial arterioles responded with vasodilation of 12.4+/-10.4% (P<0.01) in 11 arterioles of seven rats. The adhering leukocytes significantly increased in 41 venular segments of seven rats after superfusion of the neutrophil chemoattractant, N-formyl-methionine-leucine-phenylalanine solution for 15 minutes (P<0.001) but not after superfusion of only artificial cerebrospinal fluid for 15 minutes. During these experiments, no adhering leukocyte was seen in the pial arterioles. Fluorescein isothiocyanate-labeled RBCs look like shooting stars in arterioles with silicon-intensified target tube camera processing at 30 frames per second, but individual fluorescein isothiocyanate-labeled RBCs could be recognized frame by frame with the image-intensified high-speed video camera system. In 13 arterioles of four rats, the RBC velocity was 5.3+/-2.0 mm per second. This closed spinal window technique in rats is available and applicable for the study of the spinal microcirculation, such as the pathophysiology of a secondary injury.

Effect of chronic ethanol feeding on endotoxin-induced hepatic injury: role of adhesion molecules on leukocytes and hepatic sinusoid.

Endotoxin is postulated to be an important aggravating factor for alcoholic liver disease. We have previously reported that rats fed ethanol are more vulnerable to endotoxin-induced liver damage, and hepatic microcirculatory disturbance plays an important role for this liver damage by observation with an intravital microscopy. In this study, we have investigated the role of adhesion molecules in endotoxin-induced microcirculatory disturbance in chronic ethanol-fed rats. Male Wistar rats were pair-fed with ethanol liquid diet (ethanol group) or an isocaloric control diet (control group) for 6 weeks. Leukocyte adherence to the hepatic sinusoid by stimulation with lipopolysaccharides (1 mg/kg of body weight) was observed by an inverted fluorescence microscopy equipped with a silicon-intensified target camera and was found to be enhanced in ethanol-fed rats. Tumor necrosis factor-alpha and GRO/CINC-1 (rat counterpart of interleukin-8) was increased in the blood in these animals. Subsequent expression of adhesion molecules, LFA-1 beta-chain on leukocytes were demonstrated by flow cytometry, which suggests a possible involvement of leukocyte adherence to the hepatic damage in ethanol-fed animals. Preadministration of anti-rat LFA-1 beta-chain monoclonal antibody effectively suppressed leukocyte adherence to the hepatic sinusoid. These results suggest that the enhanced sequestration of neutrophils to the liver with these adhesion molecules may play a significant role in the pathogenesis of alcoholic liver disease.

Spectroscopic study of a supersonic jet of laser-heated argon plasma

A spectroscopic study of a low-pressure supersonic jet of laser-heated argon plasma is presented. The experimental set-up consisted of a high-pressure convergent nozzle and a supersonic nozzle. The supersonic nozzle was placed just behind the convergent nozzle and was connected to a low-pressure chamber. A continuous wave laser with output power of 2 kW was used to maintain the plasma in the stream of argon gas flowing from the convergent nozzle. The plasma then expanded through the supersonic nozzle. Emission spectra from the laser-sustained plasma and supersonic jet were measured with a 1.3 m focal length spectrograph and 1254 silicon intensified target (SIT) detector connected to an EG&G PARC optical multichannel analyser (OMA) III. We found that the supersonic stream of argon plasma had an electron density of - and a temperature of 6 - 7 kK.

Spectroscopic measurements of laser induced plasma during welding with CO2 laser

Results of spectroscopic measurements of laser-induced plasma under welding conditions are presented. Welding was performed with the use of a continuous-wave CO2 laser operating at a power of 2 kW. Argon or helium was used as a shielding gas. The welding metal was stainless steel or titanium. Emission spectra from plasma were measured with a spectrograph and 1254 Silicon Intensified Target detector connected to the optical multichannel analyzer. The plasma electron temperatures were determined from the relative intensities of spectral lines and the electron densities were determined either from Stark broadening of atomic line or the absolute intensity of ionic line. The distributions of the electron temperature and density over the metal surface are presented and the influence of a shielding gas on plasma parameters is demonstrated. The plasma parameters obtained were used to calculate the absorption of a laser beam in the plasma over the metal surface. No significant absorption was found in our experimental conditions.

A high-sensitivity CCD system for parallel electron energy-loss spectroscopy (CCD for EELS).

A cooled frame transfer CCD camera system was developed and tested as a parallel detector in an electron energy-loss spectrometer mounted on a transmission electron microscope. The use of a shutterless camera with a frame transfer CCD collected virtually 100% of the photon signal with a reasonably fast acquisition time. The system detective quantum efficiency was over 90% under normal experimental conditions. Because of the low channel to channel gain variations in the CCD, the signal-to-noise ratio and the detection limit were substantially better than that obtained with a silicon intensified target (SIT) camera, and direct fitting to the standard data was feasible. Quantitation at the phosphorus L edge generated from a phosphoprotein, phosvitin, showed that, under identical experimental conditions, direct fitting of spectra obtained with this CCD system gave better sensitivity than that given by the SIT camera system. Because of its larger pixel charge well, the CCD system can also operate at a much higher beam current, resulting in a significant reduction in the time required for elemental mapping at a given sensitivity.

Dynamic behaviors of growth cones extending in the corpus callosum of living cortical brain slices observed with video microscopy

During development, axons of the mammalian corpus callosum must navigate across the midline to establish connections with corresponding targets in the contralateral cerebral cortex. To gain insight into how growth cones of callosal axons respond to putative guidance cues along this CNS pathway, we have used time-lapse video microscopy to observe dynamic behaviors of individual callosal growth cones extending in living brain slices from neonatal hamster sensorimotor cortex. Crystals of the lipophilic dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil) were inserted into the cortex in vivo to label small populations of callosal axons and their growth cones. Subsequently, 400 microns brain slices that included the injection site, the corpus callosum, and the target cortex were placed in culture and viewed under low-light-level conditions with a silicon-intensified target (SIT) camera. Time-lapse video observations revealed striking differences in growth cone behaviors in different regions of the callosal pathway. In the tract, which is defined as the region of the callosal pathway from the injection site to the corresponding target cortex, growth cones advanced rapidly, displaying continual lamellipodial shape changes and filopodial exploration. Forward advance was sometimes interrupted by brief pauses or retraction. Growth cones in the target cortex had almost uniform compact shapes that were consistently smaller than those in the tract. In cortex, axons adhered to straight radial trajectories and their growth cones extended at only half the speed of those in the tract. Growth cones in subtarget regions of the callosum beneath cortical targets displayed complex behaviors characterized by long pauses, extension of transitory branches, and repeated cycles of collapse, withdrawal, and resurgence. Video observations suggested that extension of axons into cortical targets could occur by interstitial branching from callosal axons rather than by turning behaviors of the primary growth cones. These results suggest the existence of guidance cues distinct for each of these callosal regions that elicit characteristic growth cone behaviors.

Orientation imaging of microstructures

The complexity of microstructures characteristic of polycrystalline materials presents the serious investigator with many challenges. The materials engineer hopes to associate the important technological properties of these materials with specific (quantifiable) attributes of the microstructure; however, microscopy presents an overwhelming myriad of details over a wide range of scales of inquiry. Thus, the persistent question becomes: What is important in the microstructure relative to a specific property or aspect of material performance? One particular viewpoint, which stems from the modern atomistic interpretation of the structure of solids, is that for polycrystalline materials it is the spatial placement of lattice orientation that is of essential interest.The past decade has seen some remarkable progress in microdiffraction technique in conjunction with the scanning electron microscope. This progress now makes it possible to vigorously pursue the aforementioned lattice-orientational viewpoint. Since 1987 modern SIT (silicon intensified target) vidicon and CCD (charge-coupled device) cameras have been used to capture the backscattered Kikuchi diffraction (BKD) formed in stationary spot mode in the scanning electron microscope.

Nitric oxide mediates lipopolysaccharide-induced alteration of mitochondrial function in cultured hepatocytes and isolated perfused liver

The influence of endogenous nitric oxide, which is generated by stimulation with lipopolysaccharide, on the mitochondrial energization of rat hepatocytes was investigated in vitro and ex vivo. Using a fluorescence microscope equipped with a silicon intensifier target camera, we visualized fluorescence of rhodamine-123, a mitochondrial energization-sensitive fluorescence probe, in individual hepatocytes and measured the fluorescence intensity with a digital imaging processor. Although addition of Kupffer cells or lipopolysaccharide in a range of 0.1 to 1.0 microgram/ml caused no significant alteration in the fluorescence in hepatocytes, Kupffer cells plus 1.0 microgram/ml lipopolysaccharide reduced fluorescence intensity in the cocultured hepatocytes. The alteration of rhodamine-123 fluorescence in the hepatocytes induced by lipopolysaccharide-activated Kupffer cells was significantly inhibited by the addition of NG-monomethyl-L-arginine, a selective inhibitor of nitric oxide synthesis. The transportal infusion of lipopolysaccharide also decreased rhodamine-123 fluorescence in perfused rat liver. The decrease was significantly enhanced in the pericentral regions. Autofluorescence of NADH was elicited by continuous infusion of lipopolysaccharide; this reaction was also enhanced in the pericentral regions. We showed the main site of uptake of infused lipopolysaccharide in the hepatic lobule to be in the periportal regions with fluorescein isothiocyanate-labeled lipopolysaccharide. Our results indicate that the inhibition of mitochondrial energization occurs mainly in pericentral regions, which are distant from the lipopolysaccharide uptake site. The continuous administration of NG-monomethyl-L-arginine significantly attenuated the lipopolysaccharide-induced decrease in rhodamine-123 fluorescence and increase of the NADH contents of the hepatic lobule.(ABSTRACT TRUNCATED AT 250 WORDS)

Nitric oxide mediates lipopolysaccharide-induced alteration of mitochondrial function in cultured hepatocytes and isolated perfused liver

The influence of endogenous nitric oxide, which is generated by stimulation with lipopolysaccharide, on the mitochondrial energization of rat hepatocytes was investigated in vitro and ex vivo. Using a fluorescence microscope equipped with a silicon intensifier target camera, we visualized fluorescence of rhodamine-123, a mitochondrial energization–sensitive fluorescence probe, in individual hepatocytes and measured the fluorescence intensity with a digital imaging processor. Although addition of Kupffer cells or lipopolysaccharide in a range of 0.1 to 1.0 νg/ml caused no significant alteration in the fluorescence in hepatocytes, Kupffer cells plus 1.0 μg/ml lipopolysaccharide reduced fluorescence intensity in the cocultured hepatocytes. The alteration of rhodamine-123 fluorescence in the hepatocytes induced by lipopolysaccharide-activated Kupffer cells was significantly inhibited by the addition of NG-monomethyl-L-arginine, a selective inhibitor of nitric oxide synthesis. The transportal infusion of lipopolysaccharide also decreased rhodamine-123 fluorescence in perfused rat liver. The decrease was significantly enhanced in the pericentral regions. Autofluorescence of NADH was elicited by continuous infusion of lipopolysaccharide; this reaction was also enhanced in the pericentral regions. We showed the main site of uptake of infused lipopolysaccharide in the hepatic lobule to be in the periportal regions with fluorescein isothiocyanate–labeled lipopolysaccharide. Our results indicate that the inhibition of mitochondrial energization occurs mainly in pericentral regions, which are distant from the lipopolysaccharide uptake site. The continuous administration of NG-monomethyl-L-arginine significantly attenuated the lipopolysaccharide-induced decrease in rhodamine-123 fluorescence and increase of the NADH contents of the hepatic lobule. These results suggest that nitric oxide mediates the lipopolysaccharide-activated, Kupffer cell–induced inhibition of mitochondrial electron transport in hepatocytes. (HEPATOLOGY 1993;18:380–388).

Oxyradical Generation from Leukocytes during Endotoxin-Induced Microcirculatory Disturbance in Rat Mesentery-Attenuating Effect of Cetraxate

By using the intravital microscope equipped with digital imaging processor, we investigated the granulocyte-mediated oxidative burst during the endotoxin-induced microvascular derangement in rat mesentery. The leukocyte behavior after the injection of acridine orange was detected by using a silicon-intensified target camera, the erythrocyte velocity was measured by using a high-speed video camera system, and the luminol-dependent photoemission was visualized by an ultrasensitive photon-counting camera in lipopolysaccharide (LPS)-treated micro-vascular beds. At 60 min after the LPS administration, a significant leakage of FITC-labeled albumin was observed along mesenteric venules under a fluorescence microscopy. The number of sticking leukocytes increased in association with the decrease in erythrocyte velocity after starting the LPS infusion. The luminol-dependent chemiluminescence in microvascular beds gradually increased over that recorded prior to LPS exposure and was fourfold higher 60 min after the start of LPS infusion. The distribution of the photoemission clearly corresponded to the venular endothelium, to which leukocytes adhered. In blood samples taken from the mesenteric vein at 60 min after the LPS administration, a decrease in the number of granulocytes and increases of total and individual chemiluminescence activities were observed. These results suggest that LPS induces oxidative burst from granulocytes on the venular endothelium. Cetraxate, an inhibitor of proteases including plasmin, significantly inhibited the leukocyte activation and prevented alterations in microvascular hemodynamics induced by LPS in vivo, whereas it had no effect on the LPS-induced oxyradical generation from adherent leukocytes in vitro. The present study demonstrates that proteases such as plasmin may play an important role in the pathogenesis of endotoxin-induced microvascular disturbances.