The influence of local and systemic preconditioning on oxygenation, metabolism and survival in critically ischaemic skin flaps in pigs.

Stress proteins represent a group of highly conserved intracellular proteins that provide adaptation against cellular stress. The present study aims to elucidate the stress protein-mediated effects of local hyperthermia and systemic administration of monophosphoryl lipid A (MPL) on oxygenation, metabolism and survival in bilateral porcine random pattern buttock flaps. Preconditioning was achieved 24h prior to surgery by applying a heating blanket on the operative site (n = 5), by intravenous administration of MPL at a dosage of 35 microg/kg body weight (n = 5) or by combining the two (n = 5). The flaps were monitored with laser Doppler flowmetry, polarographic microprobes and microdialysis until 5h postoperatively. Semiquantitative immunohistochemistry was performed for heat shock protein 70 (HSP70), heat shock protein 32 (also termed haem oxygenase-1, HO-1), and inducible nitrc oxide synthase (iNOS). The administration of MPL increased the impaired microcirculatory blood flow in the proximal part of the flap and partial oxygen tension in the the distal part by approximately 100% each (both P<0.05), whereas both variables remained virtually unaffected by local heat preconditioning. Lactate/pyruvate (L/P) ratio and glycerol concentration (representing cell membrane disintegration) in the distal part of the flap gradually increased to values of approximately 500 mmol/l and approximately 350 micromol/l, respectively (both P<0.01), which was substantially attenuated by heat application (P<0.01 for L/P ratio and P<0.05 for glycerol) and combined preconditioning (P<0.01 for both variables), whereas the effect of MPL was less marked (not significant). Flap survival was increased from 56% (untreated animals) to 65% after MPL (not significant), 71% after heat application (P<0.05) and 78% after both methods of preconditioning (P<0.01). iNOS and HO-1 were upregulated after each method of preconditioning (P<0.05), whereas augmented HSP70 staining was only observed after heat application (P<0.05). We conclude that local hyperthermia is more effective in preventing flap necrosis than systemic MPL administration because of enhancing the cellular tolerance to hypoxic stress, which is possibly mediated by HSP70, whereas some benefit may be obtained with MPL due to iNOS and HO-1-mediated improvement in tissue oxygenation.     

Percutaneous Transhepatic Cholangiodrainage as Rescue Therapy for Symptomatic Biliary Leakage Without Biliary Tract Dilation After Major Surgery

Symptomatic biliary leakage following major upper abdominal surgery is a severe complication resulting in increased morbidity and mortality. Treatment options usually include either endoscopic intervention or surgical revision. These options may be burdened by a high perioperative risk for the patient (e.g., patients with severe disease) or simply may not be possible (e.g., nonpreserved gastroduodenal passage). In the past, percutaneous transhepatic cholangiodrainage did only seem to be a viable option for patients with dilated bile ducts. Here, we present our experience in a consecutive series of patients with symptomatic biliary leakage following major upper abdominal surgery and without dilation of the biliary system that underwent percutaneous transhepatic cholangiodrainage. Percutaneous transhepatic cholangiodrainage was feasible in 15 of 18 patients (83.3%). The procedure was technically not possible in three patients (16.7%). In 10 of the 15 patients (66.6%) with feasible percutaneous transhepatic cholangiodrainage, biliary leakage was definitely controlled without the need for surgical revision. Depending on the experience with the interventional procedure, percutaneous transhepatic cholangiodrainage should be considered as an alternative for treatment of symptomatic biliary leakage instead of immediate reoperation. Presented at the Digestive Disease Week 2005 (DDW), Chicago, IL, May 14–19, 2005 (poster presentation).     

The Significance of Histology and Morphometry in Predicting Lymph Node Metastases in Patients with Squamous Cell Carcinoma of the Vulva

The material consists of a series of 73 patients with squamous cell carcinoma of the vulva. The site and the size of the primary tumor and the histological status of the lymph nodes of the groin were known. Two pathologists evaluated nuclear hyperchromatism, nuclear polymorphism, histological differentiation, number of mitoses, inflammatory response, and vascular invasion and graded these parameters from one to three. The reliability of the histopathological grades evaluated by the κ coefficient showed considerable interobserver variation. Despite this a model which included the subjective parameter nuclear hyperchromatism could predict patients without lymph node metastases. The model consisted of patients with tumors which were not situated on the clitoris, were less than 40 mm in diameter, and exhibited only slight hyperchromatism. The model fitted 19 (26%) and 14 (19%) of the patients with two different pathologists evaluating the nuclear hyperchromatism and none of these patients had lymph node metastases. The quantitative parameter—mean nuclear volume—determined by morphometry was of no diagnostic value for the prediction of patients without groin node metastases at the time of operation.     

Increase in vascular permeability induced by leukotriene b4 and the role of polymorphonuclear leukocytes

Leukotriene B₄ (LTB₄), a metabolite of arachidonic acid, is known to be a potent chemotactic and chemokinetic substance. We have used the hamster cheek pouch microcirculation model to study the effect of LTB₄ on vascular permeability and the involvement of neutrophil granulocytes in this response. Intravascular fluorescein-labeled dextran (mol wt 150,000) was used as a tracer of macromolecular permeability. Topical application of LTB₄ (150 nM-5 M) to the hamster cheek pouch resulted in an immediate increase in adhering leukocytes in postcapillary venules and later larger venules. Leukocyte accumulation was reversible, but continued longer the higher the dose of LTB₄ used. Subsequently, a dose-dependent increase in vascular permeability was seen at postcapillary and larger venules, with a maximum 10–20 min after application; the maximum occurred later the higher the dose of LTB₄. Depletion of neutrophil granulocytes by pretreatment of the animals with antineutrophil serum obtained from immunized rabbits significantly decreased the permeability response to LTB₄, whereas the response to histamine was unaffected. These results suggest that neutrophil granulocytes play a role in LTB₄-mediated permeability increase. LTB₄ may be of importance both for the leukocyte accumulation and for the edema formation seen in inflammatory reactions.     

Enzymatic digestion of adult human articular cartilage yields a small fraction of the total available cells

We investigated whether different protocols for the digestion of adult human articular cartilage influence the cell yield and capacity to attach and proliferate in culture dishes. Chondrocyte yields were expressed as a percentage of the total number of cells in the tissue, determined both histologically (using the dissector method) and biochemically (measuring the DNA content of tissue digests). Human cartilage specimens (n = 79) were digested using different protocols based on combinations of collagenase II (CGN), trypsin/EDTA, hyaluronidase, and tosyllysylchloromethane (TLCM). Yields of viable chondrocytes were the highest within a specific range of CGN concentrations and digestion times, but always < 22% of the total available cells. The combination of CGN with trypsin/EDTA or TLCM accelerated the digestion process but did not significantly increase cell yields. The percentage of viable cells that attached to culture dishes ranged 75-85% (< 19% of the total) and was reduced by TLCM. Doubling times of attached cells were comparable in all experimental groups. Our results indicate that chondrocyte yields and capacity to attach and proliferate are not highly sensitive to the specific isolation protocol used. However, typically used cartilage digestion protocols yield only a small fraction of the total available cells, possibly introducing an uncontrolled selection of certain chondrocyte subpopulations.     

Enu mouse mutagenesis: generation of mouse mutants with aberrant plasma IgE levels

BACKGROUND: The ENU Mouse Mutagenesis Project aims at a large-scale, systematic production of mouse mutants using the alkylating agent ethyl-nitrosourea (ENU). Offspring of mutagenized mice are subjected to a multiparameter screen to detect alterations in various phenotypes with the ultimate goal of identifying novel genes relevant for the expression of the phenotype. Using this approach, we have analyzed plasma IgE concentrations to identify mouse mutants with aberrant plasma IgE levels. METHODS AND RESULTS: ENU-mutagenized male C3HeB/FeJ were mated to wild-type females to produce F1 offspring. F1 animals were analyzed for alterations in their plasma IgE concentrations that showed a dominant mode of inheritance, or bred further to screen for recessive phenotypes. Plasma IgE concentrations were determined by ELISA and a normal range for plasma IgE was established using C3HeB/FeJ wild-type animals. So far we have tested 6568 F1 animals. Repeated testing confirmed a stable aberrant IgE phenotype in 124 animals. To confirm the genetic basis of the observed phenotype, these mice were subjected to confirmation crossing. Currently we have established 9 independent mutant mouse lines (3 with high plasma IgE and 6 with plasma IgE below detection limit) that have been genetically confirmed and additional 24 variant mouse lines are currently undergoing confirmation testing. CONCLUSION: ENU mouse mutagenesis allowed us to generate and identify mouse mutants with aberrant plasma IgE levels, which may be used to characterize novel genes involved in IgE regulation and may serve as animal models for IgE-mediated diseases.     

Compound fractures of the proximal humerus

This paper stresses the importance of considering reconstructive measures when dealing with complex fractures, including fracture dislocations of the proximal humerus. Mainly, we try to use "biological" techniques with minimal exploration and internal fixation for stabilization. Indications for internal fixation are compared with prosthetic replacement and primary fusion.