Objective: We have shown that High-Intensity Focused Ultrasound (HIFU) can effectively control bleeding from injuries to solid organs such as liver, spleen, and lung. Achievement of hemostasis was augmented when a homogenate of tissue and blood was formed. The objective of this study was to investigate quantitatively the effect of homogenate production on HIFU application time for hemostasis. Possible mechanisms involved in homogenate production were also studied.Methods: Ten anesthetized rabbits had laparotomy and liver exposure. Liver incisions, 15-25 mm long and 3-4 mm deep, were made followed immediately by HIFU application. Two electrical powers of 80 and 100 W corresponding to focal acoustic intensities of 2264 and 2829 W/cm(2) respectively were used. Tissue and homogenate temperatures were measured. Smear and histological tissue sample analysis using light microscopy were performed.Results: In treatments with homogenate formation, hemostasis was achieved in 76 +/- 1.3 s (Mean +/- Standard Error Mean: SEM) at 80 W. In treatments without homogenate formation (at 80 W), hemostasis was achieved in 106 +/- 0.87 s. At 100 W, hemostasis was achieved in 46 +/- 0.3 s. The time required for homogenate formation, at 80 and 100 W were 60 +/- 2.5 and 23 +/- 0.3 s, respectively. The homogenate temperature was 83 degreesC (SEM 0.6 degreesC), and the non-homogenate tissue temperature at the treatment site was 60 degreesC (SEM 0.4 degreesC). The smear and histological analysis showed significant blood components and cellular debris in the homogenate with some intact cells.Conclusion: The HIFU-induced homogenate of blood and tissue resulted in a statistically significant shorter HIFU application time for hemostasis. The incisions with homogenate had higher temperatures as compared to incisions without homogenate. Further studies of the correlation between homogenate formation and temperature must be done. as well as studies on the long-term effects of homogenate in achieving hemostasis. (C) 2004 Elsevier B.V. All rights reserved.

Objective: High-intensity focused ultrasound (HIFU) has been shown to provide an intra-operative method for arresting active bleeding in liver, spleen, and lung. The efficacy of HIFU has been observed to increase significantly when a homogenate of blood and tissue is formed at the treatment site. Here, we report on the investigation of the HIFU parameters (power and duration) for optimal formation of the homogenate in the liver and the mechanisms involved in its formation. Methods: Ten rabbits were anesthetized, their livers exposed surgically, and incisions (1.5 cm long, 4 mm deep, and 1 cm apart) were made. HIFU was applied within 10 seconds using a solid titanium conical applicator, operating at 5.47 MHz in CW mode. Two HIFU electrical power settings of 100 and 80 Watts were empirically determined and used for achieving hemostasis, with and without the formation of the homogenate, respectively. Thermal measurements of the treatment site were also performed. A total of 58 incisions at 100 W and 74 incisions at 80 W were treated. Tissue samples were obtained for histological examination. Results: The mean HIFU application time required to achieve hemostasis was significantly shorter when homogenate was formed (46 s) compared to when no homogenate was formed (106 s) (p<0.05). The mean temperature at the treatment site was lower for non-homogenate (60 deg. C) vs. homogenate (82 deg. C) treatment (p<0.05). Histological analysis of the treatment sites with homogenate formation showed a mixture of disrupted erythrocytes, neutrophils, and lymphocytes. The mixture was mainly composed of blood with a few recognizable epithelial cells and a large concentration of fibrin. At the treatment sites with no homogenate formation, coagulative necrosis was observed with a minimal amount of fibrin and other blood products. Conclusions: The formation of homogenate appears to enhance acoustic hemostasis, resulting in short HIFU application times. The mechanism responsible for homogenate formation appears to involve both thermal (boiling) and mechanical effects (cavitation) of HIFU. Further investigations of optimal intra-operative HIFU parameters should improve the safety of the HIFU application.

High-intensity focused ultrasound (HIFU) and conventional B-mode ultrasound (US) imaging were synchronized to develop a system for real-time visualization of HIFU treatment. The system was tested in vivo in pig liver. The HIFU application resulted in the appearance of a hyperechoic spot at the focus that faded gradually after cessation of HIFU exposure. The duration of HIFU exposure needed for a hyperechoic spot to appear, was inversely related to the HIFU intensity. The threshold intensity required to produce a hyperechoic spot in liver in < is was 970 W/cm(2), in situ. At this HIFU dose, no immediate cellular damage was observed, providing a potential for pretreatment targeting. The real-time visualization method was used in hemostasis of actively bleeding internal pelvic vessels, allowing targeting and monitoring of successful treatment. Real-time US imaging may provide a useful tool for image-guided HIFU therapy. CE-mail: adasi@u.washington.edu) (C) 2001 World Federation for Ultrasound in Medicine & Biology.

BACKGROUND:Platelet-activating factor (PAF) is one of the most potent biological mediators of tissue injury. PAF acetylhydrolase (PAF-AH) is a recently isolated naturally occurring enzyme that hydrolyzes PAF and renders it inactive. We hypothesize that inhibition of PAF with PAF-AH will reduce myocardial ischemia-reperfusion (I/R) injury in vivo.METHODS AND RESULTS:The coronary ligation model was used in New Zealand white rabbits. The large branch of the marginal coronary artery was occluded for 45 minutes, followed by 2 hours of reperfusion. Fifteen minutes before reperfusion, animals were given either 2 mg/kg of vehicle or of PAF-AH. At the completion of 120 minutes of reperfusion, percentage of necrosis, degree of neutrophil infiltration, and measurements of regional contractility were assessed. Data are expressed as the mean+/-SEM and compared by Student's t test or Mann-Whitney ANOVA. Both groups of animals showed an equivalent area at risk; however, 46.7+/-11% was necrotic in the animal treated with vehicle. In contrast, 20.9+/-7.0% was necrotic in the animals treated with PAF-AH (P<0.05). Systolic shortening and wall thickness were significantly greater in those animals treated with PAF-AH at 15, 30, 60, and 120 minutes of reperfusion (P<0.05). Quantification of neutrophil infiltration showed a 62% reduction in the PAF-AH treated animals compared with those treated with vehicle alone.CONCLUSIONS:PAF-AH is a potent cardioprotective agent in an in vivo model of I/R injury.

Several mechanisms are postulated to be responsible for acoustically-induced hemostasis. Both mechanical and thermal aspects of ultrasound energy are thought to play a role. Thermal effects may be important when tissue shrinkage is necessary to close a bleeding site. A moderate temperature rise may also accelerate clotting when hypothermia and coagulopathy is present. Tissue welding via collagen restructuring may be promoted via a thermal effect. The mechanical effects of streaming and radiation pressure may assist in stopping the flow of blood out of a wound and, if directed properly, may cause insertion of blood vessel wall material into a breached region to plug the hole, and aid the natural clotting mechanism. Streaming may provide shearing forces on platelets, facilitating their activation for subsequent aggregation and adhesion to the vessel wall. Cavitation may produce tissue disruption that exposes collagen and tissue factors to platelets thus assisting in triggering clotting. These mechanisms are discussed, and supportive evidence given

Background: High-intensity focused ultrasound (HIFU) has been shown to control bleeding from liver incisions, and blood vessel punctures and incisions. The objective of the current study was to investigate the capability of HIFU to stop bleeding from splenic injuries in a pig model. Methods: Surgical incisions, 25 to 50 mm in length and 2 to 8 mm in depth, were made in the spleens of five anesthetized pigs. HIFU with a frequency of 5 MHz was applied within 5 seconds of making the incision. A total of 39 incisions and HIFU treatments were performed. Results: Bleeding from all incisions was stopped completely after HIFU treatment. The average times to control and completely arrest the hemorrhage were 28 and 55 seconds, respectively. The mechanisms of hemostasis appeared to be thermally induced coagulation necrosis of splenic tissue and occlusion of blood vessels by a mechanically induced homogenized splenic tissue. Conclusion: HIFU may provide a useful method of hemostasis for actively bleeding spleen. Because of its ability to induce hemostasis at adjustable depth, HIFU may prove to be a useful cauterization method both in the operating room and for patients who are managed nonoperatively.

BACKGROUND One proinflammatory property observed during endothelial cell activation is the expression of the neutrophil adhesion molecule E-selectin on the surface of endothelial cells. An important regulatory element in endothelial cell E-selectin expression is the nuclear localization of the transcription factor nuclear factor (NK)-kappa B, which binds to and affects the function of several genes encoding proteins mediating inflammation. METHODS AND RESULTS In this study, we investigated the ability of pyrrolidine dithiocarbamate (PDTC), an agent that inhibits the nuclear localization of NF-kappa B, to (1) block endothelial cell E-selectin expression in vitro in response to tumor necrosis factor (TNF)-alpha, interleukin (IL)-1, and lipopolysaccharide (LPS) and (2) reduce neutrophil infiltration in a rabbit model of systemic inflammation. As measured with the use of an enzyme-linked immunosorbent assay, TNF-alpha, IL-1, and LPS each induced a significant increase in surface expression of E-selectin in cultured human umbilical vein endothelial cells (HUVECs) compared with HUVECs treated with medium alone. In contrast, E-selectin surface expression was blocked in HUVECs pretreated with PDTC before TNF-alpha, IL-1, or LPS stimulation. NF-kappa B was present in HUVEC nuclei treated with TNF-alpha, whereas translocation of NF-kappa B to the nucleus was absent in TNF-alpha-treated HUVECs pretreated with PDTC. In vivo, rabbits pretreated with PDTC before LPS infusion showed significantly less neutrophil infiltration in the lungs, liver, and heart compared with animals infused with LPS alone. This correlated with a reduction in E-selectin expression in vivo. CONCLUSIONS Our data suggest that NF-kappa B regulation of gene expression in the vascular endothelium may be an important cellular mechanism in endothelial cell activation.

The rat mast cell line RBL-2H3.1 contains an 85-kDa cytosolic phospholipase A2 (cPLA2) that is very likely involved in liberating arachidonate from membrane phospholipid for the synthesis of eicosanoids following stimulation with either calcium ionophore or IgE/antigen. In this study, the intracellular location of cPLA2 was determined using immunofluorescence microscopy and immuno-gold electron microscopy. In nonstimulated cells, cPLA2 is distributed throughout the cytosol and is excluded from the nucleoplasm. Following cell activation with calcium ionophore, most of the cPLA2 translocates to the nuclear envelope, and the enzyme remains there during the entire period that ionophore is present. With IgE/antigen stimulation for 5 min, approximately 20-30% of the cPLA2 translocates to the nuclear envelope, and after 30 min of stimulation, most of the enzyme returns to the cytosol. Measurement of intracellular calcium using the dye Fura-2/AM shows that the level of calcium rises immediately after antigen is added, remains high for about 30 s, and then declines back to resting levels. Activation with calcium ionophore produces a 10-fold larger release of arachidonate than does stimulation with IgE/antigen. Thus, the results suggest that the extent of membrane binding of cPLA2 correlates with the release of arachidonate and that the site of arachidonate liberation is the nuclear envelope where many of the enzymes that oxygenate this fatty acid are located.

Although infection by group A streptococci is a model of extracellular mucosal pathogenesis, these organisms can be associated with highly invasive infections resulting in sepsis and shock. Over the last 6 yr this species has renewed its reputation as a significant cause of sepsis and has piqued interest in the mechanism by which some strains are better able to breach mucosal barriers to gain access to the bloodstream than are others. An internalization assay was developed on the basis of resistance of intracellular streptococci to penicillin and gentamicin. Experiments showed that stationary-phase, as opposed to logarithmic-phase, bacteria are efficiently internalized and can persist in cultured human cells. Electron microscopy confirmed that streptococci were contained within intracellular vacuoles. Various strains of streptococci revealed significant differences in their capacity to be internalized. Two type M1 streptococci isolated from blood infections were internalized at frequencies equal to those reported for Salmonella and Listeria monocytogenes and greater than the frequency of a clonal variant from a case of pharyngitis.

Users of the Hitachi S-900 field emission SEM attempting to do high resolution topographical studies of cell surfaces (or macromolecular assemblies) and correlative localization of colloidal gold probes are handicapped by imaging conditions related to contamination or radiation damage of the specimen surface since secondary (SE) and backscatter (BS) electron imaging must be done consecutively.We have developed a digitial acquisition system for the S-900 FESEM using an analog/digital conversion board on the VME bus of a Silicon Graphics IRIS, using the conventional and graphics memory for image storage, rather than using a frame grabber with its internal A/D and frame memory. A DT-1492-G board from Data Translation with a 250 kHz throughput was used for the 12-bit A/D circuitry. We collect data at 12-bit resolution, and use the frame buffer memory on the IRIS for separately converting, and then displaying as 8-bit (greyscale, 256 levels) SE and BS electron images.

Current concepts of the pathogenesis of lung injury and repair are derived from in vitro cellular and in vivo investigations. Studies with viable ex vivo models may offer additional insights into disease processes, since essential cellular interactions would be maintained. However, a major limiting factor has been the availability of a model that maintains normal parenchymal structure, viability, and homeostasis beyond 4 wk in serum-free conditions. We have succeeded in establishing an ex vivo lung culture system which reproducibly maintains parenchymal architecture for up to 9 wk. Our method is a simple, modified version of previously utilized techniques. Thin slices of mature murine lung were inflated with agar-defined medium and cultured on Gelfoam saturated with serum-free medium. Normal pulmonary parenchyma, with the exception of endothelial cells, was maintained for up to 60 days as assessed chronologically by light and electron microscopy. The integrity of the microvasculature and endothelial cells was lost beyond 7 days. The adult lung ex vivo culture system maintained necessary epithelial and interstitial cellular interactions in the alveolar wall without systemic circulatory influences. Future studies with this model may provide important insights in assessing the pathogenesis of many acute and chronic lung diseases and clarify existing controversies raised from in vitro and in vivo studies.

The interaction of Pseudomonas aeruginosa with a human lung pneumocyte cell line (A549) was studied. Wild-type strain PAK adhered efficiently to the A549 cells, while an isogenic mutant, carrying a mutation in the pilin structural gene, adhered at 10 to 20% of the wild-type levels. Another nonpiliated mutant of P. aeruginosa PAK, defective in the pleiotropic regulatory gene rpoN, did not adhere to A549 cells, suggesting the presence of a second, RpoN-controlled adhesin on the bacterial surface. Endocytosis of wild-type P. aeruginosa PAK by A549 cells was also demonstrated. A significant fraction of the internalized bacteria were recovered in a viable form after several hours of residence within the A549 cells. When examined by electron microscopy, intracellular bacteria were located in membranous vesicles, and no evidence of killing by lysosomal mechanisms was observed. These studies raise the possibility that during chronic respiratory tract infections in immuno-compromised patients, P. aeruginosa may persist in intracellular compartments and therefore be protected from the defense mechanisms of the host.

Microthrombi found in the pulmonary capillaries in patients dying with post-traumatic pulmonary insufficiency suggests that pulmonary microembolism (PME) may be etiologically important, but a temporal relationship has not been demonstrated. We used a modified Lim-Blaisdell model of PME to cause a severe ischemic soft tissue injury in dogs. The appearance of microaggregates (MA) in the venous circulation was measured using a laser optical scanning technique. The effect of MA on pulmonary physiologic and histologic parameters was measured and compared to control animals. In the ischemic soft tissue injury group, following restoration of local circulation, the platelet count dropped by 72% (P less than 0.00002), the number of MA increased by 800% (P less than 0.00002), the mean pulmonary artery pressure (PA) increased from 15.6 to 32 mm Hg (P less than 0.00002), and electron micrographs of lung obtained at 4 hr after ischemic insult revealed PM with severe lung injury that was consistent with a capillary membrane leak. The control group never demonstrated a significant change in platelets, MA, PA, or histologic lung injury. These findings imply that MA found in the pulmonary microcirculation are temporally related to the development of physiologic and anatomic lung abnormalities.