A surgical navigation system for aortic vascular surgery: a practical approach.

In aortic vascular surgery, a navigation system must represent the anatomical map of individual patient in order to detect the important artery. To provide a proper fit for positions along the dorsoventral axis, the spinous process was added to a currently used anatomical point set consisting of four anterior body landmarks. In addition, we attempted to reduce the registration error by compensating for alignment errors resulting from variations in tissue thickness at each landmark. The alignment values were examined using a human phantom consisting of a skeleton model with subcutaneous tissue in the semilateral position. Using this method, a phantom simulation and five clinical trials were performed. Target errors were evaluated at the orifice of the intercostal artery. In the phantom simulation, the error at the target point was 4.1 ± 2.7 mm. However, for one patient undergoing thoracoabdominal aortic aneurysm replacement surgery, the target error was 8.0 mm using the proposed method.

Serum keratan sulfate is a promising marker of early articular cartilage breakdown.

OBJECTIVES: To find serum markers that may serve as indices for an early diagnosis of degeneration or damage of the articular cartilage. METHODS: Twenty-four healthy volunteers, 19 individuals with knee trauma (KT) and 31 with knee osteoarthritis (OA) were evaluated. KT patients were divided into a group (n = 5) with an injury <2 months old (recent KT) and a group (n = 14) with that >2 months old (old KT). Articular cartilage damage was assessed using either arthroscopy or direct observation. Serum concentrations of hyaluronic acid (HA), cartilage proteoglycan aggrecan turnover epitope (CS846) and cartilage oligomeric protein (COMP) were measured using enzyme-linked immunosorbent assay kits and those of keratan sulfate (KS) and chondroitin-6-sulfate (C6S) using high-performance liquid chromatography. RESULTS: Serum KS in the recent KT group (2095 +/- 594 ng/ml) was significantly higher than that in the old KT group (1373 +/- 418 ng/ml; P = 0.021), and serum COMP in the recent KT group (1572 +/- 182 ng/ml) showed a tendency that was higher than that in the old KT group (1350 +/- 250 ng/ml; P = 0.079). Serum KS in OA patients with Kellgren and Lawrence (KL) grades 0 and I (1456 +/- 334 ng/ml) showed a tendency that was higher than that in OA patients with KL grades II, III and IV (1248 +/- 220 ng/ml; P = 0.084). CONCLUSIONS: The serum concentration of KS correlated with the damage of the articular cartilage and it was significantly increased even at an early stage after the injury.

Dynamic imaging of pancreatic diseases by contrast enhanced coded phase inversion harmonic ultrasonography.

BACKGROUND: Coded phase inversion harmonic ultrasonography, a newly available sonographic technique, enables visualisation of slow flow in minute vessels in a real time fashion with the use of a sonographic contrast agent containing monosaccharide. Our purpose was to employ this novel technique to observe microvessels in pancreatic tumours. SUBJECTS AND METHODS: Sixty five patients with suspicious pancreatic tumours received contrast enhanced coded phase inversion harmonic ultrasonography, contrast enhanced computed tomography, and endosonography. Final diagnoses based on histological findings were pancreatic ductal carcinomas in 49 patients, inflammatory pseudotumours with chronic pancreatitis in seven, and endocrine tumours in nine. For contrast enhanced coded harmonic ultrasonography, Levovist, a contrast agent, was injected intravenously as a bolus. When the first microbubble signal appeared in the pancreas, images of the ideal scanning plane were displayed in a real time continuous fashion (vessel images). Subsequently, interval delay scanning (perfusion images) was taken to demonstrate parenchymal flow. Tumour vascularity was evaluated by using the two types of imaging. Sensitivities for depicting pancreatic tumours were compared between three examinations. RESULTS: Contrast enhanced ultrasonography demonstrated tumour vessels in 67% of pancreatic ductal carcinomas, although most were relatively hypovascular compared with the surrounding pancreatic tissue. The vascular patterns of tumours obtained by contrast enhanced ultrasonography were closely correlated with those obtained by contrast enhanced computed tomography. Values for sensitivity in depicting pancreatic tumours of 2 cm or less in size were 68% for contrast enhanced computed tomography, 95% for endosonography, and 95% for contrast enhanced ultrasonography. CONCLUSION: Contrast enhanced coded phase inversion harmonic ultrasonography successfully visualised fine vessels in pancreatic tumours and may play a pivotal role in the depiction and differential diagnosis of pancreatic tumours.

The inhibitory mechanism of gap junctional intercellular communication induced by polyethylene and the restorative effects by surface modification with various proteins.

Gap junctional intercellular communication (GJIC) is a function that plays an important role in maintaining cell and tissue homeostasis and in regulating cell growth, development, and differentiation. Change in this function of V79 fibroblasts cultured on polyethylene films modified with albumin or collagen was estimated using fluorescence redistribution after photobleaching (FRAP) analysis. The GJIC function of V79 cells on nontreated polyethylene was strongly inhibited in comparison with those on a glass coverslip. When the cells were culture on collagen-immobilized polyethylene film, this function was recovered to about 70% of the cells cultured on the coverslip. However, albumin immobilization did not recover the function as much as collagen immobilization. Western blotting analysis and immunostaining of connexin 43, which is a major protein constituting gap junctional channel of these cells, revealed its abnormal expression and distribution in the cells on nontreated polyethylene, whereas its almost normal distribution was observed in the cells on collagen-immobilized polyethylene. This abnormal expression and distribution of connexin 43 induced by the surface of polyethylene may be ascribed to a strong inhibition of GJIC of V79 fibroblasts.

Studies on in vitro evaluation for the biocompatibility of various biomaterials: inhibitory activity of various kinds of polymer microspheres on metabolic cooperation.

Gap junctional intercellular communication is a function that plays an important role in maintaining cell and tissue homeostasis and in regulating cell growth, development, and differentiation. Change in this function when contacting fibroblasts with various polymer microspheres was estimated using the metabolic cooperation assay system. When the cells were in contact with the microspheres after their adhesion onto a substrate, the function did not alter. However, when they were in contact with precoated microspheres on test dishes, the function was inhibited as the quantity of microspheres increased. Moreover, the inhibition level increased as the diameters of polyethylene and polystyrene microspheres decreased. However, no inhibition was observed if precoated microspheres were composed from poly(L-lactic acid). These findings suggest that the size and the material of microspheres, and how cells recognize the microspheres, are factors affecting cell function of gap junctional intercellular communication. Therefore, estimating this function may provide valuable information about the biocompatibility of many kinds of materials even in the form of particles.

Differential activation of brain-derived neurotrophic factor gene promoters I and III by Ca2+ signals evoked via L-type voltage-dependent and N-methyl-D-aspartate receptor Ca2+ channels.

Although the brain-derived neurotrophic factor (BDNF) gene is activated by the intracellular Ca(2+) signals evoked via Ca(2+) influx into neurons, little is known about how the activation of alternative BDNF gene promoters is controlled by the Ca(2+) signals evoked via N-methyl-d-aspartate receptors (NMDA-R) and L-type voltage-dependent Ca(2+) channels (L-VDCC). There is a critical range in the membrane depolarization caused by high K(+) concentrations (25-50 mm KCl) for effective BDNF mRNA expression and transcriptional activation of BDNF gene promoters I and III (BDNF-PI and -PIII, respectively) in rat cortical culture. The increase in BDNF mRNA expression induced at high K(+) was repressed not only by nicardipine, an antagonist for L-VDCC, but also by dl-amino-5-phosphonovalerate, an antagonist for NMDA-R, which was supported by the effects of antagonists on the Ca(2+) influx. Although the promoter activations at 25 and 50 mm KCl were different, BDNF-PIII was activated by either the Ca(2+) influx through NMDA-R or L-VDCC, whereas BDNF-PI was predominantly by the Ca(2+) influx through L-VDCC. Direct stimulation of NMDA-R supported the activation of BDNF-PIII but not that of BDNF-PI. Thus, the alternative BDNF gene promoters responded differently to the intracellular Ca(2+) signals evoked via NMDA-R and L-VDCC.

Studies on the mechanisms of tumorigenesis induced by polyetherurethane in rats: production of superoxide, tumor necrosis factor, and interleukin 1 from macrophages cultured on different polyetherurethanes.

Interaction of macrophages (Mos) with polyetherurethane (PEU) was investigated to clarify the role of the Mos in the early stage of tumorigenesis of PEUs. As for the inflammatory cytokines produced from Mos, the amount of tumor necrosis factor (TNF) produced on three PEUs (PU-4, 6, and 8) was similar, but less than that on a control glass dish. Interleukin 1 (IL-1) production levels on the PEUs, especially on PU-6, also decreased in comparison with the glass dish. Superoxide (O(2)(-)) release from Mos on the PEUs was similar or more than that on the glass dishes within 24 h incubation. In particular, the O(2)(-) release on PU-6, possessing the lowest tumorigenic potential in vivo, reached the highest level among the PEUs tested. To clarify the causes that enhanced O(2)(-) release from Mos, the methanol extracts of the three PEUs and chemicals that constitute the PEUs were tested. The extracts and 1,4-butanediol did not show an effect on O(2)(-) release. However, 4, 4'-di(ethoxycarboamide) diphenylmethane, a model hard segment of PEU, was remarkably enhanced, whereas poly(tetramethyleneoxide), a soft segment of PEU, reduced the O(2)(-) release in a dose-dependent manner. From these results, although it is still unclear what affects different types of O(2)(-) production, lower tumorigenic potentials of PU-6 may be caused by O(2)(-) release from Mos, which play an important role in the tumoricidal process. Further, the low IL-1 production on PU-6 suggests that PU-6 suppresses inflammatory responses other than O(2)(-) production in vivo, resulting in lower tumorigenic potentials. On the other hand, TNF production was almost similar when Mos were cultured on three PEUs, suggesting TNF did not play a role in the different tumorigenic potentials of PEUs.

Silencer-mediated repression and non-mediated activation of BDNF and c-fos gene promoters in primary glial or neuronal cells.

Although the neuron-restrictive silencer element (NRSE/Regard) has been shown to function as a negative-acting DNA regulatory element to prevent the expression of neuron-specific genes in non-neuronal cells, little is known about its silencing effect on transcription in primary glial cells nor its effect on transcriptional activation in primary neurons. By DNA transfection in primary cultures of rat cortical neuronal or glial cells, we investigated the effect of NRSE on transcription mediated by the BDNF promoter I or c-fos promoter to which NRSE sequences derived from the SCG10 gene were linked. Transfection of plasmid DNAs to NIH3T3 fibroblasts resulted in a marked repressive effect of NRSE on BDNF promoter I- or c-fos promoter-mediated transcription. In primary neuronal cells, however, NRSE did not repress the basal promoter activities of BDNF and c-fos genes and allowed the transcriptional activation of these genes induced by membrane depolarization although NRSE slightly reduced the magnitude of BDNF promoter I activation. In contrast to neuronal cells, a marked repression of basal promoter activities of both genes was detected in primary glial culture and a two base pair-mutation of NRSE partially recovered the repression. These results indicate that NRSE negatively acts on its linked promoters in primary glial cells and does not interfere an activation of linked promoters in neuronal cells.

Inducibility of BDNF gene promoter I detected by calcium-phosphate-mediated DNA transfection is confined to neuronal but not to glial cells.

Although DNA transfection with calcium-phosphate/DNA precipitates for promoter analysis of genes has been previously applied to primary cultures of neuronal cells, it remains uncertain whether the expression of the introduced genes in glial cells, which are also in primary culture, affects the transcriptional signals obtained. Using plasmid DNA containing the brain-derived neurotrophic factor (BDNF) gene promoter I or c-fos promoter, we investigated the optimum conditions for calcium-phosphate-mediated DNA transfection in primary culture of rat cortical neuronal cells. Normalizing the firefly luciferase activity of the experimental reporter gene to the Renilla luciferase activity of the internal control increased experimental reliability. Maximum expression of firefly luciferase activity in the cells stimulated by membrane depolarization required 6-12 hrs of incubation after stimulation. Differences in the ratio of the experimental reporter gene to the internal control did not affect experimental gene expression. Under our optimal conditions, the activation of the BDNF gene promoter I was detected in neuronal but not in glial cells. Calcium-phosphate-mediated DNA transfection should be widely applicable for promoter analysis of inducible genes in neurons.

Studies on tumor-promoting activity of polyethylene: inhibitory activity of metabolic cooperation on polyethylene surfaces is markedly decreased by surface modification with collagen but not with RGDS peptide.

Tumor promotion activity of polyethylene (PE) was estimated in terms of the inhibitory potentials on the gap-junctional intercellular communication using the metabolic cooperation assay. The gap-junctional intercellular communication of test cells was inhibited on the PE film, but this inhibitory activity was markedly decreased when the surface of the PE film was immobilized with collagen. These results suggest that the in vivo tumor promotion activity of the untreated PE may be stronger than that of collagen-immobilized PE. On the other hand, surface modification with RGDS peptide, which is well known as the sequence of cell attachment domain in extracellular matrix proteins, did not reduce the promotion activity of PE film. In addition, neither modification with bovine serum albumin nor RGES peptide reduced the activity of PE film. These findings suggest that reduction of the inhibitory activity on gap-junctional intercellular communication by collagen immobilization is not simply due to improved cell adherence via the RGDS sequence but to some cell-cell recognition via collagen molecules essential for the gap-junctional intercellular communication.

Production of interleukin 1 from macrophages incubated with poly (DL-lactic acid) granules containing ovalbumin.

The production profile of interleukin 1 (IL-1) from mouse peritoneal macrophages (M phi) was determined following their incubation with poly(DL-lactic acid) (PDLLA) granules containing ovalbumin (OVA). Upon incubation, M phi produced IL-1 at a significantly high rate compared with those incubated with OVA in the free form or OVA-free granules. A simple mixture of empty granules and free OVA exhibited the same level of IL-1 production as induced by free OVA alone. IL-1 production by the granules with a fixed OVA loading increased with an increase in their amount added to M phi. When incubated with a fixed amount of granules containing OVA of different loadings, M phi produced more IL-1 with an increase in the total OVA amount, but the IL-1 production decreased at OVA loadings higher than 10%. The presence of free OVA enhanced IL-1 production with the increased addition of empty granules, but the level induced by OVA loaded in granules was higher than that by mixtures of free OVA and empty granules, when compared at a similar OVA dose, irrespective of the absolute amount of PDLLA added. These findings indicate that the sustained release of OVA from the granules is critical to enhance the OVA-induced IL-1 production, in contrast to the OVA release accompanying a large initial burst, which reduced IL-1 production. It was concluded that the direct contact of PDLLA granules with M phi and the subsequent sustained release of OVA around M phi effectively activated M phi, resulting in enhanced IL-1 production.

Adjuvant effect of biodegradable poly(DL-lactic acid) granules capable for antigen release following intraperitoneal injection.

Ovalbumin (OVA)-containing poly(DL-lactic acid) (PDLLA) granules were prepared with different conditions. Following the intraperitoneal (i.p.) immunization of mice with the granules containing OVA, production of anti-OVA IgG antibody in the mouse serum was investigated. The i.p. injection of the granules induced a strong antibody production compared with that of free OVA, irrespective of the amount of OVA released for initial a few weeks and the period of OVA release. The serum level of IgG antibody induced by the granules was retained at a high level over 16 weeks although the period of OVA release and the amount of OVA released initially were different from each other. The initial OVA release for a few weeks was essential to induce the enhanced antibody production. Comparison of mice immunization by granules with different OVA loadings but at a similar dose revealed that antibody level was higher for the granules with lower loading than for those with the higher loading. However, when the granules were injected after encapsulation into a poly(vinyl alcohol) (PVA) hydrogel tube, the difference in their antibody level became insignificant. Because PVA encapsulation did not affect the OVA release profile, this finding indicates that the injection amount of the granules seems to have influenced the antibody production. We conclude that the release profile of OVA is not always a key factor to enhance the antibody production of OVA-containing granules so far as the initial OVA controlled release is achieved.

Size effect on the antibody production induced by biodegradable microspheres containing antigen.

Poly(L-lactic acid) (PLLA) microspheres containing a model antigen, ovalbumin (OVA), were prepared by the evaporation method using double emulsion, and fractionated into different sizes by counterflow elutriation. Following the intraperitoneal (i.p.) and subcutaneous (s.c.) injection of the microspheres to mice, the titer of anti-OVA antibody in the serum was measured to assess the size effect on the profile of antibody production. OVA was released from the microspheres for 80 days, irrespective of the microsphere size. In both the s.c. and i.p. immunization, the serum level of anti-OVA IgG antibody in the mice induced by the microspheres containing OVA was higher than that of free OVA when compared at the same dose. The serum level of antibody in the mice i.p. injected with the microspheres tended to increase with the decreasing size. On the other hand, in the s.c. immunization, the microsphere size had little influence on the antibody production. It is possible that the injected microspheres tend to aggregate in the s.c. tissue, disappearing the size effect on the antibody production. Since the amount of microspheres injected increases with the decreasing size when their OVA loading is fixed, the increase in the amount will promote the interaction with immune cells, resulting in an enhanced antibody production. The cell interaction with the microspheres in the peritoneal cavity seems to be influenced by their size to a greater extent than in the s.c. tissue, probably because of their more frequent interaction with immune cells.

Studies on the mechanisms of tumorigenesis induced by polyetherurethanes in rats: leachable and biodegradable oligomers involving the diphenyl carbamate structure acted as an initiator on the transformation of Balb 3T3 cells.

The tumor-initiating activities of the methanol extracts of polyetherurethanes (PEUs) were first detected in the presence of 12-0-tetradecanyl-phorbol-13-acetate (TPA) using Balb 3T3 transformation assay. A model hard segment of PEUs, 4,4'-di(ethoxycarboamide) diphenylmethane (MDU), showed initiating activity, while chemical moieties other than the hard segment were shown to be negative in the test. The transformation assay was carried out using glass dishes half coated with two different PEUs, PU4 and PU8. In the presence of TPA, the transforming activities correlated with the tumorigenic potential in the rat implantation study on the coated surface of PEUs, but not on the uncoated glass area. From these results it was concluded that initiation was caused by the hard-segment moiety such as MDU structure derived not only from the leachable extracts but also from the biodegradable substances by the direct interaction of cells with the coated materials.

Potentiality of gelatin microsphere as immunological adjuvant.

This paper describes a new attempt to enhance the production of antibody by delivery of an antigen to phagocytic antigen-presenting cells (e.g. macrophages) using gelatin microspheres. A model protein antigen, human gamma globulin (HGG), was incorporated into microspheres composed of gelatin which have an opsonic ability for macrophage phagocytosis. Subcutaneous injection of the microspheres induced the production of HGG-specific IgG antibody in the mouse serum to a great extent compared with that of HGG in soluble form or in Freund's incomplete adjuvant (FIA) form. There was an optimal concentration of cross-linking agent (glutaraldehyde) for the highest production of antibody. When gelatin microspheres were cross-linked at lower concentrations of glutaraldehyde, they were more extensively swollen in an aqueous solution, leading to an increase in the size of hydrated microspheres because of their lower cross-linking densities. The increased size of microspheres caused a decrease in their macrophage phagocytosis, whereas the release rate of HGG from the microspheres increased as the concentration of cross-linking agent became low. The balance of the two factors, the microsphere susceptibility to macrophage phagocytosis and the rate of HGG release, seemed to affect the efficacy of gelatin microspheres to enhance the antibody production. In addition, incorporation of HGG into gelatin microspheres enhanced the delayed-type hypersensitivity reaction. Moreover, the microspheres developed a strong secondary response in comparison with FIA. The gelatin microspheres induced a minimal inflammatory response around the injection site in contrast to FIA. These findings demonstrate that the gelatin microsphere is promising as an adjuvant to enhance both humoral and cellular immune responses to antigen.

Studies on the tumor-promoting activity of polyethylene: inhibitory activity of metabolic cooperation of polyethylene films containing an antioxidant.

Tumor-promoting activity, inhibitory activity on the gap-junctional intercellular communication of polyethylene (PE) film containing a model antioxidant, 2,2'-methylene-bis(4-methyl-6-tert-butylphenol) (MBMBP), at various amounts was assessed by V79 metabolic cooperation (MC) assay. The extracts prepared from PE film containing MBMBP showed the inhibitory activities and the potencies of the inhibitory activities depended on the amount of MBMBP involved in the film, whereas the extract prepared from MBMBP-free PE film did not show any inhibitory activities. However, the inhibitory activities were observed when MC assay was carried out on the surface of the MBMBP-free film. These findings indicate that tumor-promoting activity of PE film is influenced by the surface property of the film as well as by amount and kind of additives incorporated.