Juergen Hartmut Fischer

Healing of onlay mandibular bone grafts covered with collagen membrane or bovine bone substitutes: a microscopical and immunohistochemical study in the sheep.

The objective of this study was to evaluate the role of collagen membrane and Bio-Oss coverage in healing of an onlay graft to the mandible. Twelve adult sheep each received an onlay bone graft (experiment 1), bone graft+Bio-Gide (experiment 2), and bone graft+Bio-Oss/Bio-Gide (experiment 3) on the lateral surface of the mandible. The animals were euthanized at 4, 8, 12 or 16 weeks after surgery, and findings were analysed by routine microscopy and immunohistochemistry for proliferation (Ki67) and apoptotic (Caspase-3) markers. Grafts were fully incorporated in all specimens. Pronounced resorption was observed in experiment 1. Minimal loss of graft volume was seen in experiment 2 specimens without membrane displacement. A remarkable increase in the augmented region of the mandible was observed in experiment 3. A high number of osteoclasts were expressed within the grafts during the early healing period, and thereafter declined markedly. Osteoblasts within the grafts expressed a moderate level of Ki67 at 8 weeks, which thereafter declined markedly. The strongest expression of Caspase-3 on the bone surface was observed after 16 weeks. In conclusion, the effect of collagen membrane coverage on bone graft volume maintenance was dependent on membrane stability during healing. An autogenous bone graft covered with Bio-Oss particles resulted in a remarkable increase in augmented lateral surface of the mandible. The late stage of bone graft healing was associated with a high apoptotic induction pathway of osteoblasts lining the surfaces of the new bone, demonstrated by strong positive Caspase-3 immunoreactivity.

Metabolism of canine kidneys in anaerobic ischemia and in aerobic ischemia by persufflation with gaseous oxygen

SummaryThe changes in the status of the adenylic acid-creatine phosphate system and in glycogen, glucose, and lactate were investigated in canine kidneys during preservation in anaerobic and aerobic ischemia at normo- and hypothermia. For anaerobic ischemia, the kidneys were stored without additional measures; for aerobic ischemia, the ischemic kidneys were persufflated with gaseous O2 or gas mixtures containing O2 via the renal artery or vein (orthograde and retrograde aerobic ischemia respectively). Prior to preservation, the kidneys were flushed with cell-free perfusates.Changes in the metabolic status characteristic of anaerobic ischemia were a decrease in the tissue levels of ATP, creatine phosphate, glycogen, glucose, and the sum of adenine nucleotides, a transitory increase in ADP and AMP, and an increase in the tissue level of lactate. Hypothermia caused a retardation of the anaerobic alterations; the loss of ATP was slowed down by factors of 2 and 9.5 when the temperatures were lowered from 37° to 26° and 6°C, respectively.In aerobic ischemia with orthograde and retrograde persufflation of 100% O2 (persufflation pressure = PP 60 and 30 mm Hg), deviations from a regular metabolic status developed very slowly. While the ATP-level decreased to 50% of the control value within scarcely 30 min of anaerobic ischemia at 6°C, it was almost 48 h before a similar loss of ATP occurred during orthograde aerobic ischemia at 6°C. Reduction of O2 in the persufflation gas to 40 and 21 vol-% resulted in less adequate preservation of the metabolic status. Increasing PP to 100 mm Hg for orthograde persufflation had no effect. An increase in PP to 60 mm Hg for retrograde persufflation resulted in a further delay in the metabolic deterioration. The long-term preservation of a regular metabolic status or the slow deviation of the metabolic pattern from the normal range during ischemia with O2-persufflation indicates a significantly smaller energy deficit than during storage in anaerobic ischemia. It must be attributed to a sufficient aerobiosis in the aerobically ischemic kidney, during which organogenic substrates brought in by the initial perfusion are utilized.

Cardioplegic arrest induces apoptosis signal-pathway in myocardial endothelial cells and cardiac myocytes

OBJECTIVE Myocardial ischemia-reperfusion is associated with free radical-mediated injury and may be involved in cardiac apoptosis. The purpose of our study was to investigate (1) if cardioplegia-induced ischemia-reperfusion initiates cardiac apoptosis signal pathway, and (2) if this is mediated by free radicals. METHODS We subjected 13 pigs (56+/-10 kg) to 1 h of cold crystalloid cardioplegic arrest (CA) on cardiopulmonary bypass (CPB), and collected five transmural LV biopsies: prior to CPB (baseline), at 60 min CA, at 15 and 30 min reperfusion on CPB, and at 120 min post CPB. Two additional pigs were subjected to CPB but not CA and two further pigs were neither subjected to CPB nor CA and served as sham-operated time controls. LV specimens were cut at 7 microm and immunocytochemically stained against active caspase-3 and 85 kDa poly(ADP-ribose) polymerase (PARP) as apoptosis signal-pathway key enzymes, nitrotyrosine as indicator for peroxynitrite (ONOO(-))-mediated tissue injury, and 8-iso-prostaglandin-F(2)alpha as indicator for oxygen free radical-mediated lipid peroxidation. Specimen were assessed using a scale of 0 (negative) to 3 (highly positive), and cardiomyocytes were quantitatively investigated using TV densitometry. RESULTS At 60 min CA, caspase-3 was increased by 9.2+/-3.7 gray units and remained on this level until 2 h post CPB (P</=0.003 vs. baseline); nitrotyrosine increased over time to reach a maximum of +8.5+/-8.1 gray units at 120 min post CPB (P=0.016); and there was a trend for increased 8-iso-prostaglandin-F(2)alpha at 60 min CA (+3.6+/-4.7 gray units; P=0.089). At 60 min CA, 92% of the hearts showed active caspase-3, only 42% demonstrated nitrotyrosine formation, and 58% exhibited 8-iso-prostaglandin-F(2)alpha. At 120 min post CPB, most hearts positive for caspase-3 were also positive for nitrotyrosine (83%), and 8-iso-prostaglandin-F(2)alpha (75%), but no heart showed PARP cleavage. Hearts subjected to CPB but not CA as well as time controls remained negative for all variables. CONCLUSIONS Our data show that CA initiates apoptosis signal-pathway in myocardial endothelium and myocytes; however, this did not result in apoptotic cell death as we did not find PARP cleavage. Further, the data suggest that CA-induced apoptosis signal pathway activation is not mediated by free radicals as caspase-3 activation preceded both nitrotyrosine and 8-iso-prostaglandin-F(2)alpha formation.

Function and metabolism of canine kidneys after aerobic ischemia by orthograde persufflation with gaseous oxygen

SummaryIn search for new techniques of renal preservation, kidneys were persufflatedin situ with gaseous O2 at a pressure of 90–100 mm Hg via their vascular system (orthograde aerobic ischemia). The results on post-ischemic function and blood flow, and on the changes in the renal tissue levels of the adenine nucleotides were compared to those obtained from kidneys ischemically stored without additional measures (anaerobic ischemia). The kidneys were stored for 4 hrs at 6°C. A contralateral nephrectomy was performed before the preservation period.All dogs subjected to renal anaerobic ischemia survived. After orthograde aerobic ischemia, 3 out of 4 dogs died from uremia within 8 days, 1 animal survived. Although the aerobic ischemic preservation resulted in the maintenance of a metabolic status characteristic for aerobiosis, post-ischemic renal function recovered too slowly and insufficiently on the average. Renal blood flow was within normal ranges after anaerobic ischemia, it was reduced to about one third of this after orthograde aerobic ischemia.

Impact of ischemia-reperfusion on extracellular matrix processing and structure of the basement membrane of the heart.

Purpose Acute ischemic injury is a strong inductor of cardiac remodelling, resulting in structural changes of the extracellular matrix (ECM) and basement membrane (BM). In a large animal model of ischemia-reperfusion (I/R) we investigated the post-ischemic liberation of the collagen-IV-fragments Tumstatin (TUM; 28 kDa-fragment of collagen-IV-alpha-3), Arresten (ARR; 26 kDa-fragment of collagen-IV-alpha-1) and Endorepellin (LG3, 85 kDa-fragment of perlecan) which are biologically active in angiogenesis and vascularization in the post-ischemic myocardium. Methods and Results In this blinded study, 30 pigs were randomized to 60 min of global I/R at either 4°C or 32°C or served as control. Three transmyocardial tissue samples were collected prior to ischemia and within 30 min and 150 min of reperfusion. Tissue content of TUM, ARR and LG3 was analyzed by western blotting and immunostaining. Within 150 min of mild hypothermic I/R a significantly increased tissue content of ARR (0.17±0.14 vs. 0.56±0.56; p = 0.001) and LG3 (1.13±0.34 vs. 2.51±1.71, p<0.001) was observed. In contrast, deep hypothermic I/R was not associated with a significant release of cleavage products. Cleavage of TUM remained unchanged irrespective of temperature. Increased matrix processing following mild hypothermia I/R is further supported by a >11fold elevation of creatine kinase (2075±2595 U/l vs. 23248±6551 U/l; p<0.001) in the coronary sinus plasma samples. Immunostaining demonstrated no changes for ARR and LG3 presentation irrespective of temperature. In contrast, TUM significantly decreased in the BM surrounding cardiomyocytes and capillaries after mild and deep hypothermic I/R, thus representing structural alterations of the BM in these groups. Conclusion The study demonstrates an early temperature-dependent processing of Col-IV as major component of the BM of cardiomyocytes and vascular endothelium. These observations support the protective effects of deep hypothermia during I/R. Furthermore, the results suggest an increased structural remodelling of the myocardial basement membrane with potential functional impairment during mild hypothermic I/R which may contribute to the progression to post-ischemic heart failure.

Effect of the Na+/H+ exchange inhibitor eniporide on cardiac performance and myocardial high energy phosphates in pigs subjected to cardioplegic arrest

BACKGROUND Pharmacologic Na(+)/H(+) exchange inhibition has been suggested to ameliorate cardiac performance depression associated with myocardial ischemia/reperfusion. The purpose of our experimental study was to investigate the impact of the novel Na(+)/H(+) exchange inhibitor Eniporide (EMD 96785) on cardiac performance and high energy phosphate content in a clinically relevant pig model of cardioplegic arrest. METHODS We subjected 21 pigs (47 +/- 12 [SD] kg) to cardiopulmonary bypass (CPB) and 60 minutes cold (4 degrees C) crystalloid cardioplegic arrest (Bretschneider). The pigs were randomized to receive either systemic infusion of 3 mg/kg Eniporide before cardioplegia with added 2 micromol/L Eniporide (ENI-CP+iv; n = 7); 3 mg/kg Eniporide in cardioplegia only (ENI-CP; n = 7); or no Eniporide (control; n = 7). For cardiac performance determination we measured preload recruitable stroke work and Tau, the time constant of left ventricular (LV) isovolumic relaxation using sonomicrometry and micromanometry before CPB as well as 30, 60, and 120 minutes after weaning off CPB. LV and right ventricular myocardial adenine nucleotides (ATP, ADP, and AMP), glycogen, and water content were determined at the end of the experiments. RESULTS Neither for standard hemodynamics including vascular pressures and cardiac index nor for cardiac performance factors did we find statistically significant differences between the groups. Similarly, myocardial adenine nucleotides, glycogene, and water content did not differ significantly between the groups. CONCLUSIONS In this acute study we did not find significant effects of the Na(+)/H(+) exchange inhibitor Eniporide on cardiac performance and high energy phosphate content in healthy pig hearts subjected to ischemia/reperfusion induced by crystalloid cardioplegic arrest.

Renin release after furosemide and ethacrynic acid in man. Evidence for neural reflex control mechanisms.

SummaryThe mechanisms of renin release after furosemide (F) and ethacrynic acid (EA) in man were examined.We evaluated whether acute volume shifts within the low pressure system after F induce renin release via neural pathways.Immersion in a water-bath or beta-blockade reduced the increase of plasma renin concentration after F but not after EA.It is concluded that acute renin release after F but not after EA in man is partially due to neurally mediated reflexes originating from volume receptors in the cardiopulmonary area.ZusammenfassungEs wurden die Mechanismen der Reninfreisetzung nach Furosemid und Ethacrynsäure beim Menschen untersucht. Geprüft wurde die Hypothese, ob akute Volumenverschiebungen innerhalb des Niederdrucksystems nach Furosemid zu einer neural vermittelten Reninstimulation führen. Sowohl Immersion in einem Wasserbad als auch Betablokkade reduzierte den Reninanstieg nach Furosemid nicht jedoch nach Ethacrynsäure. Es wird geschlossen, daß die akute Reninfreisetzung nach Furosemid, nicht jedoch nach Ethacrynsäure teilweise auf neuralen Reflexmechanismen — ausgehend von Volumenrezeptoren des Niederdrucksystems — beruht.

Pneumocyte Apoptosis Induction during Cardiopulmonary Bypass: Effective Prevention by Radical Scavenging Using N-Acetylcysteine

Cardiopulmonary bypass (CPB) and cardioplegic arrest are associated with pulmonary dysfunction. We sought to investigate whether pulmonary ischemia/reperfusion during standard CPB and cardioplegic arrest is associated with reactive oxygen species (ROS)-mediated pulmonary tissue injury and pneumocyte apoptosis induction, and whether ROS scavenging using N-acetylcysteine (NAC) attenuates these alterations. Twelve pigs (41 ± 8 kg) were randomized to receive either NAC (100 mg/kg prior to CPB; n = 7) or placebo (n = 5) and subjected to CPB and 60 min of cold (4°C) crystalloid cardioplegic arrest. We collected lung biopsies prior to CPB, at 60 min CPB, as well as at 30, 60, and 120 min post CPB. Lung specimens were immunocytochemically stained against nitrotyrosine, 8-isoprostaglandin-F2α, and 8-hydroxy-2′-deoxyguanosine (8-OH-dG) as indicators for ROS-mediated tissue injury and active caspase-3, an apoptosis signal pathway key enzyme. Oxidative stress markers were judged using a scale from 1 to 4 (low to intensive staining), and caspase-3-positive pneumocytes were counted per view field. In placebo, the number of caspase-3-positive pneumocytes significantly increased over time to reach a maximum at 120 min post CPB (p =. 03 vs baseline). NAC significantly prevented caspase-3 activation in pneumocytes (p =. 001 vs Placebo). Pneumocyte nitrotyrosine and 8-OH-dG staining significantly increased over time (p =. 003) in the placebo group, but decreased in the NAC group (p =. 004). In both groups staining for 8-isoprostaglandin-F2α showed no significant changes. This yields the conclusion that standard CPB and cardioplegic arrest initiate ROS-mediated tissue injury and apoptosis in pneumocytes that can be reduced by NAC. Thus, ROS scavenging using NAC may represent a novel approach to minimize lung injury associated with CPB.

Experimental immediate loading of dental implants in conjunction with grafting procedures

The aim of this study was to evaluate immediate loading (IL) of dental implants in conjunction with grafting procedures. A total of 107 IL implants were inserted in six mini pigs. Before implant placement, crestal or apical defects were created, which were treated with bone chips, phycogene hydroxyapatite (HA), bovine HA, or bovine HA enhanced with a synthetic peptide. Crestal grafts were stabilized with titanium membranes. All bridges were in function and showed signs of chewing wear after 4-month loading. Three out of 107 implants showed no osseointegration (2.7%). Most of the crestal defects showed incomplete regeneration, due to an infection of the membranes (74.3%) The difference in height between surgical and remaining defect was calculated as defect development, which was 2.3 +/- 2.08 mm for bone chips with an area of regenerated bone of 22.8 +/- 3.34% and 0.7 +/- 2.22 mm for phycogene HA with 11.3 +/- 4.36% regenerated bone. Bovine HA showed an increase of defects 1.3 +/- 2.47 mm with only 7.9 +/- 1.7% bone regeneration. Bovine HA enhanced with a peptide showed a defect development of 1.1 +/- 1.42 mm with an area of regenerated bone of 18.2 +/- 2.38%. In conclusion, local grafting procedures did not disturb the course of osseointegration for immediate loaded implants if primary stability was reached. The regeneration of apical defects was uneventful even with immediate loading. Crestal defects required membrane fixation with a careful flap elevation to avoid membrane exposure and loss of the graft.

Regulation of endothelial nitric oxide synthase (eNOS) in myocardium subjected to cardioplegic arrest.

BACKGROUND Nitric oxide (NO) production by both coronary endothelial cells and cardiomyocytes is thought to play a significant role in myocardial pathophysiology following ischemia/reperfusion (I/R). METHODS In thirteen pigs subjected to 1 hour cardioplegic arrest (CA) on CPB, left ventricular (LV) biopsies were collected prior to CPB (baseline), at 60 min CPA, at 15 and 30 min reperfusion on CPB, and at 120 min post CPB. LV specimens were immunocytochemically stained against phospho-eNOS (Ser1177), phospho-eNOS (Thr495), phosphorylated ERK1/2, and AKT/PKB. Four additional pigs without CA served as controls. Cardiomyocytes were quantitatively investigated using TV densitometry (gray units: U). RESULTS After 60 min CA phosphorylation of eNOS (Ser1177) increased significantly and remained elevated until 30 min of reperfusion. In contrast, eNOS (Thr495) phosphorylation remained unchanged during CA and throughout reperfusion. In control animals, eNOS phosphorylation remained unchanged. Akt/PKB activity significantly increased after 60 min CA and decreased thereafter. ERK1/2 activity remained unchanged during ischemia but increased during reperfusion. CONCLUSIONS ENOS activation during ischemia occurs through phosphorylation at Ser1177 mediated by Akt/PKB. ERK1/2 does not seem to be involved in myocardial eNOS regulation especially not via phosphorylation at eNOS (Thr495).