Botulinum toxin A and B raise blood flow and increase survival of critically ischemic skin flaps.

BACKGROUND: Botulinum toxin (BTX) A and B are commonly used for aesthetic indications and in neuromuscular disorders. New concepts seek to prove efficacy of BTX for critical tissue perfusion. Our aim was to evaluate BTX A and B in a mouse model of critical flap ischemia for preoperative and intraoperative application. METHODS: BTX A and B were applied on the vascular pedicle of an axial pattern flap in mice preoperatively or intraoperatively. Blood flow, tissue oxygenation, tissue metabolism, flap necrosis rate, apoptosis assay, and RhoA and eNOS expression were endpoints. RESULTS: Blood-flow measurements 1 d after the flap operation revealed a significant reduction to 53% in the control group, while flow was maintained or increased in all BTX groups (103%-129%). Over 5 d all BTX groups showed significant increase in blood flow to 166-187% (P < 0.01). Microdialysis revealed an increase of glucose and reduced lactate/pyruvate ratio and glycerol levels in the flap tissue of all BTX groups. This resulted in significantly improved tissue survival in all BTX groups compared with the control group (62% ± 10%; all P < 0.01): BTX A preconditioning (84% ± 5%), BTX A application intraoperatively (88% ± 4%), BTX B preconditioning (91% ± 4%), and intraoperative BTX B treatment (92% ± 5%). This was confirmed by TUNEL assay. Immunofluorescence demonstrated RhoA and eNOS expression in BTX groups. All BTX applications were similarly effective, despite pharmacologic dissimilarities and different timing. CONCLUSIONS: In conclusion, we were able to show on a vascular, tissue, cell, and molecular level that BTX injection to the feeding arteries supports flap survival through ameliorated blood flow and oxygen delivery.

Practice of split-thickness skin graft storage and histological assessment of tissue quality.

Storage of split-thickness skin grafts (STSGs) represents a standard procedure in burn surgery. The purpose of this study was to evaluate clinical routine of STSG preservation. Further, we aimed at investigating the effect of storage on tissue integrity and cell viability, proliferation, apoptosis and vascularization. A survey was performed among plastic surgery centres in Europe. STSGs were harvested from healthy patients and analysed by histology (HE, Verhoeff's, Masson's Trichrome, Sirius Red) and immunohistochemistry (Ki67, TUNEL, CD31). Cell viability was determined by MTT assay. The survey revealed that storage of STSGs up to 10 days is common practice. STSGs mostly were stored at 4 °C in saline-moisturized gauze. Histology showed no disintegration of the tissue or a decrease of collagen and elastic fibres. Proliferation increased to 22.5% of total cells after 3 days. On day 7 of STSG storage apoptotic cells amounted for 25% of total cells. Cell viability decreased by 50% after day 3 of storage. Even though reportedly superior methods for skin grafts storage exist, most study participants applied the simplest method of storage. Our data underscore this practice. However, a reduced cell viability after 3 days of storage may have an influence on graft healing.

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.

EPO reverses defective wound repair in hypercholesterolaemic mice by increasing functional angiogenesis.

This study aims to elucidate the effect of erythropoietin (EPO) on the microcirculation during wound healing in mice genetically depleted of apolipoprotein E (ApoE(-/-)). The skinfold chamber in mice was used for intravital microscopy, whereby an incisional wound was created within the chamber. Animals received Recormon(®) 1000 U kg(-1) body weight (BW) intra-peritoneally (i.p.) at day 1, 3, 5, 7, 9 and 11 post-wounding at a concentration of 100 Uml(-1) (n=42). Normal healing and vehicle-treated wild type animals (WT) served as controls. The microcirculation of the wound was analysed quantitatively in vivo using epi-illumination intravital fluorescence microscopy. Microtomography (micro-CT) analysis of casted wound microvessels was performed allowing three-dimensional (3D) histomorphometric analysis. Tissue samples were examined ex vivo for wound scoring and for expression analysis of EPO-Receptor (Epo-R) and endothelial nitric oxide synthase (eNOS). Upon EPO treatment, the total wound score in ApoE(-/-) mice was increased by 23% on day 3, by 26% on day 7 and by 18% on day 13 when compared to untreated ApoE(-/-) mice (all P<0.05 vs. vehicle). Improved wound healing was accompanied with a significant increase of functional angiogenetic density and angiogenetic red blood cell perfusion on days 5, 7, 9 and 11 post-wounding. 3D histomorphometric analysis revealed an increase of vessel thickness (1.7-fold), vessel volume (2.4-fold) and vessel surface (1.7-fold) (all P<0.05 vs. vehicle). In addition, improved wound healing was associated with enhanced Epo-R expression (4.6-fold on day 3 and 13.5-fold on day 7) and eNOS expression (2.4-fold on day 7) (all P<0.05 vs. vehicle). Our data demonstrate that repetitive systemic EPO treatment reverses microvascular dysfunction during wound healing in hypercholesterolaemic mice by inducing new vessel formation and by providing the wound with more oxygen.

Radial pressure waves mediate apoptosis and functional angiogenesis during wound repair in ApoE deficient mice.

This study aims to quantify by intravital microscopy and histological wound scoring the effect of radial pressure wave treatment (RPWT) on murine incisional wound healing. The dorsal skinfold chamber in mice was used for intravital microscopy, whereby an incisional wound was created within the chamber. RPWT to the wound was carried out using a ballistic pressure wave source (EMS Swiss DolorClast). Animals received a dose of 500 pulses at an energy flux rate of 0.1mJ/mm(2) and a frequency of 3Hz at day 1, 3, 5, 7, 9, and 11 post wounding. RPW treated and untreated ApoE depleted mice (ApoE(-/-)) were compared to normal healing wild type animals (WT). The microcirculation of the wound was analyzed quantitatively in vivo using epi-illumination intravital fluorescence microscopy. Tissue samples were examined ex vivo for wound scoring and immunohistochemistry. Upon RPWT total wound score in ApoE(-/-) mice was increased by 13% (not significant) on day 3, by 37% on day 7 (P<0.05), and by 39% on day 13 (P<0.05) when compared to untreated ApoE(-/-) mice. Improved wound healing was associated with an increase of functional angiogenetic density by 23% (not significant) on day 5, by 36% on day 7 (P<0.05), and by 41% on day 9 (P<0.05). Following RPWT, on day three we observed enhanced expression of capase-3 (2-fold), proliferating cell nuclear antibody (PCNA, 1,6-fold), and endothelial nitric oxide synthase (eNOS, 2.6-fold), all P<0.05. In conclusion repetitive RPWT accelerated wound healing in ApoE(-/-) mice by increasing functional neovascular density. In addition our findings strongly suggest that RPW may facilitate the linear progression of wound healing phases by fostering apoptosis.

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.

Chemical composition of hepatic lipids mediates reperfusion injury of the macrosteatotic mouse liver through thromboxane A(2).

BACKGROUND & AIMS: Chemical composition of hepatic lipids is an evolving player in steatotic liver ischemia/reperfusion (I/R) injury. Thromboxane A(2) (TXA(2)) is a vasoactive pro-inflammatory lipid mediator derived from arachidonic acid (AA), an omega-6 fatty acid (Ω-6 FA). Reduced tolerance of the macrosteatotic liver to I/R may be related to increased TXA(2) synthesis due to the predominance of Ω-6 FAs. METHODS: TXA(2) levels elicited by I/R in ob/ob and wild type mice were assessed by ELISA. Ob/ob mice were fed Ω-3 FAs enriched diet to reduce hepatic synthesis of AA and TXA(2) or treated with selective TXA(2) receptor blocker before I/R. RESULTS: I/R triggered significantly higher hepatic TXA(2) production in ob/ob than wild type animals. Compared with ob/ob mice on regular diet, Ω-3 FAs supplementation markedly reduced hepatic AA levels before ischemia and consistently blunted hepatic TXA(2) synthesis after reperfusion. Sinusoidal perfusion and hepatocellular damage were significantly ameliorated despite downregulation of heme oxygenase-1. Hepatic transcript and protein levels of IL-1ß and neutrophil recruitment were significantly diminished after reperfusion. Moreover, TXA(2) receptor blockage conferred similar protection without modification of the histological pattern of steatosis. A stronger protection was achieved in the steatotic compared with lean animals. CONCLUSIONS: Enhanced I/R injury in the macrosteatotic liver is explained, at least partially, by TXA(2) mediated microcirculatory failure rather than size-related mechanical compression of the sinusoids by lipid droplets. TXA(2) blockage may be a simple strategy to include steatotic organs and overcome the shortage of donor organs for liver transplantation.

Microvascular response to shock wave application in striated skin muscle.

BACKGROUND: This study aims to quantify by intravital microscopy the microhemodynamic response after extracorporeal shock wave application (ESWA) to the physiologic microcirculation of the mouse dorsal skinfold chamber. MATERIALS AND METHODS: ESWA was carried out using an electrohydraulic shock wave source. Two different shock wave doses of 500 and 1000 pulses at an energy flux rate of 0.08 mJ/mm(2) and a frequency of 4 Hz were compared with sham-operated animals. Microcirculatory analyses were performed at baseline (BL) and during a 3 d observation period after ESWA. The expression of caspase-3 (casp-3), proliferating cell nuclear antibody (PCNA), von Willebrand factor (vWF), and endothelial nitric oxide synthase (eNOS) were analyzed semiquantitatively by immunohistochemistry. RESULTS: ESWA provoked a significant and persistent increase of functional capillary density (FCD) throughout the observation period, reaching a maximum (140% ± 5% of BL, P < 0.05 versus sham) after 1 d when animals were treated with 1000 pulses. ESWA induced a slight increase of leukocyte rolling (∼2- to ∼3.5-fold, P < 0.05) and leukocyte adherence (∼1.5- to ∼2-fold, P < 0.05) to the endothelial lining of postcapillary venules. One day following ESWA, we observed enhanced expression of casp-3 (∼3- to ∼4-fold), PCNA (∼9- to ∼14-fold), vWF (∼11- to ∼14-fold), and eNOS (∼3-fold), all P < 0.05. CONCLUSION: This study shows that ESWA provokes a favorable persistent increase of patent capillaries, however accompanied by a transient and slight inflammatory response but also by dose-dependant apoptotic cell death. Our data suggest that ESWA might represent a noninvasive biomechanical tool to treat critically perfused and endangered tissues, but certainly warrants further investigation.

Erythropoietin requires endothelial nitric oxide synthase to counteract TNF-[alpha]-induced microcirculatory dysfunction in murine striated muscle.

In the present study, we aimed to evaluate whether erythropoietin (EPO) treatment may exert nonhematopoietic endothelial protection against TNF-[alpha]-induced microvascular inflammation and to determine the involvement of the nitric oxide (NO)-producing enzyme isoforms endothelial NO synthase (eNOS) and inducible NO synthase (iNOS). Murine dorsal skinfold chambers of wild-type (WT) animals were topically stimulated with TNF-[alpha] after pretreatment with epoetin beta (1,000 IU/kg body weight i.p.) or physiological saline. Leukocyte behavior, microvascular perfusion, and apoptosis were assessed by in vivo fluorescence microscopy. To study the involvement of NO, we compared eNOS-deficient (eNOS-/-) and iNOS-deficient (iNOS-/-) mice with WT animals. TNF-[alpha]-associated leukocyte activation, perfusion failure, and apoptosis were substantially attenuated in EPO-pretreated WT mice, which was accompanied by marked reduction of perivascular infiltration with F4/80-stained macrophages. The anti-inflammatory protective effects of EPO were abolished in eNOS-/-, but not in iNOS-/- mice, both with unaffected intercellular adhesion molecule 1 expression. However, the antiapoptotic effect of EPO was maintained in both eNOS-/- and iNOS-/- mice, indicating that this mechanism might rather be independent of NO. We conclude that EPO treatment elicits protection against TNF-[alpha]-induced microcirculatory dysfunction, depending on NO derived from endothelial cells, but not on the inducible isoform.

Temporary angiogenic transformation of the skin graft vasculature after reperfusion.

BACKGROUND: In the era of tissue engineering, the physiologic process of skin graft revascularization remains unclear, preventing the successful development of skin substitutes. Therefore, the authors developed a new in vivo model with which to visualize the process of engraftment and its microvascular architecture. The aim of this study was to specifically investigate the vascular transformations within the skin graft to gain applicable knowledge on how vascular processes during engraftment occur. METHODS: Microsurgical preparation of the modified dorsal skinfold chamber including autologous skin grafting was performed in male C57BL/6J mice (n = 10). In addition, immunohistochemistry of angiogenic factors, endothelial cells, and pericytes, and corrosion casting were performed to further characterize the specific mechanisms. RESULTS: The graft exhibited capillary widening starting at day 3, resulting in the temporary formation of spherical protrusions at the graft capillary divisions starting in the center of the graft 24 to 48 hours after revascularization. Confocal microscopy showed the simultaneous expression of CD31 and desmin. Corrosion casting and evaluation by light microscopy and scanning electron microscopy showed the three-dimensional formation of capillaries in the wound bed that connected to the preexisting capillary loops of the skin graft. CONCLUSIONS: The authors were able to show for the first time a temporary angiogenic response within the capillaries of the skin graft. This most likely represents a reaction to reperfusion allowing the supply of proangiogenic factors to the hypoxic skin graft. The detection of an angiogenic response within the graft capillaries is for the first time made possible in the newly developed model and is therefore completely novel.

Erythropoietin reduces necrosis in critically ischemic myocutaneous tissue by protecting nutritive perfusion in a dose-dependent manner.

BACKGROUND: Erythropoietin (Epo), the primary regulator of erythropoiesis, has recently been shown to exert antiinflammatory and antiapoptotic properties in neuronal and myocardial tissue. We herein studied whether Epo pretreatment can reduce cell death and ischemic necrosis in a chronic in vivo model. METHODS: C57BL/6 mice were treated daily for 3 consecutive days with either 500 IU EPO/kg body weight (bw) (group Epo 500, n = 8) or 5000 IU EPO/kg bw (group Epo 5000, n = 8) administered intraperitoneally 24 hours before surgery. Thereafter, a random pattern myocutaneous flap subjected to acute persistent ischemia was elevated and fixed into a dorsal skinfold chamber. Flap elevation in animals receiving the water-soluble vitamin E analog Trolox (6-hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxylic acid) served as a nonspecific antiinflammatory agent control group (Tro); untreated control animals (Con) received saline only. Capillary perfusion, leukocyte-endothelial cell interaction, apoptotic cell death, and tissue necrosis were determined over a 10-day observation period using intravital multifluorescence microscopy. RESULTS: Epo 5000 (44 +/- 26 cm/cm(2)) but, more noticeably, Epo 500 (116 +/- 32 cm/cm(2)) improved capillary perfusion compared with the two control groups, particularly the Con group (9 +/- 7 cm/cm(2); P < .05). The ischemia-associated leukocytic inflammation was found drastically attenuated in both Epo-pretreatment groups. Epo 500 further decreased apoptotic cell death and was effective in significantly reducing tissue necrosis (16% +/- 4% vs Tro: 48% +/- 7% and Con: 52% +/- 4%; P < .001). No angiogenic blood vessel formation could be observed in either of the Epo groups. Of interest, Epo 5000-but not Epo 500-increased systemic hematocrit. CONCLUSION: Despite the lack of neovascularization, Epo pretreatment was capable of reducing ischemic tissue necrosis by protecting capillary perfusion, ie, nutrition of the tissue. Low-dose pretreatment was more effective, a result that was most likely due to the better perfusion conditions without an increase of the hematocrit values. Thus, low-dose Epo pretreatment might represent a promising strategy to protect critically perfused ischemic tissue.

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.

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 (Nomega-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 approximately 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.

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.

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.

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.

Microdialysis of the rectus abdominis muscle for early detection of impending abdominal compartment syndrome.

OBJECTIVE: To investigate whether microdialysis is capable of assessing metabolic derangements during intra-abdominal hypertension (IAH), and whether monitoring of the rectus abdominis muscle (RAM) by microdialysis represents a reliable approach in the early detection of organ dysfunctions in abdominal compartment syndrome (ACS). DESIGN: Prospective, randomized, controlled animal study. SETTING: University animal research facility. SUBJECTS: Fifteen isoflurane-anesthetized and mechanically ventilated Sprague-Dawley rats. INTERVENTIONS: IAH of 20 mmHg was induced for 3 h and followed by decompression and reperfusion for another 3-h period (n = 10). Five sham-operated animals served as controls. Microdialysis was performed in the anterior gastric wall, liver, kidney, and RAM. The anterior cervical muscles served as distant reference. Glucose, lactate, pyruvate, and glycerol was analyzed throughout the 6-h experiment. MEASUREMENTS AND MAIN RESULTS: Prolonged IAH induced significant cardiopulmonary dysfunction and persistent abdominal organ injury. Microdialysis revealed a significant increase of lactate/pyruvate and glycerol in kidney, intestine and liver, indicating ischemia, energy failure, and cell membrane damage. In addition, at 3 h IAH glucose was significantly decreased in all organs studied. The distant reference did not show any alteration of lactate/pyruvate, glycerol, and glucose over the entire 6-h observation period. In contrast to the other organs, microdialysis of the RAM showed an early and more pronounced increase of lactate, lactate/pyruvate and glycerol already at 1 h IAH. It is noteworthy that lactate, glycerol, and glucose did not completely recover upon decompression of IAH. CONCLUSIONS: Our data suggest that continuous microdialysis in the RAM may represent a promising tool for early detecting IAH-induced metabolic derangements.

Human recombinant erythropoietin protects the striated muscle microcirculation of the dorsal skinfold from postischemic injury in mice.

Erythropoietin (EPO) has been proposed as a novel cytoprotectant in ischemia-reperfusion (I/R) injury of the brain, heart, and kidney. However, whether EPO exerts its protection by prevention of postischemic microcirculatory deterioration is unknown. We have investigated the effect of EPO on I/R-induced microcirculatory dysfunctions. We used the mouse dorsal skinfold chamber preparation to study nutritive microcirculation and leukocyte-endothelial cell interaction in striated muscle of the dorsal skinfold by in vivo fluorescence microscopy before 3 h of ischemia and during 5 days of reperfusion. Animals were pretreated with EPO (5,000 U/kg body wt) 1 or 24 h before ischemia. Vehicle-treated I/R-injured animals served as controls. Additional animals underwent sham operation only or were pretreated with EPO but not subjected to I/R. I/R significantly (P < 0.05) reduced functional capillary density, increased microvascular permeability, and enhanced venular leukocyte-endothelial cell interaction during early reperfusion. These findings were associated with pronounced (P < 0.05) arteriolar constriction and diminution of blood flow during late reperfusion. Pretreatment with EPO induced EPO receptor and endothelial nitric oxide synthase expression at 6 h of reperfusion (P < 0.05). In parallel, EPO significantly (P < 0.05) reduced capillary perfusion failure and microvascular hyperpermeability during early reperfusion and arteriolar constriction and flow during late reperfusion. EPO pretreatment substantially (P < 0.05) diminished I/R-induced leukocytic inflammation by reducing the number of rolling and firmly adhering leukocytes in postcapillary venules. EPO applied 1 h before ischemia induced angiogenic budding and sprouting at 1 and 3 days of reperfusion and formation of new capillary networks at 5 days of reperfusion. Thus our study demonstrates for the first time that EPO effectively attenuates I/R injury by preserving nutritive perfusion, reducing leukocytic inflammation, and inducing new vessel formation.

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

UNLABELLED: 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.

Hemoglobin vesicles reduce hypoxia-related inflammation in critically ischemic hamster flap tissue.

OBJECTIVES: The aim of this study was to investigate the effect of a highly viscous, left-shifted hemoglobin vesicle solution (HbV) on the hypoxia-related inflammation and the microcirculation in critically ischemic peripheral tissue. DESIGN: Randomized prospective study. SETTING: University laboratory. SUBJECTS: Twenty-four male golden Syrian hamsters. INTERVENTIONS: Island flaps were dissected from the back skin of anesthetized hamsters for assessment with intravital microscopy. 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 an injection of 25% of total blood volume of 0.9% NaCl or NaCl suspended with HbVs at a concentration of 5 g/dL (HbV5) or 10 g/dL (HbV10). MEASUREMENTS AND MAIN RESULTS: Plasma viscosity was increased from 1.32 cP to 1.61 cP and 2.14 cP after the administration of HbV5 and HbV10, respectively (both p < .01). Both HbV solutions raised partial oxygen tension (Clark-type microprobes) in the ischemic tissue from approximately 10 torr to 17 torr (p < .01), which was paralleled by an increase in capillary perfusion by > 200% (p < .01). The 50% increase in macromolecular capillary leakage found over time in the control animals was completely abolished by the HbV solutions (p < .01), which was accompanied by a > 50% (p < .01) reduction in cells immunohistochemically stained for tumor necrosis factor-alpha and interleukin-6 and in leukocyte counts, whereas no such changes were observed in the anatomically perfused, normoxic tissue. CONCLUSIONS: Our study suggests that in critically ischemic, hypoxic peripheral tissue, hypoxia-related inflammation may be reduced by a top-load infusion of HbV solutions. We attributed this effect to a restoration of tissue oxygenation and an increase in plasma viscosity, both of which may have resulted in attenuation of secondary microcirculatory impairments.