The Application of Stereological Methods for Estimating Structural Parameters in the Human Heart

This study describes and exemplifies generally applicable design‐based stereological methods for obtaining quantitative estimates of the numbers and sizes of capillaries, cardiomyocytes, and cardiomyocyte nuclei in immersion‐fixed human left ventricles (N = 6). The design‐based stereological methods are valid in all cardiac investigations onto quantifying changes in structure and function as seen under various conditions such as during development, aging, hypertrophy, and following ischemia/reperfusion. The applied principles of unbiased stereology were as follows: 1) uniform random sampling was taken at all levels, also in respect to orientations, for estimates of length and mean sizes. 2) All global structural quantities were estimated as total quantity = density × volume of the left ventricle. As an example, the left ventricle contains 1.5 × 10⁹ capillaries with a total length of just below 200 km. 3) Stereological methods were used for estimating the volume density, surface area density, and length density of capillaries and cardiomyocytes. The numerical density of cardiomyocyte nuclei and capillaries was estimated, using the optical and physical disector, respectively. 4) In all local quantities, “size” was estimated either directly, using unbiased estimators to obtain the average individual size and size distribution parameters, or indirectly, using the relationship that: average size = total quantity/total number. In the six hearts constituting this study, we observed the anticipated correlation between left ventricular volume and global estimates such as total number of capillaries. There were no correlation between local quantities and total left ventricular volume (e.g., average star volume of individual cardiomyocytes). Anat Rec, 2009. © 2009 Wiley‐Liss, Inc.     

Proposed requirements for a European Doctorate in Dentistry: a discussion document prepared by a special interest group under the auspices of the Association for Dental Education in Europe

In the Bologna process a third cycle is distinguished at the doctoral level. In documents on the Bologna process it is advocated to harmonise the structure and requirements of the doctorate, which in Europe are characterised by a wide variety. Differences exist in all possible requirements between countries, and even between schools within one country differences can be seen. In this paper an inventory is made of these differences in the dental doctorate between European countries. Moreover, the need for necessary harmonisation of requirements for a European dental doctorate is strongly advocated and a proposal is presented.     

Seconds from disaster: lessons learned from laparoscopic release of the median arcuate ligament

Median arcuate ligament syndrome (MALS) is a rare entity that manifests as abdominal pain, nausea, vomiting, and diarrhea. The median arcuate ligament is a fibrous band that connects the crura of the diaphragm. In some people, the ligament is positioned in a way that compresses the celiac axis, which in a subset of individuals causes the symptoms associated with MALS. Surgical release of the ligament can relieve these symptoms. After viewing a video that described the laparoscopic median arcuate ligament release technique at the 2006 SAGES meeting and reviewing the online video, we report our experience with two cases and discuss the lessons learned in performing the procedure within a training program. We also discuss the extent to which surgical resident participation contributes to intraoperative complications during a new and complex surgery. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Systemic administration of enamel matrix derivative to lipopolysaccharide-challenged pigs: effects on the inflammatory response

BACKGROUND: Periodontitis is the primary clinical indication for enamel matrix derivative (EMD). Recent investigations, showing that EMD inhibits the production of tumor necrosis factor-alpha (TNF-alpha) when added to human whole blood, indicate a novel role for EMD as a modulator of systemic inflammation. In the present study, we investigated the systemic effects of EMD in lipopolysaccharide (LPS)-challenged pigs. METHODS: In a preparatory study, seven pigs received a prophylactic EMD bolus injection (5 mg/kg), followed by a continuous infusion (50 mg/kg/min). Thirty minutes later, a continuous infusion of LPS (1.7 mcg/kg/h) was started. An additional 12 pigs were randomized into two groups. Six of these animals were given the same treatment, except that EMD was administered 30 min after LPS. The remainder served as controls. The groups were compared according to organ injury and function, hemodynamics, and systemic markers of inflammation. RESULTS: Prophylactic administration of EMD triggered transient hemodynamic instability in two of seven pigs. In the randomized pigs, no or only nonspecific changes were observed in biopsies from vital organs, independent of treatment. Enamel matrix derivative did not modify systemic TNF-alpha, interleukin (IL)-1 beta, or IL-6 concentrations. CONCLUSIONS: In the formulation and dosages used, EMD did not modulate the inflammatory response. No true allergic or immunotoxic reactions were seen. To be usable for systemic application, a new formulation should be developed, or the active part of the protein(s) should be identified and produced in a soluble form designed for infusion. The potential of EMD as a systemic immune modulator is still unsettled.     

Identifying approaches to improve the accuracy of shock outcome prediction for out-of-hospital cardiac arrest

BACKGROUND: Analysis of the electrocardiogram (ECG) can predict if a cardiac arrest patient in ventricular fibrillation is likely to have a return of spontaneous circulation if defibrillated. The accuracy of such methods determines how useful it is clinically and for retrospective analysis. METHODS AND RESULTS: We wanted to identify if there is a potential of improving prediction accuracy by adding peri-arrest factors to an ECG-based prediction system, or constructing a prediction system that adapts to each patient. Therefore, we analysed shock outcome prediction data with a mixed effects logistic regression model to identify if there are random effects (unexplained variation between patients) influencing the prediction accuracy. We also added information about the patients' age, sex and presenting rhythm, ambulance response time and presence of bystander CPR to the model to try to improve it by reducing the random effects. For all the six predictive features analysed random effects where present, with p-values below 10(-3). The random effect size was 73-189% of the feature effect size. Adding the peri-arrest factors to the best ECG-based model gave no significant improvement. CONCLUSIONS: The presence of random effects shows that the shock outcome prediction accuracy can be improved by explaining more of the variation between patients, for example using the approaches outlined above, and that there is within-patient correlation between samples that should be accounted for when evaluating prediction accuracy. The specific peri-arrest factors tested here did not significantly improve prediction accuracy, but other factors should be explored.     

Activation of cytokine synthesis by systemic infusions of lipopolysaccharide and peptidoglycan in a porcine model in vivo and in vitro

BACKGROUND: The incidence of gram-positive and mixed bacterial infections in surgical patients has increased, and there has been an alarming rise in the number of drug-resistant bacteria. Peptidoglycan (PepG) is a cell wall component of gram-positive bacteria that stimulates inflammatory responses both ex vivo and in vivo. The systemic effects of PepG on inflammation have not been studied in a large animal model. METHODS: Anesthetized pigs were subjected to 8-h continuous intravenous infusions of lipopolysaccharide (LPS) (4 mcg/kg/h), PepG (40 mcg/kg/h), LPS plus PepG, or saline. The concentrations of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, IL-8, and IL-10 were measured in the plasma prior to infusion (time 0) and thereafter every second hour until the end of the experiments. Heparinized whole blood samples drawn at time 0 and after a 6-h infusion of LPS or PepG were incubated ex vivo with PepG (10 mcg/mL), LPS (10 ng/mL), or a combination of PepG and LPS to study the immunologic consequences of systemic inflammation. Concentrations of TNF-alpha, IL-8, and IL-1beta were measured in the supernatant liquids. RESULTS: In vivo, there was transient upregulation of TNF-alpha after infusion of LPS, PepG, or the combination. Interleukin-6 and IL-8 were upregulated by LPS but not by PepG. In vitro studies of whole blood obtained at time 0 revealed a synergistic effect of LPS and PepG on the release of TNF-alpha. Incubation of whole blood obtained after 6 h of infusion of LPS or PepG revealed tolerance and cross-tolerance between the two bacterial components in the induction of TNF-alpha, IL-8, and IL-1beta. CONCLUSIONS: Peptidoglycan is a potent inducer of TNF-alpha in this large animal model. Peptidoglycan and LPS synergized to increase the formation of the proinflammatory cytokine TNF-alpha. The study demonstrates for the first time the development of tolerance and cross-tolerance between LPS and PepG in a large animal model. These phenomena could be of importance for the signs and symptoms of sepsis.     

Evidence that compromised K+ spatial buffering contributes to the epileptogenic effect of mutations in the human Kir4.1 gene (KCNJ10)

Mutations in the human Kir4.1 potassium channel gene (KCNJ10) are associated with epilepsy. Using a mouse model with glia-specific deletion of Kcnj10, we have explored the mechanistic underpinning of the epilepsy phenotype. The gene deletion was shown to delay K(+) clearance after synaptic activation in stratum radiatum of hippocampal slices. The activity-dependent changes in extracellular space volume did not differ between Kcnj10 mutant and wild-type mice, indicating that the Kcnj10 gene product Kir4.1 mediates osmotically neutral K(+) clearance. Combined, our K(+) and extracellular volume recordings indicate that compromised K(+) spatial buffering in brain underlies the epilepsy phenotype associated with human KCNJ10 mutations.     

Metal accumulation and metallothionein in two populations of brown trout, Salmo trutta, exposed to different natural water environments during a run-off episode

Cd, Cu and Zn concentrations were measured in ambient water as well as in gills, liver and kidney tissues of two natural populations of brown trout (Salmo trutta) during a run-off episode in two rivers with different metal compositions due to mining pollution. Metallothionein (MT) was also measured in these tissues. The two rivers, Rugla (Cu contaminated) and Naustebekken (Cd and Zn contaminated), are located in two neighboring drainage basins separated by the topographic divide near the city of Røros in the County of Sør-Trøndelag, Norway. In Rugla, the Cu concentration increased from 15 μg/l at the low water level to 41 μg/l during the run-off episode. In Naustebekken, corresponding values for Cd were 90–170 ng/l and those for Zn were 49–91 μg/l. Gill concentrations of Cu and Cd/Zn MT in both populations of native trout clearly reflect the presence of these metals in the rivers during the run-off, in accordance with the hypothesis of protection caused by MT induction. When Rugla trout were transferred to Naustebekken and vice versa, both the amounts of MT itself and the Cu contents reflected the concentration of this metal in the new environment, indicating that MT induction also protects against acutely increased metal levels. The measured levels of MT in both native and transferred trout can account for all the Cd present in the tissues, but not for all of the Cu and Zn. The capacity of MT to regulate Cd and Cu in the trout populations in their natural habitat therefore seems clearly present. Our data also indicate that the MT I and II isoforms may bind metals selectively.     

Chlamydia pneumoniae DNA in peripheral blood mononuclear cells in dialysis patients,renal transplant recipients and healthy controls

While nitric oxide (NO) is implicated as an important mediator of hypotension in sepsis and endotoxemia, its role as a mediator of tissue injury in shock is controversial. During porcine endotoxemia (lipopolysaccharide (LPS) 1.7 &#119 g·kg -1 ·h -1 i.v. for 6 h), we compared circulatory and morphological changes in the liver induced by two different NO synthase inhibitors (N G -nitro-L-arginine methyl ester, L-NAME, 25 mg·kg- 1 i.v., and aminoethyl-isothiourea, AE-ITU, 10 mg·kg- 1 i.v.), both given after 3 h. LPS induced time-dependent tissue reactions with edema, sinusoidal dilation, packing of red cells and leukocyte infiltration, progressing to endothelial cell and hepatocyte damage, formation of thrombi, and at 6 h widespread necrosis. These changes were similar in all pigs receiving LPS, regardless of treatment with NOS inhibitors. LPS caused significant increases in aspartate aminotransferase (AST), alkaline phosphatase (ALP) and alpha glutathione S-transferase ( &#102 -GST). L-NAME caused further increases in AST, ALP and &#102 -GST, while AE-ITU prevented the late increase in ALP and &#102 -GST observed in the other LPS groups. LPS reduced liver blood flow by ~ 40%. L-NAME further reduced flow by ~ 50%, while AE-ITU restored liver blood flow to baseline values. Conclusion: L-NAME in endotoxemia had detrimental effects on liver circulation, while AE-ITU improved liver blood flow and attenuated the late increase in liver enzymes. Liver morphology was unaffected within the 3-h observation time after NOS inhibition.