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Since an increase of platelet-endothelial cell interactions has been observed in mice with Antigen- induced-Arthritis (AiA) as well as an increase of NO expression, the aim of our study was to investigate in vivo the influence of NO, especially the platelet and endothelial inducible NO Synthase, on the platelet- and leukocyte endothelial cell interaction.C57/Bl6 mice and iNOS deficient mice were disposed in 6 groups (each=7). After induction of AiA, rolling and adherent fluorescence labelled platelets and leukocytes were investigated by intravital microscopy (IVM) on day 8 after AiA. Rank SUM Test and ANOVA on ranks have been performed regarding the data.All arthritic mice presented an increase in platelet and leukocyte interaction with the endothelium compared to control groups. The arthritic iNOS deficient mice showed a more intense interaction of platelets and leukocytes with the endothelium in comparison with the wild-type arthritic mice. The group using arthritic wild-type recipient and iNOS deficient donor mice showed an increase in cell-interactions, leading to an endothelial effect, compared to the group using iNOS deficient arthritic recipient and wild-type donor mice.The IVM data lead to an anti-inflammatory effect of NO, since NO followed an increase in platelet- and leukocyte- endothelial cell interaction in iNOS deficient mice with AiA. In addition, we have shown for the first time in vivo that platelet NO produced by iNOS seems to have a minor influence on the leukocyte induced tissue damage in contrast to endothelial iNOS. Therefore, selective platelet inhibition would not interfere with the protective effect of NO.

There is growing evidence that platelets play an important role in the development and maintenance of rheumatoid arthritis. Activation and adherence of platelets in the synovial microcirculation might be in part responsible for endothelial damage and activation of leukocytes. Recent findings show a direct influence of P-selectin on platelet- and leukocyte-endothelial cell interaction in mice with Antigen-induced Arthritis (AiA). P-selectin is only expressed by platelets and endothelial cells, not by leukocytes. Therefore, the aim of the present study was to investigate the differential influence of platelet and endothelial P-selectin on the extent of inflammation in AiA. AiA was induced in wild-type mice and in P-selectin-deficient mice from the same genetic background (four groups: each n = 7). Intravital fluorescence microscopy (IVM) was used to visualize platelets and leukocytes in the synovial microcirculation at day 8 after AiA. Platelets from either strain were fluorescence-labelled ex vivo and transferred into either strain. We were able to demonstrate a significant decrease of platelet- and leukocyte-endothelial cell interaction in P-selectin-deficient mice with AiA in comparison to wild-type mice with AiA. When wild-type platelets were donated into P-selectin-deficient AiA recipients, the leukocyte-endothelial cell interaction was significantly increased compared to the group consisting of P-selectin-deficient recipient and donor mice. These are the first in vivo results showing that the P-selectin stored in platelets is at least partly responsible for the leukocyte-endothelial cell interaction and the resulting tissue damage in AiA. In the future, a suppression of platelet P-selectin could potentially become a treatment option for reducing the effects of rheumatoid arthritis.

Platelets are thought to participate in the pathogenesis of chronic inflammatory diseases such as rheumatoid arthritis (RA). We showed recently an in vivo increase in platelet-endothelial cell interactions in mice with antigen-induced arthritis (AiA). The underlying mechanisms are not yet clear. The aim of this study was to investigate the impact of P-selectin in AiA by means of intravital fluorescence microscopy (IVM).C57/Bl6 mice and P-selectin-deficient mice were divided into four groups (n = 7; control/AiA per strain). The extent of AiA was assessed by measuring knee joint swelling and by histological scoring. Rolling and adherent fluorescence-labelled platelets and leucocytes were investigated by IVM.In arthritic P-selectin-deficient mice (rolling: 0.05+/-0.01; adherent: 130+/-20 mm(-2)), compared to arthritic C57/Bl6 mice (rolling: 0.20+/-0.04; adherent: 1910+/-200 mm(-2)), platelet interaction was significantly reduced (p0.05) and reached the level of both control groups without AiA. In addition, interaction of leucocytes in P-selectin-deficient arthritic animals (rolling: 0.12+/-0.06; adherent: 387+/-37 mm(-2)) was significantly decreased in comparison to arthritic C57/Bl6 animals (rolling: 0.21+/-0.06; adherent: 1492+/-284 mm(-2); p0.05). Swelling of the knee joint and histological scoring were reduced in arthritic P-selectin-deficient mice compared to arthritic C57/Bl6 mice.We have demonstrated for the first time in vivo a significant decrease in the interaction of platelets and leucocytes with the endothelium in P-selectin-deficient mice with AiA and a reduction in clinical and histological symptoms of arthritis. These findings suggest that leucocyte-endothelial cell interactions depend at least partially on platelet P-selectin and therefore platelets may be responsible for the leucocyte tissue damage in AiA.

Objectives. Growing evidence supports the substantial pathophysiological impact of platelets on the development of rheumatoid arthritis. At present there are no methods for studying these cellular mechanisms in vivo. The aim of this study was to visualize and investigate platelet-endothelial cell interaction in the knee joint of mice with antigen-induced arthritis (AiA) by means of intravital microscopy. Methods. In 14 mice (Balbc) intravital microscopic assessment was performed on day 8 after AiA induction in two groups (controls, AiA). The severity of AiA was assessed by measuring knee joint swelling and by histological scoring. Ex vivo fluorescently labelled rolling and adherent platelets and leucocyte-endothelium interactions were investigated by intravital fluorescence microscopy. Results. Swelling of the knee joint as well as histological score was significantly enhanced in arthritic animals compared with controls. In control mice intravital microscopy revealed low baseline rolling and sticking of leucocytes and fluorescently labelled platelets. AiA induced a significant increase in the fraction of rolling leucocytes (3 times) and rolling platelets (6 times) compared to the control group. Furthermore, AiA induction resulted in a significantly enhanced number of adherent leucocytes (3-fold) and adherent platelets (12-fold) in comparison with control animals. Conclusions. Platelet kinetics were directly analysed using intravital microscopy in the arthritic microcirculation in vivo for the first time. We provide the first evidence that platelets accumulate in arthritic vessels, indicating platelet activation due to AiA. Platelet recruitment and subsequent activation might play an important role in the pathogenesis of rheumatoid arthritis.

The aim of this study was to establish a simple and safe method of anaesthesia for intravital microcirculatory observations in small laboratory animals. The usefulness of isoflurane inhalation anaesthesia has been investigated in different strains of mice commonly used in experimental medicine. These were the hairless (hr/hr, n = 12), the BALB/c ( n = 12) and the nude mouse (nu/nu, n = 3). Anaesthesia was maintained by mask inhalation of isoflurane vaporized at concentrations of up to 4% in the induction phase, at 1.5% during acute surgical procedures and at 0.8-1.3% during prolonged experimental observations. Isoflurane was vapoured in a N2O/O2 mixture and saturated with 32-36% FiO2. During observations the body temperature was kept constant at 37°C. The tail artery was cannulated for monitoring of mean arterial blood pressure (MAP) and heart rate (HR). To maintain the body fluid balance, isotonic saline was administered at a constant rate of 0.2 ml/h. Arterial blood samples were drawn for blood-gas analysis at the end of the experiments. All animals survived the anaesthesia protocol lasting between 3 and 6.5 h. During isoflurane inhalation, no breathing complications or changes in systemic circulatory parameters were observed. Mean values of MAP and HR were 79± 3 mmHg and 486± 13 min-1, respectively, over the entire observation period. A moderate acidosis was recorded in animals under isoflurane anaesthesia, with alterations of arterial blood pH, paO2 and pCO2 values (7.29± 0.06, 130± 19 mmHg and 35.6± 4.7 mmHg, respectively). In conclusion, inhalation anaesthesia with isoflurane is useful for experimental studies in the mouse due to (1) the simplicity of administration of the anaesthetic, (2) the rapid induction of anaesthesia, (3) easy control of the depth of anaesthesia, (4) the low percentage of complications, and (5) stable MAP and HR during observations lasting several hours. The proposed technique is especially suitable for observations of the microcirculation under intravital fluorescence microscopy.

Background. The aim of our study was to investigate the role of inducible nitric oxide synthase (iNOS)-derived nitric oxide (NO) production in different stages of murine antigen-induced arthritis (AiA). Methods. Clinical, histological and microcirculatory parameters (measured by intravital fluorescence microscopy) were assessed in the knee joint during acute and chronic AiA after inhibition of iNOS with L-N 6 -(1-iminoethyl)lysine (L-NIL). Plasma concentrations of NO - 2 and NO - 3 were evaluated by the Griess reaction and the expression of iNOS, P- and E-selectin, intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule I (VCAM-1) by immunohistochemistry. Results. In both stages of the disease, plasma concentrations of NO, and NOR were increased and iNOS was expressed. In the acute phase, swelling, leucocyte adhesion, leucocyte infiltration and expression of adhesion molecules were increased in arthritic animals treated with L-NIL in comparison with untreated arthritic animals. In the chronic phase, no change in the disease parameters could be detected after L-NIL treatment. Conclusion. Increased NO production induced by iNOS during the acute phase of AiA can be regarded as a protective response in the prevention of further leucocytic infiltration and joint destruction, whereas it seems to play a subordinate role in chronic AiA.

BACKGROUND: The aim of this study was to investigate changes in the blood microcirculation of skin, subcutaneous tissue, and striated muscle, and the venous and lymphatic outflow from hind limb, after a standardized mechanical trauma. METHODS: Trauma, defined as 50% of the minimal energy needed for tibia fracture (3.7 J/g), was applied to the leg of hairless mice. Intravenously injected fluorescein isothiocyanate-dextran 150 kDa and Rhodamine-6G were used for intra-vital fluorescence microscopy of blood vessels. Lymphatics were stained with fluorescein isothiocyanate-dextran injected into the footpad. A computer-assisted analysis system allowed measurement of the functional capillary density (FCD), vessel diameters, velocity of blood flow, and edema value expressed as extravasation index (EV). The percentage of slowly rolling and sticking leukocytes in postcapillary venules was estimated. RESULTS: At the site of injury, trauma resulted in significant reduction of FCD in skin, subcutaneous tissue, and striated muscle. There were no significant differences in the vessel diameter (skin subcutaneous and muscle arterioles and venules, and superficial saphenous artery and vein) or velocity of blood flow (subcutaneous tissue and muscle venules). The EV increased significantly in muscle venules and was higher in muscles, subcutaneous tissue, and superficial saphenous veins than in controls (nonsignificantly). An increased percentage of slowly rolling and sticking leukocytes was noted in the superficial saphenous vein at the site of injury and proximal to it. The lymphatics remained patent, with faster visualization and increased summarized cross-sectional areas in traumatized extremities. CONCLUSION: Early changes occurring in soft tissues in response to mechanical injury were characterized by reduction in FCD of skin and muscles, and less in subcutis; increased EV, reflecting leakage of macromolecules; increased percentage of slowly rolling and sticking leukocytes; maintenance of lymphatic vessel continuity; and increased lymph formation and flow rate.

Background: A newly developed observation chamber has been designed for comfortable limb immobilization during intravital microscopic analysis, which permits direct, repeated, long-lasting observations of the microcirculation in the various hindleg soft tissues. Methods: Experiments were performed under inhalation isoflurane/nitrous oxide anesthesia. Intravenously injected fluorescein isothiocyanate (FITC)-dextran (M r 150,000) and Rhodamine 6G (Sigma, St. Louis, MO) allowed for visualization of both microcirculatory phenomena in arterioles, capillaries, and venules and macrocirculatory structures as superficial saphenous artery and vein. Skin microcirculation analysis was performed from the epidermal side (group A, n = 7), whereas observation of deeper situated tissues was performed after oval skin excision on the medial surface of the tibial area (group B, n = 7). FITC-dextran (M r 150,000; group C, n = 8) injected into the foot pad permitted visualization of venous, arterial, and accompanying lymphatic vessels. With the aid of a computer-assisted microcirculation analysis system, functional capillary density, vessel diameter, edema formation, and leukocyte-endothelial cell interactions were evaluated. The ratio of rolling leukocytes, given as percentage of all leukocytes passing the vessel segment during a 30-second observation interval, and the number of sticking leukocytes per square millimeter of endothelial surface were determined. Results: This new model allows the analysis of the complex in vivo changes of macro- and microcirculatory parameters in the different (venous, arterial, lymphatic) vessels of the covering tissues (skin and muscle) of the mouse hindleg. Conclusion: The potential applications of this technique include the study of mechanical trauma, ischemia-reperfusion injury, and tissue compression mimicking both acute and prolonged venous stasis on both the microcirculatory and macrocirculatory levels in the different tissue compartments.

The microcirculation and leukocyte-endothelial cell interaction in synovial tissue of an inflamed joint are known to play a crucial role in the pathogenesis of rheumatoid arthritis. The aim of this study was to characterize the in vivo changes in the microvasculature and in leukocyte-endothelial cell interactions in the mouse synovial tissue using intravital fluorescence microscopy in three stages of antigen-induced arthritis. The expression of E- and P-selectin and ICAM-1 were also studied using immunohistochemistry.Antigen-induced arthritis (AiA) was produced in Balb/c mice. The severity of arthritis at three different phases was quantified using a clinical and histological score. For the intravital fluorescence microscopy measurements, the patella tendon was partially resected for visualization of the intraarticular synovial tissue of the knee joint. The number of rolling and adherent leukocytes, functional capillary density (FCD) and RBC velocity were quantitatively measured in synovial microvessels. Expression of ICAM-1, E- and P-selectin was assessed by immunohistochemistry.There was a significant increase in the leukocyte rolling fraction in postcapillary venules in the acute phase of AiA (from 0.26 +/- 0.05 in controls to 0.45 +/- 0.04 8 d after AiA induction). The number of leukocytes adherent to the endothelium was significantly elevated in all phases of arthritis (from 121 +/- 27 in controls to 376 +/- 62 mm2 63 d after AiA-induction). Functional capillary density was significantly enhanced in the acute (332 +/- 15 cm/cm2) and intermediate phases (320 +/- 15 cm/cm2) compared to control values (227 +/- 15 cm/cm2). Arthritis resulted in a distinct increase in the expression of ICAM-1 on the synovial endothelium in all phases of AiA. E- and P-selectin expression were detected only in the acute phase.Our model provides new insights into the microcirculatory changes which occur in the synovial tissue of an arthritic joint.

A novel model for the investigation of the microcirculation in synovial tissue of the mouse knee joint is presented. The mouse knee joint was exposed on a specially designed plexiglass stage with a slight flexion. After partial resection of the skin, the patella tendon was cut transversally, which allowed for visualization of the "Hoffa's fatty body", an intraarticular fatty tissue containing synovial cells on the interior surface of the joint. An intravital fluorescence microscope was adjusted to observe the microcirculation of this intraarticular synovial tissue without opening of the joint capsula. For staining of the plasma, fluorescein isothiocyanate (FITC)-dextran was used, and for the staining of leukocytes rhodamine 6G was used. The tissue investigated presents with a high-density honeycomb-like capillary network, containing some postcapillary venules and a few arterioles. The following parameters were assessed off-line using a computer-assisted microcirculation analysis system: flow and diameter of arterioles and postcapillary venules, as well as functional capillary density. Moreover, leukocyte-endothelial cell interaction was quantified by counting the number of rolling cells and cells adhering to the endothelium in postcapillary venules. As an indication of endothelial leakage, macromolecular extravasation was also assessed. To validate the model, we investigated these parameters at three time points during an observation period of 60 min. There was no change in functional capillary density, nor in vessel diameter after 60 min of observation. Moreover, there was neither a change in the number of rolling cells, nor in the number of cells adhering to the endothelium nor in extravasation of FITC-dextran, thus indicating the stability of the preparation. The new model allows the quantitative analysis of the intraarticular microcirculation of the synovial fatty tissue in vivo. It provides insight into the dynamics of synovial microcirculation and leukocyte-endothelial cell interaction in acute or chronic joint inflammation.

Inflammation is associated with the invasion of leukocytes into affected tissues and with the up-regulation of platelet activation and adhesion. Assuming that leukocyte accumulation is linked to platelet aggregation, the aim of our study was to examine the effects of selective platelet inhibition by the glycoprotein (GP) IIb/IIIa receptor antagonist Tirofiban on the leukocyte-endothelial cell interaction.We used the model of antigen-induced arthritis (AiA) to induce inflammatory changes in the synovial microcirculation. Ex vivo labelled platelets and in vivo fluorescence-labelled leukocytes were visualized by intravital microscopy (IVM). C57/Bl6 mice were allocated to four groups; two control groups with saline or Tirofiban and two groups with AiA that also received either saline or Tirofiban (0.5 microg/g BW) intravenously.There was no significant change in platelet- or leukocyte- endothelial cell interaction in the endothelium in healthy control animals. In contrast, after selective inhibition of platelets, the platelet- and leukocyte-endothelial cell interaction was significantly reduced in arthritic mice and reached the level of the healthy control groups.Selective platelet inhibition by Tirofiban resulted in reduced leukocyte-endothelial cell interactions in AiA. Consequently, platelets contribute to leukocyte adhesion in AiA via GPIIb/IIIa and therefore platelet inhibition could become an additional therapy option in chronic arthritic disease.

The aim of this study was to investigate whether all sizes of wear particles are capable of provoking inflammatory responses and whether there are different responses among different particle sizes. The knees of 40 female Balb/c mice were injected with polystyrene particles of three different diameters, 0.5 microm, 2.0 microm, and 75 microm, using a 0.1% vol/vol concentration. Seven days after particle injection, assessment of the synovial microcirculation using intravital microscopy, and histologic examination, were done. All the mice injected with polystyrene particles had enhanced leukocyte-endothelial cell interactions and histologic scores regardless of particle size when compared with control animals injected with sterile phosphate buffered saline. Polystyrene particles 0.5 microm in size provoked stronger membrane thickening and increased leukocyte-endothelial cell interactions than 75-microm particles. The fraction of rolling leukocytes was enhanced in the 2.0-microm particle group when compared with the 75-microm particle group. These results indicate that polystyrene particles of all sizes (0.5 microm, 2.0 microm, and 75 microm) are capable of inducing an inflammatory response. Small particles (0.5 microm, 2.0 microm) seem to provoke a stronger inflammatory response than larger particles (75 microm) in conditions with equal particle volume.

There is controversy about the effects of cyclooxygenase-2 (COX-2) on adhesion molecules and the microvasculature in inflamed tissue. Thus, the aim of this study was to assess COX-2-expression in Antigen-induced Arthritis (AiA) and to investigate the effects of selective COX-2 inhibition by Celecoxib (4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl] benzenesulfonamide) (CXB), on synovial microcirculation and adhesion molecule expression in arthritic as well as healthy mice.Balb/c mice were allocated to 4 groups; 2 control groups with saline or CXB and 2 groups with AiA which also received saline or CXB (30 mg/kg BW in 0.3 ml solution). The severity of arthritis was assessed by changes in the transverse joint diameter On day 14 after AiA-induction, the patella tendon of the left knee joint was microsurgically resected and intravital fluorescence microscopy on synovial tissue was performed. Finally, the knee joint was removed for histology and immunohistochmistry.COX-2-expression in the inflamed synovium was demonstrated by immunohistochemistry. Application of Celecoxib resulted in a significant reduction in the rolling leukocyte fraction as well as in the number of leukocytes adherent to the endothelium (0.25 +/- 0. 1 and 96 +/- 34 cells/mm2 respectively) in comparison to the untreated animals with AiA (0.44 +/- 0.03 and 206 +/- 22 cells/mm2 respectively). Additionally, CXB-treated arthritic animals showed significantly less knee joint swelling and reduced adhesion molecule expression.In the present study, COX-2 expression in the synovial tissue of mice with AiA could be demonstrated. Selective COX-2 inhibition with CXB resulted in reduced leucocyte-endothelial cell interactions and decreased adhesion molecule expression. Evidence for a protective role of COX-2 in mouse AiA was not found.

The purpose of the present study was to examine changes in the synovial microcirculation as well as synovial tissue responses to exposure to titanium, polymethylmethacrylate (PMMA), ceramic (Al(2)O(3)), cobalt-chromium alloy (Co-Cr), and polyethylene (PE) particles in an in vivo model. The particulate biomaterials were injected into the left knee joint of female Balb/c mice and assessment of the synovial microcirculation using intravital fluorescence microscopy as well as histological evaluation of the synovial tissue response were performed on day 7 after particle administration. Intravital microscopic measurements revealed that all tested biomaterials caused significantly (p < 0.05) enhanced leukocyte-endothelial cell interactions and an increase of functional capillary density compared to controls. In the histological examination PMMA, Al(2)O(3), PE, and Co-Cr particles provoked significantly (p < 0.05) enhanced inflammatory tissue responses in comparison to tissue from control animals. Titanium particles showed significantly (p < 0.05) less leukocyte-endothelial cell interactions than the other particulate biomaterials and caused significantly (p < 0.05) minor membrane thickening compared to PE and PMMA particles. In conclusion, all tested particulate biomaterials were capable of inducing inflammatory responses in the present study. Our data suggest that titanium particles may cause less leukocyte activation and inflammatory tissue responses than other particulate biomaterials used in total joint arthroplasty.

Nitric oxide production by the inducible NO-synthase in the synovium and chondrocytes is known to be enhanced during chronic joint inflammation and aseptic loosening of joint prostheses. Due to the distinct side effects of non-selective NO-inhibitors on the macro- and microhemodynamics, we investigated the in vivo changes after selective (N-iminoethyl-L-lysine (NIL)) versus non-selective NO-synthase inhibition (NG-nitro-L-arginine methyl ester (L-NAME)) in the synovium of the mouse knee joint. Our results show a significant decrease in the functional capillary density and an increase in the leukocyte accumulation after L-NAME injection. In contrast, NIL did not alter the microhemodynamics or the leukocyte-endothelial cell interaction in the synovium, indicating its potential use for therapeutic selective inhibition of iNOS in joint inflammation.

Objective: To investigate the effects of ibandronate, a novel aminobisphosphonate, on inflammation as well as leukocyte-endothelial cell interaction in mouse antigen-induced arthritis (AiA). Material and Treatment: 36 Balb/c mice were subcutaneously injected with 160 μg/kg of ibandronate once per day beginning at day 7 until day 13 after induction of AiA. Methods: The severity of arthritis was assessed by changes of the transverse knee joint diameter. For the intravital fluorescence microscopy measurements on day 14 after AiA induction, the patella tendon was partly resected to visualize the intraarticular synovial tissue of the knee joint. The number of rolling and adherent leukocytes as well as red blood cell (RBC) velocity and functional capillary density (FCD) were quantified in synovial microvessels. Furthermore, leukocyte infiltration in the synovium was determined in histological sections with an established score. Results: Both fractions of rolling leukocytes (p = 0.016) as well as number of extravasated leukocytes (p = 0.004) were enhanced in control animals treated with ibandronate in comparison to animals which received saline. Arthritic animals with and without ibandronate treatment revealed an increased FCD (p = 0.006, p = 0.008), enhanced number of rolling ( p = 0.002, p = 0.001) and adherent leukocytes (p = 0.009, p = 0.007) and greater swelling of the left knee joint (p = 0.002, p = 0.001) when compared to control animals. No significant differences between arthritic animals and arthritic animals treated with ibandronate were found in any of the parameters assessed including leukocyte adherence, FCD, histology, and knee joint swelling. Conclusion: Ibandronate treatment of healthy mice was associated with an enhanced fraction of rolling leukocytes and increased numbers of extravasated leukocytes indicating a proinflammatory effect on the synovial microcirculation. In animals with a preexisting antigen-induced arthritis, however, ibandronate did not induce an exacerbation of joint inflammation and leukocyte adherence.

Although it is now widely recognized that the inflammatory response to implant wear particles plays an important role in aseptic loosening of total joint replacements, the precise mechanisms of this process remain unclear. The aim of this study was to establish an animal model for the study of the adverse response to particulate wear debris and the effects on the synovial microcirculation as well as the leukocyte-endothelial cell interaction in the murine knee joint in vivo. Balb/c mice were injected with 50 microl of a 0.5-microm polystyrene particle suspension (0.1% v/v) into the knee joint. The severity of the inflammatory response was evaluated at days 1, 2, 3, 5, 7 (acute), 21 (intermediate), and 63 (chronic) after particle injection. Histological examination as well as assessment of the synovial microcirculation using intravital microscopy was performed. For the intravital microscopy measurements, the patella tendon was partially resected for visualization of the synovial tissue of the knee joint and the fluorescent markers FITC-dextran and rhodamine 6G were injected intravenously. There was a significantly enhanced leukocyte-endothelial cell interaction beginning at day 3 after particle injection with a maximum in the acute phase (days 5-7) and a subsequent decline in the intermediate (day 21) and chronic (day 63) phases. Functional capillary density was significantly increased from day 3 until day 21 after particle application. The histological examination showed an inflammatory reaction that complied widely with the temporal course of the microvascular parameters and resembled the histological appearance of the synovial-like membrane around loose joint prostheses. A novel model was established for the qualitative and quantitative investigation of the particle-induced inflammatory response in the joint environment. It was shown for the first time that there is a significantly enhanced leukocyte-endothelial cell interaction in the synovial tissue after intra-articular particle injection. This model seems to be suitable for further investigations, e.g., dealing with the biocompatibility of different particle materials.

Prolonged immobilization of severely ill, bed-ridden patients results in formation of pressure ulcers following inappropriate tissue blood perfusion, and local activation of leukocyte - endothelial cell interactions (L/EC). Various treatment modalities were implemented to improve local microcirculation with controversial results. The aim of our study was to investigate the influence of heparin and buflomedil, drugs improving the microcirculation, on local skin blood perfusion using intravital fluorescent microscopy (IVM) technique.Experiments were carried out on 24 male hairless mice under inhalatory isofluran anaesthesia. Intravenous injection of FITC-dextran (150 kD, 5%) allowed to visualize capillaries during IVM, after immobilisation of hind limb, in an observation chamber. Observations were performed after i.v. injection of heparin (66 IU/kg b.w., n = 8) or buflomedil (6 mg/kg b.w., n = 8) 30, 120 and 210 minutes after chamber installation. Observations were recorded in 30 sec sequences on S-VHS tapes and evaluated using special software. Functional capillary density (FCD), defined as a total length of red cells perfused capillaries per observation field (expressed in cm/cm(2)), postcapillary venule diameters, and number of sticking leukocytes per 0.2 mm vessel length during 30 sec observation time, served as parameters of skin blood perfusion and activation of L/EC interactions.A statistically significant decrease of FCD from 152.7 +/- 38.5 to 100.7 +/- 36.7 cm/cm (2) (p0.05) was observed in control animals during 210 min lasting observation. Administration of heparin prevented decrease in FCD occurring in control animals during intravital microscopy, whereas buflomedil was found ineffective. Both drugs induced a nonsignificant reduction in the number of sticking leukocytes, whereas no changes in postcapillary venule diameters could be observed.The results suggest a protective effect of heparin in clinical therapeutic doses against impairment of skin perfusion during IVM. This observation may justify a trial on the effects of heparin in prevention of development of pressure ulcers.

Production of nitric oxide by the inducible NO synthase (iNOS) is known to be enhanced in chronic joint inflammation and osteoarthritis as well as aseptic loosening of joint prostheses. Initial studies yielded promising results after inhibition of the nitric oxide synthase (NOS). However, the effect of NOS inhibition has not been studied at the site of the primary function of NO, the microcirculation of the synovium in vivo. Using our recently developed model for the in vivo study of synovial microcirculation in the mouse knee joint, the effects of selective versus nonselective inhibition of iNOS were investigated by means of intravital fluorescence microscopy. After resection of the patella tendon, the synovial fatty tissue was exposed for intravital microscopy. Diameter of arterioles, functional capillary density (FCD), diameter of venules, venular red blood cell velocity and leukocyte-endothelial cell interaction were quantitatively analyzed before, and 10 and 60 min after intravenous injection of NOS inhibitors [selective iNOS inhibitor N-iminoethyl-L-lysine (L-NIL), and nonselective NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME)]. Our results demonstrate that L-NAME causes a significant decrease in the arteriolar diameter and FCD associated with an increase in the leukocyte accumulation in the synovium in vivo. In contrast, L-NIL neither altered the microhemodynamics nor the leukocyte-endothelial cell interaction in the synovium, indicating its potential use for selective inhibition of iNOS in joint inflammation. Using our method, further studies will provide new insights into the unknown effect of NOS inhibition on the synovial microvasculature in inflammatory joint disease in vivo.

Insufficient detoxification caused by nitric oxide-related inhibition of cytochrome P450 may be important for metabolism of numerous drugs, including vecuronium. The present study investigated the pharmacodynamics and pharmacokinetics of vecuronium in rats with inflammatory liver dysfunction.Male Sprague-Dawley rats (n = 56) were randomly allocated into two groups: In the sepsis group, liver inflammation was established by injection of 56 mg/kg heat-killed Corynebacterium parvum; control rats received the solvent. At day 4, groups were subdivided according to treatment with the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine (250 mg/kg) or placebo. The aminopyrine breath test was performed to assess cytochrome P450 activity. Rats were anesthetized with propofol and mechanically ventilated. Duration of action of vecuronium (1.2 mg/kg) was measured by evoked mechanomyography (stimulation of the sciatic nerve, contraction of the gastrocnemius muscle). In seven rats of each subgroup a 50% neuromuscular blockade was established by a continuous vecuronium infusion. Vecuronium plasma levels were measured and plasma clearance of vecuronium was calculated. Nitric oxide synthesis was assessed by measuring nitrite/nitrate serum levels.In sepsis/placebo rats, vecuronium-induced neuromuscular blockade was prolonged (144% of contro/placebo), vecuronium plasma levels at 50% neuromuscular blockade were increased (122% of control/placebo), and plasma clearance was decreased (68% of control/placebo). N(G)-monomethyl-L-arginine therapy in rats with sepsis improved cytochrome P450 activity and plasma clearance of vecuronium, shortened duration of action of vecuronium, but did not alter the elevated vecuronium plasma levels.A systemic inflammatory response syndrome with liver dysfunction results in decreased sensitivity to and a decreased elimination of vecuronium. Modulation of nitric oxide synthesis may be a strategy that can be used in the future to improve xenobiotic metabolism in sepsis.

Sequestration of neutrophils and release of histotoxic mediators are considered important for the development of pathologic alterations of the lung defined as adult respiratory distress syndrome. Mechanisms of inflammatory lung injury caused by abdominal sepsis were investigated using the colon ascendens stent peritonitis (CASP) model that closely mimics the human disease. In the CASP model, a continuous leakage of intraluminal bacteria into the peritoneal cavity is induced by implantation of a stent in the ascending colon, generating a septic focus. In contrast to the cecal ligation and puncture model of peritonitis, survival of mice following CASP surgery is dependent on IFN-gamma, but independent of tumor necrosis factor (TNF). Here we show that the systemic inflammation induced by CASP surgery results in a rapid and profound increase of lung vascular permeability that was associated with the activation and recruitment of neutrophils to the lung. Activation of circulating granulocytes was characterized by increased production of serine proteinases and reactive oxygen metabolites, as well as elevated expression of cell surface Mac-1. Expression of MIP-2, KC, MIP-1alpha and E-selectin mRNA in lung was strongly increased within 3 h following CASP surgery, whereas up-regulation of IP-10, MCP-1 and P-selectin was delayed. In contrast, induction of RANTES, LIX, ICAM-1 and VCAM-1 mRNA was weak or not detectable after CASP surgery. Importantly, recruitment of leukocytes to the lung was normal in lipopolysaccharide-resistant mice, and was not affected by antibody neutralization of TNF or the chemokines MIP-2 and KC.

Inflammatory stimulation of the liver induces nitric oxide (NO) biosynthesis and suppression of detoxication. In this study the effect of NO biosynthesis on cytochrome P-450 (CYP) enzyme activity was investigated by comparing in vivo and in vitro assays. To establish liver inflammation, CD rats were injected with Corynebacterium parvum (C. parvum) suspension. After 5 days NO biosynthesis was highly induced as indicated by increased NO2- plus NO3- serum concentrations. At the same time the aminopyrine breath test (ABT), measuring CYP activity in vivo, was reduced to 42% and the in vitro assay of aminopyrine turnover was suppressed to 12% of NaCl- injected controls. When C. parvum-injected animals were treated with the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA), CYP activities significantly improved with an ABT of 76% and an in vitro aminopyrine turnover of 47% of controls. Neither C. parvum injections nor L-NMMA treatment resulted in a significant change of CYP protein concentrations. These data indicate that suppression of xenobiotic metabolism can be attenuated by inhibition of NO biosynthesis during an ongoing process of inflammation.

This article deals with a method of osteosynthesis which was tried out in a model experiment on Wistar rats. In this method, which may be termed "bone welding with ultrasound", bone fragments are glued together, using ultrasound and n-butyl-2-cyanoacrylates. The load capacities of glued and welded bones were compared in an in-vitro trial. The course of the in-vivo trial was followed up clinically and roentgenologically, and the preparations obtained after termination of an observation period of 6 weeks were then subjected to histological processing.

In two groups of six beagle dogs a standardized bone defect of 1 cm was replaced by a) "Kieler Spongiosa", and b) autologous bone material, prepared according to the formula of Urist. Instead of mechanical insufficiency an impressive osteogenetic potency and periosteosis was noticed in group b.

Bone induction by implantation of decalcified bone is described by many authors. This statement was proffed by implantation of decalcified pig bone in the abdominal wall muscle of rats. An enchondral osteogenesis was found following the implantation in a time intervall of 12 weeks according to the results of many other authors.