Claudio Contaldo

Aggravation of viral hepatitis by platelet-derived serotonin.

More than 500 million people worldwide are persistently infected with hepatitis B virus or hepatitis C virus. Although both viruses are poorly cytopathic, persistence of either virus carries a risk of chronic liver inflammation, potentially resulting in liver steatosis, liver cirrhosis, end-stage liver failure or hepatocellular carcinoma. Virus-specific T cells are a major determinant of the outcome of hepatitis, as they contribute to the early control of chronic hepatitis viruses, but they also mediate immunopathology during persistent virus infection. We have analyzed the role of platelet-derived vasoactive serotonin during virus-induced CD8+ T cell–dependent immunopathological hepatitis in mice infected with the noncytopathic lymphocytic choriomeningitis virus. After virus infection, platelets were recruited to the liver, and their activation correlated with severely reduced sinusoidal microcirculation, delayed virus elimination and increased immunopathological liver cell damage. Lack of platelet-derived serotonin in serotonin-deficient mice normalized hepatic microcirculatory dysfunction, accelerated virus clearance in the liver and reduced CD8+ T cell–dependent liver cell damage. In keeping with these observations, serotonin treatment of infected mice delayed entry of activated CD8+ T cells into the liver, delayed virus control and aggravated immunopathological hepatitis. Thus, vasoactive serotonin supports virus persistence in the liver and aggravates virus-induced immunopathology.

NONO couples the circadian clock to the cell cycle

Mammalian circadian clocks restrict cell proliferation to defined time windows, but the mechanism and consequences of this interrelationship are not fully understood. Previously we identified the multifunctional nuclear protein NONO as a partner of circadian PERIOD (PER) proteins. Here we show that it also conveys circadian gating to the cell cycle, a connection surprisingly important for wound healing in mice. Specifically, although fibroblasts from NONO-deficient mice showed approximately normal circadian cycles, they displayed elevated cell doubling and lower cellular senescence. At a molecular level, NONO bound to the p16-Ink4A cell cycle checkpoint gene and potentiated its circadian activation in a PER protein-dependent fashion. Loss of either NONO or PER abolished this activation and circadian expression of p16-Ink4A and eliminated circadian cell cycle gating. In vivo, lack of NONO resulted in defective wound repair. Because wound healing defects were also seen in multiple circadian clock-deficient mouse lines, our results therefore suggest that coupling of the cell cycle to the circadian clock via NONO may be useful to segregate in temporal fashion cell proliferation from tissue organization.

Functional results of angular-stable plate fixation in displaced proximal humeral fractures

INTRODUCTION The availability of angular-stable plate/screw systems led to a euphoric use of these implants for the treatment of displaced proximal humerus fractures. The high implant costs seem to be justified by a potentially improved outcome. PATIENTS AND METHODS Thirty one patients (20 female, 11 male, mean age: 62+/-16 years) with two-, three- and four-part proximal humerus fractures (Neer classification) were operated using the proximal humeral internal locking system (PHILOS). The mean follow-up time was 19+/-3 postoperative months (range: 340-720 days). Functional results (Constant score, UCLA-score) were analysed and compared to an equivalent historic control group of 60 patients operated for the same fracture types using two one-third tubular plates. Additionally, total implant costs for each technique were compared. RESULTS Complications in the PHILOS group included one implant failure with refracture, one secondary dislocation, two cases of subacromial impingement, and two cases of partial avascular necrosis of the humeral head. The mean Constant score (age- and sex-matched) was 80+/-11% for the affected side and 104+/-13% for the healthy side. The UCLA scores were excellent in 10%, good in 67%, and fair in 23% of the patients. Complication rate and functional results did not differ significantly from the control group treated with one-third tubular plates. Implant costs were significantly higher for the PHILOS group (684+/-40 Euro vs. 158+/-20 Euro, p<0.05). CONCLUSION Our study showed similar functional results using either plate. Although the PHILOS plate may provide important advantages in specific situations, such as osteoporotic bone, its use as a standard must be carefully judged under the economic aspect of the significant higher implant costs.

Prevention of reperfusion injury and microcirculatory failure in macrosteatotic mouse liver by omega‐3 fatty acids

Macrovesicular hepatic steatosis has a lower tolerance to reperfusion injury than microvesicular steatosis with an abnormally high ratio of omega‐6 (n‐6): omega‐3 (n‐3) polyunsaturated fatty acids (PUFAs). We investigated the influence of PUFAs on microcirculation in steatotic livers and the potential to minimize reperfusion injury in the macrosteatotic liver by normalization of PUFAs. Ob/ob mice were used as a model of macrovesicular hepatic steatosis and C57/Bl6 mice fed a choline‐deficient diet for microvesicular steatosis. Steatotic and lean livers were subjected to 45 minutes of ischemia and 3 hours of reperfusion. Hepatic content of omega‐3 and omega‐6 PUFAs was determined. Microcirculation was investigated using intravital fluorescence microscopy. A second group of ob/ob mice was supplemented with dietary omega‐3 PUFAs and compared with the control diet–fed group. Microcirculation, AST, and Kupffer cell activity were assessed. Macrosteatotic livers had significant microcirculatory dysfunction correlating with high omega‐6: omega‐3 PUFA ratio. Dietary omega‐3 PUFA resulted in normalization of this ratio, reduction of intrahepatic lipids, and decrease in the extent of macrosteatosis. Defective microcirculation was dramatically ameliorated with significant reduction in Kupffer cell activity and protection against hepatocellular injury both before ischemia and after reperfusion. Conclusion: Macrosteatotic livers disclosed an abnormal omega‐6: omega‐3 PUFA ratio that correlates with a microcirculatory defect that enhanced reperfusion injury. Thus, protective strategies applied during or after ischemia are unlikely to be useful. Preoperative dietary omega‐3 PUFAs protect macrosteatotic livers against reperfusion injury and might represent a valuable method to expand the live liver donor pool. (HEPATOLOGY 2007;45:855–863.)

A new model for studying the revascularization of skin grafts in vivo: the role of angiogenesis.

Background: Models of skin graft revascularization are based mostly on histologic evaluations but lack the possibility of analyzing the vascular biology in vivo. The aim of the present study was therefore to develop an animal model that allows continuous monitoring of the microcirculation during skin graft healing. Methods: Skin and subcutaneous tissue were removed from the back of dorsal skinfold chamber preparations in mice, leaving one layer of striated muscle and subcutaneous tissue as a wound bed (n = 5). A corresponding full-thickness skin graft was harvested from the groin and sutured into the defect in the back of the chamber. To study graft healing, repetitive intravital microscopy was performed during the first 10 days after engraftment. Results: Capillary widening in the wound bed appeared at day 1 after grafting and increased until day 4. Capillary buds and sprouts first appeared at day 2. Blood filling of autochthonous graft capillaries occurred at day 3, resulting in almost complete restoration of the original skin microcirculation on day 5. This was achieved by interconnections between the microvasculature of the wound bed and the skin graft through a temporary angiogenic response. In principle, angiogenic blood vessel growth originated in the wound bed and was directed toward the graft. Conclusions: This new model allows for repetitive analysis of the microcirculation during skin graft healing. It provides ideal in vivo conditions to further delineate the exact mechanisms of blood vessel interconnection during the complex process of angiogenesis, and may also allow study of the vascularization of tissue-engineered skin substitutes.

The effect of perfluorocarbon-based artificial oxygen carriers on tissue-engineered trachea.

The biological effect of the perfluorocarbon-based artificial oxygen carrier (Oxygent) was investigated in tissue-engineered trachea (TET) construction. Media supplemented with and without 10% Oxygent were compared in all assessments. Partial tissue oxygen tension (PtO(2)) was measured with polarographic microprobes; epithelial metabolism was monitored by microdialysis inside the TET epithelium perfused with the medium underneath. Chondrocyte-DegraPol constructs were cultured for 1 month with the medium before glycosaminoglycan assessment and histology. Tissue reaction of TET epithelial scaffolds immersed with the medium was evaluated on the chick embryo chorioallantoic membrane. Oxygent perfusion medium increased the TET epithelial PtO(2) (51.2 +/- 0.3 mm Hg vs. 33.4 +/- 0.3 mm Hg at 200 microm thickness; 12.5 +/- 0.1 mm Hg vs. 3.1 +/- 0.1 mm Hg at 400 microm thickness, p < 0.01) and decreased the lactate concentration (0.63 +/- 0.08 vs. 0.80 +/- 0.06 mmol/L, p < 0.05), lactate/pyruvate (1.87 +/- 0.26 vs. 3.36 +/- 10.13, p < 0.05), and lactate/glucose ratios (0.10 +/- 0.00 vs. 0.29 +/- 0.14, p < 0.05). Chondrocyte-DegraPol in Oxygent group presented lower glycosaminoglycan value (0.03 +/- 0.00 vs. 0.13 +/- 0.00, p < 0.05); histology slides showed poor acid mucopolysaccharides formation. Orthogonal polarization spectral imaging showed no difference in functional capillary density between the scaffolds cultured on chorioallantoic membranes. The foreign body reaction was similar in both groups. We conclude that Oxygent increases TET epithelial PtO(2), improves epithelial metabolism, does not impair angiogenesis, and tends to slow cartilage tissue formation.

Decreasing gut wall glucose as an early marker of impaired intestinal perfusion

Objective:The aim of this study was to assess the microcirculatory and metabolic consequences of reduced mesenteric blood flow. Design:Prospective, controlled animal study. Setting:The surgical research unit of a university hospital. Subjects:A total of 13 anesthetized and mechanically ventilated pigs. Interventions:Pigs were subjected to stepwise mesenteric blood flow reduction (15% in each step, n = 8) or served as controls (n = 5). Superior mesenteric arterial blood flow was measured with ultrasonic transit time flowmetry, and mucosal and muscularis microcirculatory perfusion in the small bowel were each measured with three laser Doppler flow probes. Small-bowel intramucosal Pco2 was measured by tonometry, and glucose, lactate (L), and pyruvate (P) were measured by microdialysis. Measurements and Main Results:In control animals, superior mesenteric arterial blood flow, mucosal microcirculatory blood flow, intramucosal Pco2, and the lactate/pyruvate ratio remained unchanged. In both groups, mucosal blood flow was better preserved than muscularis blood flow. During stepwise mesenteric blood flow reduction, heterogeneous microcirculatory blood flow remained a prominent feature (coefficient of variation, ∼45%). A 30% flow reduction from baseline was associated with a decrease in microdialysis glucose concentration from 2.37 (2.10–2.70) mmol/L to 0.57 (0.22–1.60) mmol/L (p < .05). After 75% flow reduction, the microdialysis lactate/pyruvate ratio increased from 8.6 (8.0–14.1) to 27.6 (15.5–37.4, p < .05), and arterial–intramucosal Pco2 gradients increased from 1.3 (0.4–3.5) kPa to 10.8 (8.0–16.0) kPa (p < .05). Conclusions:Blood flow redistribution and heterogeneous microcirculatory perfusion can explain apparently maintained regional oxidative metabolism during mesenteric hypoperfusion, despite local signs of anaerobic metabolism. Early decreasing glucose concentrations suggest that substrate supply may become crucial before oxygen consumption decreases.

New generation of hemoglobin-based oxygen carriers evaluated for oxygenation of critically ischemic hamster flap tissue

Objectives:The aim of this study was to investigate and compare the effects of a traditionally formulated, low-viscosity, right-shifted polymerized bovine hemoglobin solution and a highly viscous, left-shifted hemoglobin vesicle solution (HbV-HES) on the oxygenation of critically ischemic peripheral tissue. Design:Randomized, prospective study. Setting:University laboratory. Subject:A total of 40 male golden Syrian hamsters. Interventions:Island flaps were dissected from the back skin of anesthetized hamsters. The flap included a critically ischemic, hypoxic area that was perfused via a collateralized vasculature. One hour after completion of the preparation, the animals received a 33% blood exchange with 6% hydroxyethyl starch 200/0.5 (HES, n = 9), HbV suspended in HES (HbV-HES, n = 8), or polymerized bovine hemoglobin solution (n = 9). Measurements and Main Results:Three hours after the blood exchange, microcirculatory blood flow (laser-Doppler flowmetry) was increased to 262% of baseline for HbV-HES (p < .01) and 197% for polymerized bovine hemoglobin solution (p < .05 vs. baseline and HbV-HES). Partial tissue oxygen tension (bare fiber probes) was only improved after HbV-HES (9.4 torr to 14.2 torr, p < .01 vs. baseline and other groups). The tissue lactate/pyruvate ratio (microdialysis) was elevated to 51 in the untreated control animals, and to 34 ± 8 after HbV-HES (p < .05 vs. control) and 38 ± 11 after polymerized bovine hemoglobin solution (not significant). Conclusions:Our study suggests that in critically ischemic and hypoxic collateralized peripheral tissue, oxygenation may be improved by normovolemic hemodilution with HbV-HES. We attributed this improvement to a better restoration of the microcirculation and oxygen delivery due to the formulation of the solution.

In vivo visualization of the origination of skin graft vasculature in a wild-type/GFP crossover model

INTRODUCTION Skin substitutes are increasingly produced in tissue engineering, but still the understanding of the physiological skin revascularization process is lacking. To study in vivo conditions we recently introduced a mouse model, in which we already characterized the angiogenic changes within the wound bed and the skin graft. The aim of this study was to identify the origination of the vasculature during skin graft revascularization in vivo and to track vessel development over time. METHODS We created a crossover wild-type/GFP skin transplantation model, in which each animal carried the graft from the other strain. The preparation of the modified dorsal skin fold chamber including cross-over skin grafting was performed in male C57BL/6J wild-type mice (n=5) and C57BL/6-Tg(ACTB-EGFP)1O sb/J mice (n=5). Intravital microscopy in 12 areas of wild-type and GFP skin grafts was performed daily over a time period of 10 days. RESULTS Graft reperfusion started after 48-72 h. After reperfusion GFP-positive structures from the wound bed were visible in the graft capillaries with the highest density in the center of the graft. Overall, we observed a replacement of existing graft capillaries with vessels from the wound bed in 68% of the vessels. Of note, vessel replacement occurred in almost 100% of graft vessels in the periphery. Additionally, vessels within the graft showed a temporary angiogenic response between days 3-8, which originated predominantly from the autochthonous graft vasculature, but also contained already grown-in vessels from the wound bed. CONCLUSIONS These in vivo data indicate an early in-growth of angiogenic bed vessels into the existing vascular channels of the graft and subsequent centripetal replacement. Additionally we observed a temporary angiogenic response of the autochthonous capillaries of the skin graft with contribution from bed vessels. These findings further support the theory that sprouting angiogenesis from the wound bed in combination with the replacement of existing graft vessels are the key factors in skin graft taking. Thus, manufacturing of skin substitutes should be aimed at providing pre-formed vascular channels within the construct to improve vascularization.

Erythropoietin enhances oxygenation in critically perfused tissue through modulation of nitric oxide synthase

The aim of this study was to investigate the effect of human recombinant erythropoietin (EPO) on the microcirculation and oxygenation of critically ischemic tissue and to elucidate the role of endothelial NO synthase in EPO-mediated tissue protection. Island flaps were dissected from the back skin of anesthetized male Syrian golden hamsters including a critically ischemic, hypoxic area that was perfused via a collateralized vasculature. Before ischemia, animals received an injection of epoetin beta at a dose of 5,000 U/kg body weight with (n = 7) or without (n = 7) blocking NO synthase by 30 mg/kg body weight l-NAME (N&ohgr;-nitro-l-arginine methyl ester hydrochloride). Saline-treated animals served as control (n = 7). Ischemic tissue damage was characterized by severe hypoperfusion and inflammation, hypoxia, and accumulation of apoptotic cell nuclei after 5 h of collateralization. Erythropoietin pretreatment increased arteriolar and venular blood flow by 33% and 37%, respectively (P < 0.05), and attenuated leukocytic inflammation by ∼75% (P < 0.05). Furthermore, partial tissue oxygen tension in the ischemic tissue increased from 8.2 to 15.8 mmHg (P < 0.05), which was paralleled by a 21% increased density of patent capillaries (P < 0.05) and a 50% reduced apoptotic cell count (P < 0.05). The improved microcirculation and oxygenation were associated with a 2.2-fold (P < 0.05) increase of endothelial NO synthase protein expression. Of interest, l-NAME completely abolished all the beneficial effects of EPO pretreatment. Our study demonstrates that, in critically ischemic and hypoxic collateralized tissue, EPO pretreatment improves tissue perfusion and oxygenation in vivo. This effect may be attributed to NO-dependent vasodilative effects and anti-inflammatory actions on the altered vascular endothelium.