Combined oral administration of L-arginine and tetrahydrobiopterin in a rat model of pulmonary arterial hypertension.

Alterations in the nitric oxide (NO) pathway play a major role in pulmonary arterial hypertension (PAH). L-arginine (LA) and tetrahydrobiopterin (BH4) are main substrates in the production of NO, which mediates pulmonary vasodilation. Administration of either LA or BH4 decrease pulmonary artery pressure (PAP). A combined administration of both may have synergistic effects in the therapy of PAH. In a telemetrically monitored model of unilateral pneumonectomy and monocrotaline-induced PAH, male Sprague-Dawley rats received either LA (300 mg/kg; n = 15), BH4 (20 mg/kg; n = 15), the combination of LA and BH4 (300 mg/kg, 20 mg/kg; n = 15), or vehicle (control group; n = 10) from day 28 after monocrotaline induction. Therapy was orally administered once daily over consecutive 14 days. LA, BH4, or both equally lowered PAP, increased pulmonary vascular elasticity, restored spontaneous locomotoric activity, prevented body weight loss and palliated small vessel disease of severely pulmonary hypertensive rats. BH4 substitution lowered asymmetric dimethylarginine levels sustainably at 60 min after administration and downregulated endothelial NO synthase mRNA expression. No significant survival, macro- and histomorphologic or hemodynamic differences were found between therapy groups at the end of the study period. Administration of LA and BH4 both mediated a decrease of mean PAP, attenuated right ventricular hypertrophy and small vessel disease in monocrotaline-induced pulmonary hypertensive rats, though a combined administration of both substances did not reveal any synergistic therapy effects in our animal model.

New mechanical thrombectomy model in the rabbit: A feasibility study.

BACKGROUND AND PURPOSE: Thrombolytic therapy represented the gold standard for the treatment of ischemic stroke. Its drawbacks include increased bleeding risk and low recanalization rates. Mechanical thrombectomy is a new promising therapy option. Devices used for this procedure were substantially improved during recent years. New devices require extensive preclinical invivo testing. We therefore sought to translate the commonly used pig model to a simplified and cheaper rabbit model. MATERIALS AND METHODS: We performed thromboembolisation in eight intubated and sedated adult female New Zealand white rabbits. The thrombus was created by careful rotation of autologous blood in a silicone tube and addition of barium sulfate for radiopacity. We injected the artificial thrombus via a catheter through the cannulated femoral artery. After thromboembolisation, 2D-DSA was performed to evaluate location and thrombus dimensions. COMPARISON WITH EXISITING METHOD(S): None. RESULTS: No complications or mortality were observed in our series. In seven cases (87.5%) the location of the thromboembolism was the maxillary artery and in one case (12.5%) the thrombus reached the occipital artery. The radiopaque thrombus had a length of 7.0±4.55mm invivo. Vessel diameters in angiographic evaluation were 2.44±0.21mm for the common carotid artery and 2.1±0.16mm for the maxillary artery. CONCLUSIONS: The novel small animal model for mechanical thrombectomy in rabbit is technically feasible and cheap. It offers comparable vessel diameters to cranial arteries and closely mimics human coagulation system.

Validation for 2D/3D registration. I: A new gold standard data set.

PURPOSE: In this article, the authors propose a new gold standard data set for the validation of two-dimensional/three-dimensional (2D/3D) and 3D/3D image registration algorithms. METHODS: A gold standard data set was produced using a fresh cadaver pig head with attached fiducial markers. The authors used several imaging modalities common in diagnostic imaging or radiotherapy, which include 64-slice computed tomography (CT), magnetic resonance imaging using T1, T2, and proton density sequences, and cone beam CT imaging data. Radiographic data were acquired using kilovoltage and megavoltage imaging techniques. The image information reflects both anatomy and reliable fiducial marker information and improves over existing data sets by the level of anatomical detail, image data quality, and soft-tissue content. The markers on the 3D and 2D image data were segmented using ANALYZE 10.0 (AnalyzeDirect, Inc., Kansas City, KN) and an in-house software. RESULTS: The projection distance errors and the expected target registration errors over all the image data sets were found to be less than 2.71 and 1.88 mm, respectively. CONCLUSIONS: The gold standard data set, obtained with state-of-the-art imaging technology, has the potential to improve the validation of 2D/3D and 3D/3D registration algorithms for image guided therapy.

Animal experiments with ultra-high molecular weight polyethylene (UHMW-PE) stabilised with alpha-tocopherol used for articulating surfaces in joint endoprostheses.

Numerous investigations proved the impressive suitability of alpha-tocopherol as a stabilizer for ultra-high molecular weight polyethylene (UHMW-PE) used for endoprostheses. Regarding the biocompatibility of this new biomaterial, in-vitro celltoxicity tests gave no hint for a cyto- or genotoxic activity. In this study, animal experiments are carried out to further ensure the biocompatibility of this biomaterial. Thin UHMW-PE-films (20 x 6 x 0.23 mm3) were implanted subcutaneously into rats. Morphology and reactivity of surrounding connective tissue against either pure UHMW-PE material or UHMW-PE containing alpha-tocopherol were studied at timed intervals (2 weeks, 3 month, and 6 month after operation) in 3 groups of animals, each group comprising 10 animals. Parallel to these tests, material changes were investigated in these 3 groups of 10 animals at the same intervals after operation with the help of infrared spectroscopy (FTIR). Within the implantation time, no noteworthy oxidative degradation could be observed. The amount of lost alpha-tocopherol due to diffusion is low enough to ensure a lifetime stabilisation of the UHMW-PE. The implants were all well tolerated and definitely encapsulated already 2 weeks after operation. Presence or absence of alpha-tocopherol in the implants did not evince morphological differences. Therefore, negative consequences were not manifest in the presence of alpha-tocopherol.

Administration of proteolytic enzymes bromelain and trypsin diminish the number of CD4+ cells and the interferon-gamma response in Peyer's patches and spleen in endotoxemic balb/c mice.

Recent publications revealed that bromelain exerts a marked effect on T-cell response by inhibiting T-cell signal transduction. These experimental studies may help to explain former clinical investigations showing that Phlogenzym (PHL), a preparation consisting of the proteases bromelain and trypsin and the antioxidant rutosid, ameliorate certain diseases with an underlying inflammatory process. In this study, we showed that orally administered PHL significantly reduced lymphocyte subpopulations in Peyer's patches (PPs) of healthy and endotoxemic mice. Similarly, the number of splenic lymphocytes in endotoxin-boostered mice was significantly lowered by PHL. The effect of PHL was more pronounced on T cells than on B cells leading especially to a diminution of CD4+ cells. Moreover, PHL pretreatment decreased IFN-gamma mRNA in PPs and spleen of endotoxemic mice. These results reveal that PHL may ameliorate inflammatory process by reducing the number of CD4+ cells and by diminishing INF-gamma mRNA levels.

Activation of nuclear factor-kappa B significantly contributes to lumen loss in a rabbit iliac artery balloon angioplasty model.

BACKGROUND: To investigate the contribution of inflammation to postangioplasty lumen loss, we used an adenoviral gene therapy approach to inhibit the central inflammatory mediator nuclear factor-kappaB (NF-kappaB) by overexpression of its natural inhibitor, IkappaBalpha. METHODS AND RESULTS: The adenovirus carrying human IkappaBalpha was applied immediately after balloon dilatation by a double-balloon catheter in a rabbit iliac artery restenosis model. Immunohistochemistry of IkappaBalpha revealed that mainly smooth muscle cells of the media but also cells of the adventitia were transduced and expressed the transgene IkappaB alpha for >/= 8 days. At this time point, intercellular adhesion molecule-1 (30%) and monocyte chemotactic protein-1 (50%) expression, as well as recruitment of macrophages into the wounded area (90%), were significantly reduced in IkappaB alpha-treated vessels. In addition, expression of inhibitor of apoptosis proteins was reduced and the percentage of apoptotic cells was increased compared with control-treated contralateral vessels. Animals killed 5 weeks after treatment exhibited a significantly reduced degree of lumen narrowing (P<0.02) on the side treated with adenovirus IkappaBalpha. The lumen gain of approximately 40% was due to positive remodeling. CONCLUSIONS: From these data, we conclude that balloon angioplasty-induced activation of NF-kappaB contributes to lumen loss likely via induction of an inflammatory response and a decrease in the rate of apoptosis. These data show for the first time that inflammation mediated by NF-kappaB is involved in postangioplasty lumen narrowing. Specific and more potent inhibitors of NF-kappaB might therefore be a useful therapeutic measure to improve clinical outcome after balloon dilatation.

Oral feeding with glutamine prevents lymphocyte and glutathione depletion of Peyer's patches in endotoxemic mice.

OBJECTIVE: To determine the effect of oral glutamine feeding on lymphocyte subpopulations and glutathione metabolism in Peyer's patches (PPs) of healthy and endotoxemic mice. SUMMARY BACKGROUND DATA: Recent data indicate that nutrients both maintain nitrogen and energy balances and modulate cell and organ function. In particular, glutamine has an impact on gut and immune function. This is of special importance in the perioperative phase. METHODS: Female Balb/c mice were fed a glutamine-enriched diet or a control diet for 10 days. On day 7 25 microg lipopolysaccharide (LPS) or saline was injected. On day 3 after the challenge, mice were killed, total cell yield was determined, and lymphocyte subpopulations (total T cells, CD4+, CD8+ cells, and B cells) were analyzed by flow cytometry. One experimental group was treated with buthionine sulfoximine, a specific inhibitor of glutathione synthesis. The glutathione content in PPs was measured by high-performance liquid chromatography. RESULTS: Glutamine administration led to a significant increase in total cell yield, including T and B cells, in PPs. The LPS-induced reduction of T cells (-45%) and of B cells (-30%) was significantly lower in glutamine-treated mice. Endotoxemia caused a 42% decrease of glutathione in control animals, but not in glutamine-treated animals. As with LPS, buthionine sulfoximine also lowered lymphocyte numbers and glutathione content of the PPs. CONCLUSIONS: Administration of glutamine prevents LPS-stimulated lymphocyte atrophy in PPs, possibly by increasing the glutathione content in the PPs. Therefore, oral glutamine supply seems to be a suitable approach for improving intestinal immunity in immunocompromised patients.

Biocompatibility and performance of the Wallstent and several covered stents in a sheep iliac artery model.

PURPOSE: To compare the biocompatibility and performance of various stent-grafts to those of a bare stent in an ovine model with a subchronic (3 months) endpoint. MATERIALS AND METHODS: Three different types of stent-grafts (ePTFE/nitinol, n = 8; polyester/nitinol, n = 8; and polycarbonate urethane/cobalt-alloy, n = 8) and a bare stent as a control (Ni-Co-Ti-steel-alloy, n = 8) were implanted in the iliac arteries in eight female sheep. One type of each stent-graft was implanted per animal, two implants at each side. The implantation sites for each type varied from animal to animal. Angiographic control and intravascular ultrasound (IVUS) imaging were performed before and after implantation, after 2 months, and before explantation at 3 months and were used to characterize patency and to assess intimal hyperplasia. After 3 months, the implants were retrieved and subjected to histologic evaluation (after methacrylate embedding, cutting, and histologic staining) to characterize the biologic response. RESULTS: Implantation was technically successful in all procedures. At 2 and 3 months after implantation, all segments in which stents had been implanted were patent. Marked neointima formation was found in the polyester-covered stent-graft that showed significant luminal narrowing of 50%, compared to the ePTFE-covered (24%) and polycarbonate urethane-covered endoprostheses (22%), as well as the bare stent (Wallstent; 9%; P < .001). A minimal inflammatory vessel wall reaction was demonstrated for the polyester-covered and ePTFE-covered endoprostheses; the polycarbonate urethane-covered stent-graft's response was demonstrable but not significantly different from that of the Wallstent. At 3 months, the ePTFE-covered stent-graft showed incomplete (>90%) endothelial coverage; in the other endoprostheses, complete but partially immature endothelialization was found. CONCLUSION: All stent-grafts induced an inflammatory vessel wall reaction with neointimal hyperplasia. The polyester-covered endoprosthesis caused a marked reaction with 50% luminal stenosis. Endothelialization was retarded with the ePTFE-covered stent-graft. The bare stent performed best in regard to neointimal formation and caused the least inflammatory response.

Influence of enteral diets supplemented with key nutrients on lymphocyte subpopulations in Peyer's patches of endotoxin-boostered mice.

BACKGROUND AND AIMS: This study was undertaken to compare the effect of different key nutrients on lymphocyte subsets of Peyer's patches (PP) and spleen in endotoxemic mice. METHODS: Female Balb/c mice were fed over a period of 10 days either with an isocaloric and isonitrogenous control diet (Control), a glutamine enriched diet (Diet I) or a diet containing glutamine, arginine, glycine, and n-3 fatty acids (Diet II). On day 7 the mice were challenged intraperitoneally with 25 microg LPS. The lymphocyte subpopulations (B cells, T cells, CD4+ and CD8+) of PP and spleen were analysed by flow cytometry. Glutathione content of small intestinal mucosa and spleen was determined by HPLC and luminal small intestinal IgA by ELISA. RESULTS: Both experimental diets increased the number of B and T cells in the PP and that of T cells in the spleen (P<0.01). Glutathione content in PP and spleen was higher under administration of key nutrients (P<0.05). Diet II reduced luminal small intestinal IgA content in comparison to the two other groups. CONCLUSION: The addition of arginine, glycine and n-3 fatty acids to a glutamine supplemented diet does not enhance lymphocyte numbers in PP and spleen, but reduces intestinal IgA content.

Influence of short-term protein malnutrition of mice on the phenotype and costimulatory signals of lymphocytes from spleen and Peyer's patches.

The objective of this study was to investigate the impact of short-term protein malnutrition (PM) on immunoglobulin A (IgA) production and on the number and phenotype of lymphocytes in Peyer's patches (PP) and in the spleen. Balb/c mice were fed for 4, 7, or 10 d with a protein-deficient diet (0.1% protein). We determined B lymphocytes (CD40(+)), T lymphocytes (CD3(+)), T-helper (CD4(+)), and T-suppressor (CD8(+)) cells and the expression of costimulatory signals B7.1 (CD80) and B7.2 (CD86) on B cells and their counter receptors CD28 and CTLA-4 on T cells by fluorescence-activated cell-sorting analysis. Luminal IgA concentration in the small intestine was determined by an enzyme-linked immunosorbent assay. Four days of PM caused a significant reduction in the number of mononuclear cells in the spleen (5.6 x 10(7) +/- 1 x 10(7) versus 2. 4 x 10(7) +/- 0.5 x 10(7), P < 0.001) and the PP (13 x 10(6) +/- 3 x 10(6) versus 8.6 x 10(6) +/- 2 x 10(6), P < 0.01). There was a relative increase of T cells in the spleen and a relative increase of B cells in the PP. Luminal IgA content of small intestine was significantly reduced after 4 d of PM (242 +/- 55 microg versus 173 +/- 39 microg, P < 0.05) and remained at about this level until day 10 of PM. Four days after PM, the costimulatory signals B7.1 and B7. 2 on B cells were upregulated in the PP but markedly downregulated in the spleen, which was inversely related to the expression of the counter receptor CD28 on T-helper cells. We conclude that short-term PM increases the activation of B cells in the PP but reduces the relative number and activation state of splenic B cells. Only 4 d of PM caused a systemic and intestinal immunodepression, as indicated by a markedly decreased content of mononuclear cells in the PP and the spleen.

[Ovarian tissue banking--primary results]. / 'Ovarian Tissue Banking' - erste Ergebnisse.

Lifelong hormone replacement therapy and infertility often follow oncological therapy in young women due to a dramatic reduction of the primordial follicle reserve. Reimplantation and function of frozen ovarian tissue slices were successfully demonstrated in animal models, but the durability is limited. Cryopreservation of a whole ovary could reestablish an almost normal ovarian function. Experiments with porcine ovaries showed histological viability. Cryopreservation and retransplantation of sheep ovaries should demonstrate success by hormonal response and pregnancy.

Acidic glycoproteins accumulate in calcified areas of bioprosthetic tissue.

BACKGROUND AND AIMS OF THE STUDY: This study was performed to evaluate the role of collagen fibrils, cellular elements and matrix proteins in calcification of glutaraldehyde (GA)-fixed bioprosthetic material. METHODS: Lyoplant (lyophilized bovine pericardium, type I collagen) was processed according to three protocols. DF-group (double fixation): after conventional fixation in GA (0.5% for 72 h; storage 0.25%) Lyoplant was implanted subcutaneously into rats for 5 days. Specimens were explanted and re-fixed (conventional fixation), followed by autologous or homologous reimplantation in rats for 21 or 63 days. CF-group (conventional fixation): Lyoplant patches were conventionally fixed (as DF-group), kept in 0.9% saline for one week, and then autologously and homologously reimplanted. GF-group (high-concentration GA): Lyoplant patches were processed (as GF-group) but 0.5% GA was used for tissue storage. Explanted specimens were studied by light microscopic histochemistry; calcium contents were measured by atomic absorption spectroscopy. RESULTS: Severe calcification occurred in the DF-group without differences between autologous and homologous reimplantation. In CF- and GF-groups, calcification was negligible, but immunologic response against homologous implants was accompanied by increased calcium content. Histologic characterization of matrix material in calcified areas revealed oxyphilic glycoproteins, identified as sialoglycans. CONCLUSIONS: Modification of extracellular matrix by GA is assumed as essential to cause calcification of bioprosthetic material. Calcium deposition and accumulation of sialoglycans are simultaneous events. A specific role of this glycoprotein for calcification has to be considered.

Endothelialization of biosynthetic vascular prostheses after laser perforation.

OBJECTIVE: This study was undertaken to investigate the feasibility of transmural capillary ingrowth into the inner surface of biosynthetic vascular prostheses (Omniflow, BioNova, Melbourne, Australia) through perforations created by an excimer laser, thus inducing an endothelial cell coverage. METHOD: Biosynthetic vascular prostheses (Omniflow, 10 cm length, 6 mm diameter) were perforated with an excimer laser (diameter of the holes 50 to 100 microm, distance 4 mm) and implanted into the carotid arteries of eight sheep. They were compared to untreated Omniflow prostheses implanted at the contralateral side. Three months after implantation the prostheses were explanted and evaluated by gross morphology, histologic examination, scanning electron microscopy, and immunohistochemical staining for factor VIII to identify endothelial cells. RESULTS: All grafts remained patent. Gross morphologic examination revealed no significant difference in the thrombus-free surface between perforated and untreated prostheses. However, scanning electron microscopy showed endothelial cells in the midgraft portion of all perforated prostheses, whereas collagen fibers, fibrin meshwork, and activated platelets formed the inner layer in six of eight untreated Omniflow prostheses. Transmural capillary ingrowth in the laser group was verified by positive factor VIII staining for endothelial cells in the laser channels. CONCLUSION: Spontaneous endothelialization of biosynthetic vascular prostheses can be achieved by transmural capillary ingrowth through perforations in the wall of the prostheses in an experimental sheep model.

[Lyme borreliosis in South Germany. Epidemiologic data on the incidence of cases and on the epidemiology of ticks (Ixodes ricinus) carrying Borrelia burgdorferi]. / Lyme-Borreliose in Süddeutschland. Epidemiologische Daten zum Auftreten von Erkrankungsfällen sowie zur Durchseuchung von Zecken (Ixodes ricinus) mit Borrelia burgdorferi.

Among 2403 ticks (Ixodes ricinus) tested in 1985 for Borrelia 328 (13.6%) were carriers (adults about 20%, nymphs about 10%, larvae about 1%). The highest prevalence of infected ticks was among adult ticks in the Isar region north of Munich (33.8%). Among 9383 persons whose serum had been examined by fluorescence serology in 1985 and 1986, 1035 (11%) had raised Borrelia-specific IgG and/or IgM antibodies greater than or equal to 1:64. In 18.7% only IgM antibodies were demonstrated. Among 375 proven cases there were 78 with erythema migrans, 211 with neurological signs, 48 with Lyme arthritis and 36 with acrodermatitis. Seasonal incidence peaks were in June-August for erythema migrans, July-September for neurological signs, with no clear-cut seasonal peaks with Lyme arthritis and acrodermatitis. The incubation time for 80% of cases of each abnormality was 5-29 days for erythema migrans, 20-59 for neurological signs and 2-8 months for Lyme arthritis. Erythema migrans was most frequent among those aged 30-60 years, neurological signs among children and juveniles up to 20 years and those aged 40 to 70 years, Lyme arthritis among those aged 30-60 years, and acrodermatitis among those aged 40-80 years. Significantly more women than men developed acrodermatitis.