Biomedical subjects

Michael D Menger

Publications and source records attributed to Michael D Menger.

At least 73 records · Page 4Linked to original sources

PubMed2004

p-[123I]iodo-L-phenylalanine for detection of pancreatic cancer: basic investigations of the uptake characteristics in primary human pancreatic tumour cells and evaluation in in vivo models of human pancreatic adenocarcinoma.

Pancreatic cancer is associated with the worst 5-year survival rate of any human cancer. This high mortality is due, in part, to difficulties in establishing early and accurate diagnosis. Because most tumours share the ability to accumulate amino acids more effectively than normal tissues and any other pathology, assessment of amino acid transport in tumour cells using radiolabelled amino acids has become one of the most promising tools for tumour imaging. This study investigated the potential of p-[(123)I]iodo-L-phenylalanine (IPA) for detection of pancreatic cancer by single-photon emission tomography. IPA affinity for pancreatic tumour was investigated in human pancreatic adenocarcinoma PaCa44 and PanC1 cells, followed by analysis of the underlying mechanisms of tracer accumulation in neoplastic cells. Thereafter, IPA was evaluated for targeting of pancreatic tumours using SCID mice engrafted with primary human pancreatic adenocarcinoma cells, as well as in acute inflammation models in immunocompetent mice and rats. IPA accumulated intensively in human pancreatic tumour cells. Radioactivity accumulation in tumour cells following a 30-min incubation at 37 degrees C/pH 7.4 varied from 41% to 58% of the total loaded activity per 10(6) cells. The cellular uptake was temperature and pH dependent and predominantly mediated by specific carriers for neutral amino acids, namely the sodium-independent and L-leucine-preferring (L-system) transporter and the alanine-, serine- and cysteine-preferring (ASC-system) transporter. Protein incorporation was less than 8%. Biodistribution studies showed rapid localization of the tracer to tumours, reaching 10%+/-2.5% to 15%+/-3% of the injected dose per gram (I.D./g) in heterotopic tumours compared with 17%+/-3.5% to 22%+/-4.3% I.D./g in the orthotopic tumours, at 60 and 240 min post injection of IPA, respectively. In contrast, IPA uptake in the gastrointestinal tract and areas of inflammation remained moderate and decreased with time. Excellent tumour detection was obtained by gamma camera imaging. The specific and high-level targeting of IPA to tumour and the negligible uptake in the gastrointestinal tract and areas of inflammation indicate that p-[(123)I]iodo-L-phenylalanine is a promising tracer for differential diagnosis of pancreatic cancer.

Adenocarcinoma↗

PubMed2004

Improvement of rat liver graft quality by pifithrin-alpha-mediated inhibition of hepatocyte necrapoptosis.

Early graft dysfunction due to ischemia reperfusion injury remains a major clinical challenge in liver transplantation. Because apoptosis may contribute to graft dysfunction, we studied whether transient inhibition of p53 is capable of improving graft quality by reducing apoptotic cell death. Rat livers were harvested and stored for 24 hours or 48 hours in a 4 degrees C solution containing either pifithrin-alpha (PFT-alpha), a specific p53-inhibitor, or the vehicle dimethyl-sulfoxide. Storage was followed by 2-hour reperfusion with 37 degrees C Krebs-Henseleit buffer in an isolated liver perfusion system. Besides caspase-3 activation, apoptosis was quantified using fluorescence microscopy and hematoxylin-eosin histology. Trypan blue allowed for assessment of cell membrane damage, indicating both secondary apoptosis and primary necrosis. Bile flow, oxygen consumption, K(+)-excretion and enzyme release served as indicators of overall graft quality. Upon 2-hour reperfusion, livers developed procaspase activation as well as a mixture of apoptotic and necrotic cell death, representing necrapoptosis. In livers that had been stored for 48 hours, necrapoptotic injury was more pronounced compared with that after 24-hour storage. PFT-alpha effectively attenuated caspase activation as well as hepatocellular apoptosis and necrosis. Attenuation of both modes of cell death by PFT-alpha was associated with improved liver function, metabolism, and integrity. Experiments with the caspase inhibitor z-VAD-fmk confirmed that apoptosis is one mode of cell death in cold ischemia reperfusion. In conclusion, inhibition of p53-dependent apoptosis by PFT-alpha reduces hepatic preservation-reperfusion injury and improves primary organ function and metabolism. Fortification of the preservation solution with PFT-alpha may represent a promising and easily applicable approach to mitigate reperfusion injury in liver transplants.

Animals↗

PubMed2004

Vascular microenvironment in gliomas.

Structural and functional abnormalities of the vascular microenvironment determine pathophysiological characteristics of gliomas, such as loss of blood-brain barrier function, tumor cell invasiveness, or permselectivity for large molecules. Moreover, the effectiveness of various therapeutic strategies critically depends upon the successful transvascular delivery of molecules. In order to shed more light on the vascular microenvironment in gliomas, a variety of experimental and clinical techniques have been applied to study the glioma microvasculature, including histology, vascular corrosion casts, microangiography, autoradiography, tracer washout techniques, magnetic resonance imaging, as well as intravital fluorescence microscopy. This review summarizes the characteristic features of vascular morphology, angio-architecture, tumor perfusion, microvascular permeability, and microvessel-related immunological competence in gliomas. An improved understanding of the vascular microenvironment in gliomas will help in the future to optimize glioma imaging and to improve delivery of vectors for gene therapy or encapsulated drug carriers for pharmacotherapy in patients.

Animals↗

PubMed2004

Experimental liver metastasis: standards for local cell implantation to study isolated tumor growth in mice.

Experimental hepatic metastasis of colorectal tumors is frequently studied by local intrahepatic tumor cell implantation. However, although a variety of factors of the implantation procedure may markedly influence tumor growth characteristics, standards are not defined yet. Herein, we studied the effect of different modes of cell implantation on tumor growth and angiogenesis by in vivo fluorescence microscopy and histology seven days after grafting colorectal CT26.WT tumor cells into the left liver lobe of syngeneic BALB/c mice. We demonstrate that (i) radial growth of cells implanted within the central area of the lobe is inhibited by a regularly observed fissura which crosses at midline the surface of the lobe; (ii) cells suspended during implantation in RPMI show an uncontrolled overwhelming growth 40-fold of those suspended in PBS; (iii) cell implantation in 100 microl and 20 microl suspension medium is significantly more complicated by rupture of the liver capsule, uncontrolled intraparenchymal cell spread and recoil of the cells through the injection canal compared to cells suspended in 10 microl; (iv) the frequency of metastasis within the injection canal and at the puncture site is significantly reduced using 32G compared to 27G or 29G needles; (v) occlusion of the puncture site by acrylic glue or electric coagulation completely abolishes peritoneal tumor spread compared to no treatment or gentle compression by cotton gauze. We conclude that a standardized growth of isolated metastases is best achieved by implanting CT26.WT cells in a 10 microl PBS blister subcapsularly into the paramedian area of the lower surface of the left liver lobe, using a 32-gauge needle and closing the puncture site with acrylic glue.

Adenocarcinoma↗

PubMed2004

Advanced hepatic tissue destruction in ablative cryosurgery: potentials of intermittent freezing and selective vascular inflow occlusion.

Recent studies indicate that cryosurgery represents a promising approach to treat non-resectable liver tumors. To improve parenchymal tissue destruction, a variety of modifications of the freeze-thaw procedure have been suggested, including repetitive freezing and portal-triad cross-clamping. The aim of the present study was to analyze whether intermittent freezing by application of a double freeze-thaw procedure or selective vascular inflow occlusion are more effective than a single freeze-thaw cycle to achieve complete hepatic tissue destruction. Using a porcine model, intrahepatic cryolesions were induced by freezing the hepatic tissue for a total of 15 min (n=6, SF). Additional animals (n=6) underwent a double freeze-thaw cycle of 7.5 min each (DF). A third group of animals (n=6) was treated by a single 15-min freeze-thaw cycle during selective vascular inflow occlusion (VO-SF). Seven days after freezing, DF did not change the volume of the cryolesion (25.4+/-1.7 cm(3)) compared to SF (29.9+/-3.7 cm(3)), however, resulted in enhanced destruction of hepatocyte nuclear morphology (DF-score: 2.4+/-0.2 versus SF-score: 1.1+/-0.3; p<0.05) and attenuated leukocyte infiltration within the margin of the cryolesion (DF-score: 1.5+/-0.2 versus SF-score: 2.8+/-0.1; p<0.05). VO-SF was also effective to significantly enhance destruction of hepatocyte nuclear morphology (2.8+/-0.1; p<0.05 versus SF), but, additionally, markedly increased the volume of the cryolesions (43.3+/-5.3 cm(3); p<0.05 versus SF and DF). Interestingly, VO-SF further increased the number of apoptotic cells, while leukocyte infiltration (2.3+/-0.3) was not affected compared to that after SF-treatment. Thus, our data indicate that both DF and VO-SF are effective to enhance parenchymal cell destruction within the margin of the cryolesion. VO-SF additionally increases the volume of the lesion and may therefore be most attractive for successful clinical application.

Animals↗

PubMed2004

Reoxygenation results in cell death of human alveolar epithelial cells.

BACKGROUND: The functional response of isolated alveolar epithelial cells (AECs) to ischemia/reperfusion injury (I/R) is incompletely understood. Using a cell culture model, we investigated the tolerance of human type II alveolar cells (ATII) to hypoxia and subsequent reoxygenation. METHODS: Cell cultures of A549 cells (human lung adenocarcinoma) and primary ATII were incubated in 95% N(2)/5% CO(2) saturated medium at 37 degrees C for 48 hours or 72 hours. The hypoxic medium was subsequently exchanged to normoxic medium at 37 degrees C. Lactate dehydrogenase (LDH) release and mitochondrial viability, as assessed by WST-1 metabolism, were determined during both hypoxia and reoxygenation. A549 cells and ATII maintained under normoxic conditions served as controls. RESULTS: Before reoxygenation, after 48 or 72 hours of hypoxia, WST-1 metabolism in A549 cells was significantly reduced (p < 0.05), but LDH release remained low in both cell types. Reoxygenation after 48 h of hypoxia was associated with recovery of WST-1 metabolism and an only minimal increase in LDH release. Reoxygenation after 72 hours of hypoxia, in contrast, induced marked injury in both A549 cells and primary ATII as indicated by significantly reduced WST-1 metabolism and a dramatic increase of LDH release compared with normoxic controls (p < 0.05). CONCLUSIONS: Viability of alveolar cell lines and primary ATII is maintained during hypoxia for up to 72 hours. Reoxygenation after 72 hours of hypoxia results in rapid development of injury and cell death in both cell types.

Cell Death↗

PubMed2004

The delay of rearterialization after initial portal reperfusion in living donor liver transplantation significantly determines the development of microvascular graft dysfunction.

BACKGROUND/AIMS: Graft reperfusion in liver transplantation is usually performed by initial portal reperfusion (IPR) and delayed rearterialization. Its influence on graft microcirculation is unknown. This study aimed to assess sinusoidal perfusion in dependence to this reperfusion technique during human living-donor liver transplantation. METHODS: Hepatic microcirculation was measured both in the donor and the recipient (n=14) by using the orthogonal polarization spectral imaging technique. By using initial portal reperfusion, the mean time interval between portal venous and hepatic arterial reperfusion was 27.7+/-13.3 min. RESULTS: Hepatic nutritive perfusion, as given by the functional sinusoidal density and the volumetric blood flow, was found significantly decreased during portal reperfusion when compared to baseline. Rearterialization resulted in hyperperfusion of individual sinusoids at a decreased density of the sinusoidal network. Interestingly, the time interval between portal venous and hepatic arterial reperfusion significantly correlated with the changes of the liver grafts' microcirculation. CONCLUSIONS: The study indicates graft microcirculatory dysfunction as a major determinant of postischemic liver injury. Moreover, microvascular impairment was significantly influenced by the interval between portal venous and hepatic arterial reperfusion, which suggests the reinforcement of the pathomechanism of injury involving hypoxia and rapid graft rewarming due to initial portal reperfusion.

Adult↗

PubMed2004

Technetium-99m labeled 1-(4-fluorobenzyl)-4-(2-mercapto-2-methyl-4-azapentyl)-4-(2-mercapto-2-methylpropylamino)-piperidine and iodine-123 metaiodobenzylguanidine for studying cardiac adrenergic function: a comparison of the uptake characteristics in vascular smooth muscle cells and neonatal cardiac myocytes, and an investigation in rats.

In developing technetium-99m-based radioligands for in vivo studies of cardiac adrenergic neurons, we compared the uptake characteristics of the (99m)Tc-labeled 1-(4-fluorobenzyl)-4-(2-mercapto-2-methyl-4-azapentyl)-4-(2-mercapto-2-methylpropylamino)-piperidine ((99m)Tc-FBPBAT) with those of the clinically established meta-[(123)I]iodobenzylguanidine ((123)I-MIBG) in rat vascular smooth muscle cells and neonatal cardiac myocytes. Furthermore, the cardiac and extracardiac uptake of both radiopharmaceuticals was assessed in intact rats and in rats pretreated with various alpha- and beta-adrenoceptor drugs, and adrenergic reuptake blocking agents. The uptake of (99m)Tc-FBPBAT and (123)I-MIBG into vascular smooth muscle cells and neonatal cardiac myocytes was rapid; more than 85% of the radioactivity accumulation into the cells occurring within the first 3 minutes. Radioactivity uptake after a 60-minute incubation at 37 degrees C (pH 7.4) varied from 15% to 65% of the total loaded activity per million cells. In all cases, (99m)Tc-FBPBAT showed the higher uptake, relative to (123)I-MIBG, at any given cell concentration. The cellular uptake of (99m)Tc-FBPBAT was lower at 4 degrees C and 20 degrees C than at 37 degrees C. In contrast, the (123)I-MIBG uptake was only slightly temperature dependent. Inhibition experiments confirmed that the cellular uptake of (123)I-MIBG is mediated by the uptake-I carrier, whereas alpha(1)- and beta(1)-adrenoceptors were predominantly involved in the uptake of (99m)Tc-FBPBAT into the cardiovascular tissues. Biodistribution studies in rats showed that (99m)Tc-FBPBAT accumulated in myocardium after intravenous injection. Radioactivity in rat heart amounted to 2.32% and 1.91% of the injected dose per gram at 15 and 60 minutes postinjection, compared with 3.10% and 2.21% injected dose per gram of tissue (%ID/g) in the experiment with (123)I-MIBG, respectively. Prazosin, urapidil, and metoprolol were as effective as treatment with other adrenergic drugs in lowering cardiac uptake of (99m)Tc-FBPBAT. Uptake reduction was more pronounced in myocardium than in other adrenergic-rich organs, including the lung, spleen, kidney, and adrenals, suggesting that the (99m)Tc-FBPBAT uptake in myocardium specifically reflects a high degree of alpha(1)/beta(1)-receptor binding to cardiac adrenergic neurons. In comparison, reduction of cardiac and pulmonary uptake of (123)I-MIBG was effective after pretreatment of rats with desipramine and reserpine, confirming distinct neuronal binding sites for (99m)Tc-FBPBAT and (123)I-MIBG. (99m)Tc-FBPBAT was excreted via urine and to a lower degree via feces. Urine analysis 6 hours p.i. revealed that more than 40% of the total excreted radioactivity was unmetabolized (99m)Tc-FBPBAT. In conclusion, the uptake of (99m)Tc-FBPBAT in rat myocardium specifically reflects binding to cardiac adrenergic neurons. The (99m)Tc-FBPBAT uptake appears to be predominantly mediated via the alpha(1)/beta(1)-adrenoceptor pathway. These data indicate that (99m)Tc-FBPBAT, like (123)I-MIBG, may be suitable for mapping cardiac adrenergic innervation by SPET, especially for alpha(1)/beta(1)-adrenoceptors as target in numerous heart diseases.

3-Iodobenzylguanidine↗

PubMed2004

Aprotinin inhibits local platelet trapping and improves tissue destruction in hepatic cryosurgery.

BACKGROUND: During the last decade, cryosurgery became an interesting alternative in the treatment of nonresectable liver neoplasms. The freeze-thaw procedure, however, may be associated with life-threatening thrombocytopenia due to local platelet trapping, and success of neoplasm ablation may be compromised by inadequate parenchymal cell destruction. METHODS: Because aprotinin is capable of inhibiting the initiation of both coagulation and fibrinolysis, we studied-by whole body scintigraphy of Indium-111-labeled platelets and histomorphology in a porcine model of hepatic cryosurgery-whether this serine protease inhibitor is effective in attenuating platelet trapping and in improving tissue destruction. RESULTS: Fifteen minutes of cryotherapy (-168 degrees C at the tip of the cryoprobe) induced a 30 +/- 4 cm(3) cryolesion, which presented with massive platelet trapping (14.0 +/- 1.7% cryolesion activity/whole body activity) and incomplete parenchymal cell destruction (0.9 +/- 0.3; score of hepatocyte nuclear destruction within the margin of the cryolesion). Aprotinin treatment with 500,000 IU initial bolus injection and additional 500,000 IU infusion over 3 hours did not affect the size of the cryolesion (29 +/- 3 cm(3)) but reduced local platelet activity (1.9 +/- 1.9%; P<.001) and induced hepatocyte nuclear destruction (3.0 +/- 0.0; P<.001). CONCLUSIONS: Thus, our study indicates that aprotinin inhibits cryoablation-associated platelet trapping and improves tissue destruction. The serine protease inhibitor may represent a valuable adjunct in cryosurgery of hepatic neoplasms.

Animals↗

PubMed2004

Microvascular in vivo assessment of reperfusion injury: significance of prostaglandin E(1) and I(2) in postischemic "no-reflow" and "reflow-paradox".

BACKGROUND: Microvascular ischemia-reperfusion (I/R) injury is characterized by failure of capillary perfusion ("no-reflow") and reoxygenation-associated phenomena ("reflow-paradox"), including activation of leukocyte-endothelium interaction with cytotoxic mediator-induced loss of endothelial integrity. The objectives of this study were to elucidate the impact of both prostaglandins E(1) (PGE(1)) and I(2) (PGI(2)) in microvascular reperfusion injury, with special focus on the distinct pathophysiology of no-reflow- and reflow-paradox phenomena. MATERIALS AND METHODS: By use of the hamster dorsal skinfold preparation and in vivo fluorescence microscopy, the microcirculation of a striated skin muscle was assessed before 4 h of pressure-induced ischemia and 0.5, 2, and 24 h after onset of reperfusion. RESULTS: I/R was characterized by enhanced leukocyte-endothelium interaction in postcapillary venules, increase of macromolecular leakage, and reduction of functional capillary perfusion (P < 0.05). Intravenous 2-h infusion of PGE(1), starting with onset of reperfusion, reduced leukocyte adhesion and macromolecular leakage in postcapillary venules during early reperfusion (P < 0.05), while 6-h infusion, given during ischemia and early reperfusion, showed no significant effects. PGI(2) infusion also attenuated postischemic leukocyte adhesion, which was significant by a 6-h prolonged administration (P < 0.05), but did not influence the increase of microvascular permeability. Both prostaglandins were unable to prevent the postischemic failure of capillary perfusion (no-reflow). CONCLUSIONS: Both prostaglandins did not significantly influence postischemic no-reflow phenomena, but appeared as potent inhibitors of reflow-paradox under the experimental circumstances of this study.

Alprostadil↗

PubMed2004

Heme oxygenase does not contribute to control of basal vascular tone in isolated blood-perfused rat lung.

BACKGROUND: Vasoconstriction in pulmonary ischemia-reperfusion injury may involve dysfunction of the physiologic vasodilation of pulmonary arteries. Little is known of the relative importance of heme oxygenase (HO)/carbon monoxide (CO)-dependent vs nitric oxide synthase (NOS)/nitric oxide (NO)-dependent vasodilation of the pulmonary vasculature. We evaluated the significance of HO function on basal pulmonary vascular resistance (PVR) and compared it with the function of NOS. METHODS: Using an isolated blood-perfusion model, lungs of Lewis rats were assigned to 3 groups (n = 6/group). After stabilization, either an inhibitor of HO (tin-protoporphyrin-9 [SnPP-9]) or an inhibitor of NOS (NG-nitro-L-arginine methylester [L-NAME]) was added to the perfusate (50 micromol/liter and 1 mmol/liter as the final concentration, respectively). Lungs receiving saline served as controls. Gas exchange, hemodynamic and respiratory functions and the levels of cyclic 3',5'-guanosine monophosphate (cGMP) in the perfusate were measured. RESULTS: Inhibition of NOS by L-NAME resulted in a significant (p < 0.01) increase in PVR (DeltaPVR: 0.110 +/- 0.012 cm H(2)O/ml. min) within 5 minutes. In contrast, PVR was minimally affected by SnPP-9 (DeltaPVR: 0.005 +/- 0.005 cm H(2)O/ml. min), which was comparable to control lungs (DeltaPVR: 0.012 +/- 0.005 cm H(2)O/ml. min). The level of cGMP in the perfusate 5 minutes after drug application was markedly, but not significantly, lower in the L-NAME group (1.67 +/- 0.74 nmol/liter) when compared with controls (2.69 +/- 0.89 nmol/liter) and SnPP-9-treated lungs (2.65 +/- 0.66 nmol/liter). CONCLUSIONS: NOS but not HO contributes to the control of basal vascular tone in the rat lung.

Animals↗

PubMed2004

Microhemodynamic and cellular mechanisms of activated protein C action during endotoxemia.

OBJECTIVE: To characterize microcirculatory actions of activated protein C in an endotoxemia rodent model that allows in vivo studies of microvascular inflammation and perfusion dysfunction. DESIGN: Animal study using intravital microscopy. SETTING: Animal research facility. SUBJECTS: Male Syrian golden hamsters, 6-8 wks old with a body weight of 60-80 g. INTERVENTIONS: In skinfold preparations, endotoxemia was induced by intravenous administration of 2 mg/kg endotoxin (lipopolysaccharide, Escherichia coli). Intravital microscopy allowed quantitative analysis of arteriolar and venular leukocyte adhesion and functional capillary density (cm) that served as a measure of microvascular perfusion failure. Activated protein C (APC group, n = 8, 24 microg/kg intravenously) was substituted continuously during 8 hrs after lipopolysaccharide, whereas endotoxemic buffer-treated animals (control, n = 7) served as controls. MEASUREMENTS AND MAIN RESULTS: Lipopolysaccharide increased leukocyte adhesion and decreased functional capillary density to 50% of baseline values (p <.01 vs. baseline). Activated protein C treatment inhibited (p <.05) lipopolysaccharide-mediated leukocytic response and attenuated (p <.05) endotoxic perfusion failure in nutritive capillaries. CONCLUSIONS: Activated protein C-induced protection from lipopolysaccharide-mediated microcirculatory dysfunction was characterized in vivo for the first time. The impressive modification of leukocyte cross-talk indicates systemic anti-inflammatory activated protein C effects on leukocytes and the endothelium, subsequently improving capillary perfusion. These actions could represent the in vivo mechanism of activated protein C interactions observed in patients with severe sepsis.

Animals↗

PubMed2003

Combined inhibition of VEGF and PDGF signaling enforces tumor vessel regression by interfering with pericyte-mediated endothelial cell survival mechanisms.

Destruction of existing tumor blood vessels may be achieved by targeting vascular endothelial growth factor (VEGF) signaling, which mediates not only endothelial cell proliferation but also endothelial cell survival. In this study, however, intravital microscopy failed to demonstrate that targeting of VEGFR-2 (by the tyrosine kinase inhibitor SU5416) induces significant regression of experimental tumor blood vessels. Immunohistochemistry, electron microscopy, expression analyses, and in situ hybridization provide evidence that this resistance of tumor blood vessels to VEGFR-2 targeting is conferred by pericytes that stabilize blood vessels and provide endothelial cell survival signals via the Ang-1/Tie2 pathway. In contrast, targeting VEGFR-2 plus the platelet-derived growth factor receptor (PDGFR)-beta system (PDGFR-beta) signaling (by SU6668) rapidly forced 40% of tumor blood vessels into regression, rendering these tumors hypoxic as shown by phosphorescence quenching. TUNEL staining, electron microscopy, and apoptosis blocking experiments suggest that VEGFR-2 plus PDGFR-beta targeting enforced tumor blood vessel regression by inducing endothelial cell apoptosis. We further show that this is achieved by an interference with pericyte-endothelial cell interaction. This study provides novel insights into the mechanisms of how 1) pericytes may provide escape strategies to anti-angiogenic therapies and 2) novel concepts that target not only endothelial cells but also pericyte-associated pathways involved in vascular stabilization and maturation exert potent anti-vascular effects.

Animals↗

PubMed2003

Experimental models to study microcirculatory dysfunction in muscle ischemia-reperfusion and osteomyocutaneous flap transfer.

BACKGROUND: During the past decade, experimental studies have provided convincing evidence that microcirculatory dysfunction plays a pivotal role in the manifestation of tissue injury in ischemia-reperfusion and osteomyocutaneous flap transfer. The study of the mechanisms of injury, however, requires sophisticated experimental in vivo models. With the use of microsurgical techniques, osteomyocutaneous flap transfer can successfully be performed in rat hind limbs, allowing in vivo fluorescent microscopic analysis of post-ischemic microcirculatory dysfunction in all tissues involved, including periosteum, striated muscle, subcutis and skin. The drawback of this "acute" model is that the period of analysis is restricted to a few hours only. METHOD: To overcome this limitation, the "chronic" dorsal skinfold chamber preparation, containing striated muscle and subcutis, can be used. This model allows one to study microcirculatory dysfunction after both tourniquet-induced and pressure-induced ischemia-reperfusion-induced tissue injury over a period of up to 3 weeks. RESULTS: With the use of these models, recent investigations have demonstrated that ischemia-reperfusion and osteomyocutaneous flap transfer are associated with capillary perfusion failure (no-reflow), mediated by intravascular hemoconcentration, endothelial swelling and endothelin (ET)-1-mediated microvascular constriction. In addition, post-ischemic reperfusion provokes an inflammatory response (reflow paradox) in post-capillary venules, which is characterized by beta2-integrin-mediated and intercellular adhesion molecule (ICAM)-1-mediated leukocyte adhesion and vascular hyperpermeability, which results in interstitial edema formation. Treatment studies have produced evidence that isovolemic hemodilution and heat shock protein induction are successful in ameliorating capillary no-reflow, while blockade of adhesion molecules, inactivation of oxygen radicals and, also, induction of heat shock proteins, are capable of reducing the post-ischemic inflammatory response. CONCLUSION: These experimental results not only demonstrate the importance of the use of advanced in vivo methods to delineate pathophysiological mechanisms in complex disease models, but may also provide a basis for potential prospective randomized trials to test the benefit for the patient in the daily clinical routine.

Animals↗

PubMed2003

Protective skeletal muscle arteriolar vasomotion during critical perfusion conditions of osteomyocutaneous flaps is not mediated by nitric oxide and endothelins.

BACKGROUND: After flap surgery, vasomotion, defined as oscillation of the arteriolar diameter, may protect tissue during critical perfusion conditions. The mechanisms that regulate vasomotion are still unclear; therefore, we studied the incidence of vasomotion in peripheral tissue and whether nitric oxide or endothelins are involved in regulation of vasomotion. MATERIALS AND METHODS: In Sprague-Dawley rats, an osteomyocutaneous flap was prepared. To induce critical perfusion conditions, we reduced arterial blood flow supplying the flap to 0.15 ml/min. Seven animals received NG-nitro-L-arginine methyl ester (L-NAME), a nitric oxide-synthase inhibitor, and six animals bosentan, an endothelin A/B receptor antagonist. Microcirculation of muscle, skin, subcutis and periosteum was assessed by intravital microscopy before and after drug application. RESULTS: In all animals, reduction of arterial blood supply induced arteriolar vasomotion in muscle (100%), but not in periosteum, subcutis and skin. Vasomotion was found to be affected by neither L-NAME (frequency 2.6+/-0.2 versus 2.4+/-0.2 cycles/min; amplitude 67+/-19 versus 55+/-20%; share of dilation period in vasomotion cycle 59+/-2 versus 58+/-3%) nor bosentan (1.8+/-0.1 versus 1.7+/-0.1 cycles/min; 60+/-10 versus 64+/-6%; 50+/-2 versus 53+/-1%). CONCLUSIONS: Our study indicates that during critical perfusion conditions, arteriolar vasomotion develops only in muscle, not in skin, subcutis and periosteum, and that nitric oxide and endothelins are not involved in the regulation of this protective vascular response.

Animals↗

PubMed2003

Noninvasive in vivo analysis of the human hepatic microcirculation using orthogonal polorization spectral imaging.

BACKGROUND: Analysis of hepatic microvascular perfusion in humans by direct imaging has been impossible so far. Orthogonal polarization spectral (OPS) imaging represents a new technology that combines simultaneous epi-illumination of the subject with linearly polarized light and noninvasive imaging of the microcirculation by reflectance spectrophotometry. The aim of this study was to evaluate the feasibility of studying the human hepatic microcirculation by OPS imaging in vivo and to define microcirculatory parameters for physiologic conditions. METHODS: The hepatic microcirculation was analyzed in four different regions of both liver lobes in 11 healthy individuals undergoing partial liver resection for living-donor liver transplantation. The optical probe was gently positioned on the liver surface and sequences of at least 20 sec per measurement were recorded by a charge-coupled device camera on videotape. Microhemodynamic parameters were quantified off-line by single-frame and frame-to-frame analysis using a computer-assisted image analysis system. RESULTS: OPS images of the hepatic microcirculation showed an acceptable quality with good resolution. Quantitative analysis revealed a sinusoidal red blood cell velocity of 0.97+/-0.43 mm/sec, a sinusoidal diameter of 8.8+/-0.9 microm, a sinusoidal volumetric blood flow of 58.2+/-9.6 pL/sec, an intersinusoidal distance of 22.6+/-2.5 microm, and a mean functional sinusoidal density of 391+/-30 cm-1. Apart from the sinusoidal red blood cell velocity, all data of the parameters studied matched the pattern of normal distribution. CONCLUSIONS: OPS imaging enabled for the first time direct in vivo visualization and quantification of the human hepatic microcirculation, providing significant insight into microvascular physiology of the human liver, to the extent that these data can be considered to represent physiologic values for human hepatic microcirculation.

Adult↗

PubMed2003

Improvement of microvascular graft equilibration and preservation in non-heart-beating donors by warm preflush with streptokinase.

Using in situ fluorescence microscopy with Sprague Dawley rats, we studied the hypothesis of compromised microvascular kidney perfusion on organ harvest in non-heart-beating donors (NHBDs), and we evaluated the potential benefit of an additional preflush with saline solution containing streptokinase. Aortal flush of NHBD kidneys solely with University of Wisconsin solution resulted in a significantly (P <0.05) reduced functional capillary density (FCD) with increased perfusion heterogeneity compared with kidneys of heart-beating controls. This was associated with an increased lactate dehydrogenase (LDH) release on 24 hr postpreservation rinse of the grafts (76.7+/-18.9 U/L). Warm preflush with low-viscosity Ringer's lactate (RL) solution alone did not influence the decreased renal FCD and the postpreservation LDH release (76.2+/-29.1 U/L). In contrast, the addition of streptokinase to the RL preflush solution resulted in a significant (P <0.05) improvement of FCD with values not statistically different from those of heart-beating controls. This was associated with an attenuation of perfusion heterogeneity and a significantly lowered postpreservation LDH release (17.0+/-2.5 U/L). Furthermore, in transplanted and reperfused NHBD kidney grafts, the use of streptokinase-supplemented RL for preflush during organ harvest significantly (P <0.05) reduced early manifestation of tubular necrosis (29%+/-8%) when compared with kidneys preflushed exclusively with University of Wisconsin solution (56%+/-4%). Thus, we conclude that kidney harvest from NHBDs is prone to severe microvascular perfusion deficits, which are likely to preclude successful preservation of organ integrity during cold storage. Temporary fibrinolytic preflush with streptokinase may represent a feasible tool to improve microvascular graft equilibration, which effectively protects the renal integrity during both cold storage and posttransplant reperfusion.

Adenosine↗

PubMed2003

Blockade of in vivo VEGF-KDR/flk-1 signaling does not affect revascularization of freely transplanted pancreatic islets.

BACKGROUND: The aim of this study was to analyze the role of vascular endothelial growth factor (VEGF) binding to its high-affinity receptor kinase insert domain (KDR)-containing receptor/fetal liver kinase (flk)-1 in mediating revascularization of freely transplanted pancreatic islets in vivo. METHODS: Isolated pancreatic islets were syngeneically transplanted into dorsal skinfold chambers of Syrian hamsters. Animals were treated daily with the VEGF-KDR/flk-1 antagonist SU5416 (25 mg/kg intraperitoneally) or received vehicle for control. Intravital fluorescence microscopy and computer-assisted off-line analysis were used to study islet graft angiogenesis and revascularization during days 6, 10, and 14 after transplantation. RESULTS: In controls, islets were revascularized within 10 to 14 days after transplantation. SU5416 treatment did not affect revascularization, inasmuch as both functional capillary density and size of revascularized endocrine tissue did not differ from that of vehicle-treated controls. CONCLUSION: Because blockade of VEGF-KDR/flk-1 function does not affect islet revascularization, we conclude that VEGF signaling through its high-affinity receptor KDR/flk-1 is not an essential prerequisite for the process of new-vessel formation in freely transplanted islets of Langerhans.

Animals↗