Jan P. Roesner

A Multicenter Trial of Remote Ischemic Preconditioning for Heart Surgery

BACKGROUND Remote ischemic preconditioning (RIPC) is reported to reduce biomarkers of ischemic and reperfusion injury in patients undergoing cardiac surgery, but uncertainty about clinical outcomes remains. METHODS We conducted a prospective, double-blind, multicenter, randomized, controlled trial involving adults who were scheduled for elective cardiac surgery requiring cardiopulmonary bypass under total anesthesia with intravenous propofol. The trial compared upper-limb RIPC with a sham intervention. The primary end point was a composite of death, myocardial infarction, stroke, or acute renal failure up to the time of hospital discharge. Secondary end points included the occurrence of any individual component of the primary end point by day 90. RESULTS A total of 1403 patients underwent randomization. The full analysis set comprised 1385 patients (692 in the RIPC group and 693 in the sham-RIPC group). There was no significant between-group difference in the rate of the composite primary end point (99 patients [14.3%] in the RIPC group and 101 [14.6%] in the sham-RIPC group, P=0.89) or of any of the individual components: death (9 patients [1.3%] and 4 [0.6%], respectively; P=0.21), myocardial infarction (47 [6.8%] and 63 [9.1%], P=0.12), stroke (14 [2.0%] and 15 [2.2%], P=0.79), and acute renal failure (42 [6.1%] and 35 [5.1%], P=0.45). The results were similar in the per-protocol analysis. No treatment effect was found in any subgroup analysis. No significant differences between the RIPC group and the sham-RIPC group were seen in the level of troponin release, the duration of mechanical ventilation, the length of stay in the intensive care unit or the hospital, new onset of atrial fibrillation, and the incidence of postoperative delirium. No RIPC-related adverse events were observed. CONCLUSIONS Upper-limb RIPC performed while patients were under propofol-induced anesthesia did not show a relevant benefit among patients undergoing elective cardiac surgery. (Funded by the German Research Foundation; RIPHeart ClinicalTrials.gov number, NCT01067703.).

The fibrin-derived peptide Bβ15- 42 is cardioprotective in a pig model of myocardial ischemia-reperfusion injury

Objective:The fibrin-derived peptide B&bgr;15–42 has been shown to reduce infarct size in rodent models of ischemia-reperfusion injury. To increase its potential for translation into the clinic, we studied the effects of B&bgr;15–42 in pigs, whose coronary anatomy is similar to that of humans. In addition, we evaluated the pharmacokinetics and safety of B&bgr;15–42 in several species, including humans. Design:Animal study and phase I trial. Setting:University hospital and contract research laboratories. Subjects:Pigs/healthy volunteers. Interventions:Male farm-bred Landrace pigs were subjected to 1 hr of left anterior descending coronary artery occlusion followed by 3 hrs of reperfusion. At the time of reperfusion, B&bgr;15–42 (2.4 mg/kg, n = 6) or random peptide (control; 2.4 mg/kg, n = 6) was administered as an intravenous bolus. As a positive control, pigs were subjected to ischemic preconditioning (n = 6). Cardiac damage and hemodynamics were recorded. Biodistribution and pharmacokinetics of B&bgr;15–42 were determined in rats and dogs. In a phase I trial involving 30 male healthy volunteers, pharmacokinetics and safety were tested in a randomized, double-blinded, placebo-controlled, parallel-group, single ascending dose study. Measurements and Main Results:B&bgr;15–42 and ischemic preconditioning significantly reduced myocardial infarct size and troponin I levels. B&bgr;15–42 also reduces interleukin-6 levels, underlining its anti-inflammatory properties. Furthermore, in humans, the pharmacokinetics of the peptide B&bgr;15–42 were comparable to those of animals, and no serious adverse effects were observed. Conclusions:B&bgr;15–42 elicits cardioprotection in pigs and is clinically safe in phase I testing of humans. This study confirms the new concept of a pathogenic role of fibrin derivatives in myocardial reperfusion injury, which can be inhibited by peptide B&bgr;15–42.

Drug detection in breath: effects of pulmonary blood flow and cardiac output on propofol exhalation

AbstractBreath analysis could offer a non-invasive means of intravenous drug monitoring if robust correlations between drug concentrations in breath and blood can be established. In this study, propofol blood and breath concentrations were determined in an animal model under varying physiological conditions. Propofol concentrations in breath were determined by means of two independently calibrated analytical methods: continuous, real-time proton transfer reaction mass spectrometry (PTR-MS) and discontinuous solid-phase micro-extraction coupled with gas chromatography mass spectrometry (SPME-GC-MS). Blood concentrations were determined by means of SPME-GC-MS. Effects of changes in pulmonary blood flow resulting in a decreased cardiac output (CO) and effects of dobutamine administration resulting in an increased CO on propofol breath concentrations and on the correlation between propofol blood and breath concentrations were investigated in seven acutely instrumented pigs. Discontinuous propofol determination in breath by means of alveolar sampling and SPME-GC-MS showed good agreement (R2 = 0.959) with continuous alveolar real-time measurement by means of PTR-MS. In all investigated animals, increasing cardiac output led to a deterioration of the relationship between breath and blood propofol concentrations (R2 = 0.783 for gas chromatography-mass spectrometry and R2 = 0.795 for PTR-MS). Decreasing pulmonary blood flow and cardiac output through banding of the pulmonary artery did not significantly affect the relationship between propofol breath and blood concentrations (R2 > 0.90). Estimation of propofol blood concentrations from exhaled alveolar concentrations seems possible by means of different analytical methods even when cardiac output is decreased. Increases in cardiac output preclude prediction of blood propofol concentration from exhaled concentrations. FigureExperimental setup for simultaneous real-time (PTR-MS) and discontinuous (SPME-GC-MS) drug determination in the breath of acutely instrumented pigs (A). In order to assess the influence of hemodynamic variables pulmonary artery blood flow was determined by means of Doppler-measurement (B).

Bβ15-42 (FX06) reduces pulmonary, myocardial, liver, and small intestine damage in a pig model of hemorrhagic shock and reperfusion

Objective:The fibrin-derived peptide Bβ15-42 (also called FX06) has been shown to reduce myocardial infarct size following ischemia/reperfusion. Hemorrhagic shock (HS) followed by volume resuscitation represents a similar scenario, whereby a whole organism is vulnerable to reperfusion injury. Design:We subjected male farm-bred landrace pigs (∼30 kg) to HS by withdrawing blood to a mean arterial pressure of 40 mm Hg for 60 minutes. Pigs were then resuscitated with shed blood and crystalloids for 60 minutes, and at this time, FX06 (2.4 mg/kg, n = 8) or vehicle control (phosphate buffered saline; 2.4 mg/kg, n = 7) was injected as an intravenous bolus. Setting:University hospital laboratory. Subjects:Anesthetized male farm-bred landrace pigs. Measurements and Main Results:Data are presented as mean ± sd. Five hours after resuscitation, controls presented acute lung injury (Pao2/Fio2-ratio <300 mm Hg; extra-vascular lung water index (marker for lung injury): 9.0 ± 1.8 mL/kg) and myocardial dysfunction/damage (cardiac index: 4.3 ± 0.25 L/min/m2; stroke volume index: 30 ± 6 mL/m2; cardiac TnT levels: 0.58 ± 0.25 ng/mL). In contrast, FX06-treated animals showed significantly improved pulmonary and circulatory function (Pao2/Fio2-ratio >*400 mm Hg; extra-vascular lung water index: *5.2 ± 2.1 mL/kg, cardiac index: *6.3 ± 1.4 L/min/m2; stroke volume index: *51 ± 11 mL/m2; cardiac TnT levels: *0.11 ± 0.09 ng/mL; *p < 0.05). Also, tissue oxygenation (tpO2; mm Hg) was significantly improved during reperfusion in FX06-treated pigs when compared with controls (liver 51 ± 4 vs. *65 ± 4; serosa 44 ± 5 vs. *55 ± 7; mucosa 14 ± 4 vs. *26 ± 4). Finally, FX06 reduced accumulation of myeloperoxidase-positive cells (mainly neutrophils) in myocardium, liver, and small intestine and reduced interleukin-6 plasma levels (*p < 0.05; compared with controls). Conclusion:We conclude that in a pig model of HS and reperfusion, administration of FX06 during reperfusion protects shock- susceptible organs such as heart, lung, liver, and small intestine.

Toll-Like Receptor 2–Blocking Antibodies Promote Angiogenesis and Induce ERK1/2 and AKT Signaling via CXCR4 in Endothelial Cells

Objective—Toll-like receptor 2 (TLR2) inhibition by function blocking antibodies (ABs) is associated with enhanced preservation of endothelial cell function during vascular disease. In the present study, we investigated the capacity of TLR2-blocking ABs to modulate the angiogenic response of endothelial cells in vitro and in vivo. Approach and Results—Incubation of endothelial cells with mono- or polyclonal anti-TLR2 ABs resulted in increased tube formation, sprouting, and migration of endothelial cells compared with controls. In a mouse model of hindlimb ischemia, using TLR2-deficient or anti-TLR2 AB–treated wild-type mice resulted in increased new capillary formation and enhanced reperfusion. The effects of anti-TLR2 ABs were similar to those exerted by stromal cell–derived factor-1, and we show that anti-TLR2 ABs yet not TLR2 ligands lead to comparable activation of extracellular signal–regulated kinase1/2 and AKT but not p38 mitogen-activated protein kinase as activation of the CXCR4 canonical signal transduction pathways by stromal cell–derived factor-1. Immunoprecipitation of TLR2 revealed that anti-TLR2 ABs initiate an association of TLR2 with CXCR4 and mitogen-activated protein kinase activation. The proangiogenic properties of anti-TLR2 ABs were abolished by both G-protein inhibition and CXCR4 knockdown in endothelial cells. Conclusions—Our results provide evidence for a proangiogenic effect of TLR2-blocking ABs on endothelial cells in vitro and in vivo. They identify a novel molecular mechanism linking TLR2 to angiogenic processes that is independent from the activation of inflammatory cascades and further support the concept of a beneficial effect of TLR2 inhibition for endothelial cell function in vascular disease.

Therapeutic injection of parp inhibitor ino-1001 preserves cardiac function in porcine myocardial ischemia and reperfusion without reducing infarct size

Pharmacological protection from myocardial reperfusion injury, despite plenty of approaches, has still not been realized in humans. We studied the putative infarct size (IS)-sparing capacity of poly(ADP-ribose)polymerase inhibitor, INO-1001, and focused on cardiac functional recovery during reperfusion. Male farm-bred Landrace pigs were subjected to 1-h left anterior descending coronary artery occlusion followed by 3 h of reperfusion (control). Infarct size was determined by triphenyltetrazolium chloride/Evans blue staining. Plasma markers of myocardial injury (troponin T, creatine kinase, lactate dehydrogenase) were determined upon protocol completion. Cardiac function was continuously assessed via pulmonary and femoral artery catheters. INO-1001 (1 mg/kg) was administered upon reperfusion in the treatment group. As a positive control, untreated pigs were subjected to ischemic preconditioning (10-min left anterior descending coronary artery occlusion followed by 15-min reperfusion before the intervention). Ischemic preconditioning reduced myocardial damage reflected by a smaller IS and lower plasma markers of myocardial injury. INO-1001 did not reduce IS but significantly improved functional recovery (increased stroke volume, cardiac index, and mixed venous oxygen saturation) during reperfusion compared with vehicle-treated control and ischemic preconditioning. Although we could not confirm the IS-sparing capacities of poly(ADP-ribose)polymerase inhibitor, INO-1001, the drug holds the potential of hemodynamic improvement during reperfusion.

The standardized creation of a lumbar spine vertebral compression fracture in a sheep osteoporosis model induced by ovariectomy, corticosteroid therapy and calcium/phosphorus/vitamin D-deficient diet.

INTRODUCTION Vertebral compression fractures (VCFs) are one of the most common injuries in the aging population presenting with an annual incidence of 1.4 million new cases in Europe. Current treatment strategies focus on cement-associated solutions (kyphoplasty/vertebroplasty techniques). Specific cement-associated problems as leakage, embolism and the adjacent fracture disease are reported adding to open questions like general fracture healing properties of the osteoporotic spine. In order to analyze those queries animal models are of great interest; however, both technical difficulties in the induction of experimental osteoporosis in animal as well as the lack of a standardized fracture model impede current and future in vivo studies. This study introduces a standardized animal model of an osteoporotic VCF type A3.1 that may enable further in-depth analysis of the afore mentioned topics. MATERIAL AND METHODS Twenty-four 5-year-old female Merino sheep (mean body weight: 67 kg; range 57-79) were ovariectomized (OP1) and underwent 5.5 months of weekly corticosteroid injections (dexamethasone and dexamethasone-sodium-phosphate), adding to a calcium/phosphorus/vitamin D-deficient diet. Osteoporosis induction was documented by pQCT and micro-CT BMD (bone mineral density) as well as 3D histomorphometric analysis postoperatively of the sheep distal radius and spine. Non osteoporotic sheep served as controls. Induction of a VCF of the second lumbar vertebra was performed via a mini-lumbotomy surgical approach with a standardized manual compression mode (OP2). RESULTS PQCT analysis revealed osteoporosis of the distal radius with significantly reduced BMD values (0.19 g/cm(3), range 0.13-0.22 vs. 0.27 g/cm(3), range 0.23-0.32). Micro-CT documented significant lowering of BMD values for the second lumbar vertebrae (0.11 g/cm(3), range 0.10-0.12) in comparison to the control group (0.14 g/cm(3), range 0.12-0.17). An incomplete burst fracture type A3.1 was achieved in all cases and resulted in a significant decrease in body angle and vertebral height (KA 4.9°, range: 2-12; SI 4.5%, range: 2-12). With OP1, one minor complication (lesion of small bowel) occurred, while no complications occurred with OP2. CONCLUSIONS A suitable spinal fracture model for creation of VCFs in osteoporotic sheep was developed. The technique may promote the development of improved surgical solutions for VCF treatment in the experimental and clinical setting.

Hydrocortisone reduces the beneficial effects of toll-like receptor 2 deficiency on survival in a mouse model of polymicrobial sepsis.

Introduction Toll-like receptors (TLRs) play a crucial role in early host defense against microorganisms. Toll-like receptor 2 (TLR2) polymorphisms have a prevalence of 10%; functional defects of TLR2 are associated with higher susceptibility toward gram-positive bacteria, and TLR2 deficiency has been associated with an impaired adrenal stress response. In the present study, we compared endogenous corticosterone production of wild-type (WT) and TLR2-deficient (TLR2−/−) mice and analyzed survival after hydrocortisone therapy during sepsis induced by cecal ligation and puncture (CLP). Methods Male C57BL/6J (WT); and B6.129-Tlr2tm1Kir/J (TLR2−/−) mice were subjected to CLP or sham operation and randomly assigned to postoperative treatment with either hydrocortisone (5 mg/kg) or vehicle (n = 10 mice/group). Survival was documented for an observation period of 48 h. Endogenous corticosterone production following hydrocortisone treatment and lipoteichoic acid (LTA) exposure, interleukin 6 (IL-6) and IL-1&bgr; plasma levels, and blood counts were determined following sham operation or CLP using another n = 5 mice/group. Statistical analysis was performed using analysis of variance/Bonferroni. Results TLR2−/− mice exhibited a lack of suppression and an attenuated increase in endogenous corticosterone production following hydrocortisone or LTA treatment, respectively. After CLP, TLR2−/− mice exhibited an uncompromised adrenal stress response, higher IL-6 levels, and increased survival compared with WT controls (75 vs. 35%; P < 0.05). Hydrocortisone therapy of TLR2−/− mice completely abolished this advantage (decrease in survival to 45%, P < 0.05 vs. vehicle-treated TLR2−/− mice) and was associated with decreased IL-1&bgr; plasma concentrations. Conclusions Toll-like receptor 2 deficiency is associated with an uncompromised adrenal stress response and increased survival rates during polymicrobial sepsis. Hydrocortisone treatment increases mortality of septic TLR2−/− mice, suggesting that hydrocortisone therapy might be harmful for individuals with functional TLR2 polymorphisms.

Accurate and continuous measurement of oxygen deficit during haemorrhage in pigs

OBJECTIVE Haemorrhagic shock can cause organ failure and high mortality. Uncontrolled bleeding, a predetermined bleeding volume or blood pressure controlled bleeding are traditionally used to study haemorrhagic shock. These models are influenced by compensatory mechanisms preventing accurate knowledge about the severity of cellular insult. We describe the use of a method for continuous measurement of oxygen deficit during haemorrhage in pigs. METHODS We defined a cumulative oxygen deficit of approximately 100mL/kg as the primary endpoint for severe haemorrhage. For continuous assessment of oxygen deficit a metabolic monitor (Deltatrac II, Datex-Ohmeda Instrumentation Corp., Helsinki, Finland) was used. Data are presented as mean+/-SD; (*)P<0.05 was considered to be significant. RESULTS 17 out of 22 anaesthetised male pigs achieved a mean cumulative oxygen deficit of 106+/-3 mL/kg (range: 95-117 mL/kg) by withdrawing an average blood volume of 47+/-6 mL/kg over 1h. Mean arterial blood pressure (MAP) fell from 83+/-19 to 22+/-7mmHg (baseline versus shock), heart rate increased from 83+/-7 to 147+/-37min(-1). Venous base excess changed from 4.8+/-2.4 to -12.5+/-3.4 mmol/L and venous lactate increased from 1.5+/-0.4 to 13.3+/-2.4 mmol/L after haemorrhage. Two pigs (11%) died during the haemorrhagic shock phase. The traditional method of assessing haemorrhage (measuring blood volume lost) showed only a poor correlation with heart rate (r=0.3872; P=0.1540), MAP (r=0.3901; P=0.1505), mixed venous oxygen saturation (svO(2); r=0.0944; P=0.7379) or cardiac index (CI; r=0.2101; P=0.4523). Cumulative oxygen deficit correlated significantly better with heart rate (r=0.7175; P=0.0026), MAP (r=0.5039; P=0.0556), svO(2) (r=0.7084; P=0.0031) or CI (r=0.6260; P=0.0125). CONCLUSION We describe a model to study haemorrhagic shock based on the cumulative oxygen deficit. We believe that the use of a metabolic monitor to measure oxygen deficit in our model represents an improvement on the current available methods to study the effects of haemorrhagic shock.

Cementless Titanium Mesh Fixation of Osteoporotic Burst Fractures of the Lumbar Spine Leads to Bony Healing: Results of an Experimental Sheep Model

Introduction. Current treatment strategies for osteoporotic vertebral compression fractures (VCFs) focus on cement-associated solutions. Complications associated with cement application are leakage, embolism, adjacent fractures, and compromise in bony healing. This study comprises a validated VCF model in osteoporotic sheep in order to (1) evaluate a new cementless fracture fixation technique using titanium mesh implants (TMIs) and (2) demonstrate the healing capabilities in osteoporotic VCFs. Methods. Twelve 5-year-old Merino sheep received ovariectomy, corticosteroid injections, and a calcium/phosphorus/vitamin D-deficient diet for osteoporosis induction. Standardized VCFs (type AO A3.1) were created, reduced, and fixed using intravertebral TMIs. Randomly additional autologous spongiosa grafting (G1) or no augmentation was performed (G2, n = 6 each). Two months postoperatively, macroscopic, micro-CT and biomechanical evaluation assessed bony consolidation. Results. Fracture reduction succeeded in all cases without intraoperative complications. Bony consolidation was proven for all cases with increased amounts of callus development for G2 (58.3%). Micro-CT revealed cage integration. Neither group showed improved results with biomechanical testing. Conclusions. Fracture reduction/fixation using TMIs without cement in osteoporotic sheep lumbar VCF resulted in bony fracture healing. Intravertebral application of autologous spongiosa showed no beneficial effects. The technique is now available for clinical use; thus, it offers an opportunity to abandon cement-associated complications.