Establishing a brain-death donor model in pigs.

INTRODUCTION: An animal model that imitates human conditions might be useful not only to monitor pathomechanisms of brain death and biochemical cascades but also to investigate novel strategies to ameliorate organ quality and functionality after multiorgan donation. METHODS: Brain death was induced in 15 pigs by inserting a catheter into the intracranial space after trephination of the skull and augmenting intracranial pressure until brain stem herniation. Intracranial pressure was monitored continuously; after 60 minutes, brain death diagnostics were performed by a neurologist including electroencephalogram (EEG) and clinical examinations. Clinical examinations included testing of brain stem reflexes as well as apnoe testing; then intensive donor care was performed according to standard guidelines until 24 hours after confirmation of brain death. Intensive donor care was performed according to standard guidelines for 24 hours after brain death. RESULTS: Sixty minutes after brain-death induction, neurological examination and EEG examination confirmed brain death. Intracranial pressure increased continuously, remaining stable after the occurrence of brain death. All 15 animals showed typical signs of brain death such as diabetes insipidus, hypertensive and hypotensive periods, as well as tachycardia. All symptoms were treated with standard medications. After 24 hours of brain death we performed successful multiorgan retrieval. DISCUSSION: Brain death can be induced in a pig model by inserting a catheter after trephination of the skull. According to standard guidelines the brain-death diagnosis was established by a flat-line EEG, which occurred in all animals at 60 minutes after induction.

Establishing a donation after cardiac death model in pigs.

INTRODUCTION: Due to the lack of human donors, several strategies have sought to expand the organ pool. Efforts to characterize donation after cardiac death (DCD) have included studies of cell viability, histological and immunohistochemical changes, and oxidative stress, which is known to negatively impact graft survival. A large animal model would be useful for these inquiries. Therefore, we sought to establish a DCD animal model in pigs. METHODS: We simulated non-heart-beating donation Maastricht II and III conditions in 24 pigs. Cardiac fibrillation was induced using 9-V direct current. After various times of ventricular fibrillation (1-10 minutes) with no mechanical and/or medical treatment to achieve cardiac output, reanimation was performed for 30 minutes prior to multiorgan donation. Then, a neurological status was performed. Blood samples were obtained at defined times tissue samples were stored in liquid nitrogen and subsequently embedded in paraffin and subjected to further analysis. RESULTS: We established a DCD pig model in our laboratory by inducing cardiac fibrillation. Up to now, only DCD donation according to the Maastricht criteria II and III has been performed, but establishing all Maastricht criteria of DCDs seems to be feasible. CONCLUSION: A DCD model in pigs enables us to characterize organ quality more precisely as well as evaluate amelioration of storage conditions and donor treatments in a large-animal model.

Increased expression of vascular endothelial growth factor in human skeletal muscle in response to short-term one-legged exercise training.

The effects of short-term exercise training on vascular endothelial growth factor (VEGF) and one of its regulatory transcription factors, the hypoxia inducible factor 1 (HIF-1) subunit, were studied in eight healthy males. Muscle and blood samples were obtained before the 1st, and 24 h after the 7th training session. VEGF and HIF-1 mRNA were analysed using RT-PCR, VEGF mRNA localization with in situ hybridization and VEGF protein with ELISA. Concurrent increases in VEGF mRNA and protein levels were observed in skeletal muscle, and the mRNA was expressed within the skeletal muscle fibres and in cells in the interstitium. These data support the idea of a pretranslational regulation of exercise-induced changes in VEGF mRNA, and indicate that increased VEGF protein expression is an early event in skeletal muscle adaptation to training. Furthermore, different cell types may act as sources for the production of angiogenic factors in response to exercise. The levels of HIF-1 mRNA subunits did not change, suggesting no change in HIF-1 mRNA transcript levels in the regulation of training-induced VEGF expression. In contrast to increased tissue VEGF expression, the arterial and femoral venous plasma levels of VEGF were decreased by training, which may indicate an exercise-induced enhancement of the peripheral uptake of VEGF.

Molecular adaptations in human skeletal muscle to endurance training under simulated hypoxic conditions.

This study was performed to explore changes in gene expression as a consequence of exercise training at two levels of intensity under normoxic and normobaric hypoxic conditions (corresponding to an altitude of 3,850 m). Four groups of human subjects trained five times a week for a total of 6 wk on a bicycle ergometer. Muscle biopsies were taken, and performance tests were carried out before and after the training period. Similar increases in maximal O(2) uptake (8.3-13.1%) and maximal power output (11.4-20.8%) were found in all groups. RT-PCR revealed elevated mRNA concentrations of the alpha-subunit of hypoxia-inducible factor 1 (HIF-1) after both high- (+82.4%) and low (+78.4%)-intensity training under hypoxic conditions. The mRNA of HIF-1alpha(736), a splice variant of HIF-1alpha newly detected in human skeletal muscle, was shown to be changed in a similar pattern as HIF-1alpha. Increased mRNA contents of myoglobin (+72.2%) and vascular endothelial growth factor (+52.4%) were evoked only after high-intensity training in hypoxia. Augmented mRNA levels of oxidative enzymes, phosphofructokinase, and heat shock protein 70 were found after high-intensity training under both hypoxic and normoxic conditions. Our findings suggest that HIF-1 is specifically involved in the regulation of muscle adaptations after hypoxia training. Fine-tuning of the training response is recognized at the molecular level, and with less sensitivity also at the structural level, but not at global functional responses like maximal O(2) uptake or maximal power output.

Exercise-induced expression of angiogenesis-related transcription and growth factors in human skeletal muscle.

mRNA expression of vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), and hypoxia-inducible factor (HIF) subunits HIF-1alpha and HIF-1beta in human skeletal muscle was studied during endurance exercise at different degrees of oxygen delivery. Muscle biopsies were taken before and after 45 min of one-legged knee-extension exercise performed under conditions of nonrestricted or restricted blood flow (approximately 15-20% lower) at the same absolute workload. Exercise increased VEGF mRNA expression by 178% and HIF-1beta by 340%, but not HIF-1alpha and FGF-2. No significant differences between the restricted and nonrestricted groups were observed. The exercise-induced increase in VEGF mRNA was correlated to the exercise changes in HIF-1alpha and HIF-1beta mRNA. The changes in VEGF, HIF-1alpha, and HIF-1beta mRNAs were correlated to the exercise-induced increase in femoral venous plasma lactate concentration. It is concluded that 1) VEGF but not FGF-2 gene expression is upregulated in human skeletal muscle by a single bout of dynamic exercise and that there is a graded response in VEGF mRNA expression related to the metabolic stress and 2) the increase in VEGF mRNA expression correlates to the changes in both HIF-1alpha and HIF-1beta mRNA.

A stochastic PCR approach for RNA quantification in multiple samples.

When studying the effect of various treatments on gene expression in humans, one occasionally is faced with the problem of detecting small changes in transcript levels in minute tissue samples. In addition, interindividual variations can be quite large and may even be the major source of variation (1). Therefore, numerous samples usually have to be analyzed to detect such small variations in gene expression.

Fibre-type specific expression of fast and slow essential myosin light chain mRNAs in trained human skeletal muscles.

The fibre-type specific expression patterns of fast and slow isoforms of essential (alkali) myosin light chains (ELC) was analysed in trained, untrained and pathological human muscles. Biopsies from m. vastus lateralis of moderately trained and untrained persons, as well as highly trained endurance and strength athletes were analysed, by in situ hybridization, for the expression of the 'fast' ELC 1f/3f and the 'slow' ELC 1 sb. We wanted to investigate if changes in the fibre-type specific ELC mRNA pattern could be used as markers for training adaptation, especially, if the mRNA of the slow ELC 1sb isoform would appear in type IIA fibres as a result of endurance training (Baumann et al. 1987). We found the fast/slow ELC expression patterns in the fibre types to be remarkably stable. Physiological stress, even high training loads, did not affect it. No IIA fibres expressing ELC 1sb mRNA were found. They could be detected, however, in pathological muscle samples, where fast/slow ELC patterns not found in normal muscles were frequent. Our data suggest that in healthy muscles, only a subset of the theoretically possible combinations of myosin heavy and light chain isoforms are expressed at the level of their mRNAs.

Expression of fos and jun genes in human skeletal muscle after exercise.

It is believed that the induction of the fos and jun gene family of transcription factors might be at the origin of genetic events leading to the differential regulation of muscle-specific genes. We have investigated the effect of a 30-min running bout in untrained subjects on the expression of the mRNAs of all members of the fos and jun gene families, including c-fos, fosB, fosBdel, fra-1, and fra-2 as well as c-jun, junB, and junD. While the fos family members were transiently upregulated 10- to 20-fold (an exception being fra-2) the induction of the jun family members was up to 3-fold only. The induction of c-fos could also be demonstrated at the protein level. Both c-fos and c-jun mRNAs were coinduced in muscle fiber nuclei. The induction was not restricted to a particular fiber type, as expected from established muscle fiber recruitment schemes, but followed a "patchy" pattern confined to certain regions of the muscle. The signals leading to the expression of these immediate early genes are therefore unclear.

Fiber-type-specific expression of essential (alkali) myosin light chains in human skeletal muscles.

We studied the expression patterns of the essential (alkali) myosin light-chain isoforms in adult human skeletal muscles, using in situ hybridization and single-fiber protein analysis. In analogy to other species, we found that the fiber type-specific expression of essential myosin light chains is regulated via the availability of the respective mRNAs in a given fiber. In contrast to other species, the slow isoform 1sa was only expressed in the most oxidative Type I fibers (Subtype IA) in addition to 1sb. These fibers also contained high levels of carbonic anhydrase III. Within the fibers, the essential myosin light-chain mRNAs were located preferentially in the perinuclear regions and to a lesser extent in the intermyofibrillar spaces, a distribution that excludes cotranslational assembly of these light chains into the myofibrils as the main mechanism. In comparing leg and shoulder muscles, we found less distinct fiber typing in the expression patterns of the essential myosin light chains in the leg muscles than in muscles from the shoulder region.

Hsp70 expression in human skeletal muscle after exercise.

Prolonged exercise of a sufficiently high intensity is thought to create physiological stress and to disturb cellular homeostasis, ultimately inducing cellular adaptations which enable the organism to better deal with any future exercise challenge. Heat shock proteins (hsp) are expressed when cells are exposed to different types of stress. In this study, we have investigated whether the expression of the heat inducible form of hsp70 is increased in human skeletal muscle cells after a single bout of exercise. Five untrained subjects performed an exercise bout at their individual anaerobic threshold for 30 min on a treadmill. Hsp70 mRNA concentration was significantly increased by a factor of four at 4 min post-exercise. Similarly high levels were also observed 30 min and 3 h after the end of exercise. Hsp70 protein concentration, on the contrary, did not change within 3 h after cessation of exercise. Thus, a single exercise bout in humans is able to increase the steady state concentration of hsp70 mRNA, but is probably not sufficient to have an effect on the already high basal level of its protein. The analysis of hsp70 mRNA is potentially useful as a method to detect stress in tissues with a high basal level of heat shock proteins.

mRNAs of enzymes involved in energy metabolism and mtDNA are increased in endurance-trained athletes.

Improvements in endurance capacity by training are associated with structural and biochemical adaptations of working muscles that affect the mitochondrial compartment. We investigated whether the 1.8-fold higher mitochondrial volume density in a group of endurance-trained athletes compared with untrained subjects was reflected by higher steady-state levels of mRNAs coding for components of the oxidative phosphorylation pathway using a quantitative polymerase chain reaction approach. We found that mitochondrially encoded RNAs (cytochrome-c oxidase subunit I, NADH reductase subunit 6, 16S rRNA), as well as nuclear-encoded RNAs (cytochrome-c oxidase subunit IV, succinate dehydrogenase, fumarase) are all increased coordinately in the athletes (1.54- to 1.94-fold). In addition, mitochondrial (mt) DNA concentration was also 1.55-fold higher in the trained athletes, whereas genomic DNA was not changed. Our findings thus show similar RNA expression of mitochondrially encoded genes in sedentary and endurance-trained subjects, whereas pretranslational control mechanisms account for higher levels of nuclear-encoded RNAs in the athletes.

An efficient polymerase chain reaction approach for the quantitation of multiple RNAs in human tissue samples.

We describe a PCR quantitation approach that we have set up to study gene expression in human skeletal muscle biopsies. It is characterized by the independent standardization of the individual steps and ignores attempts to control for the efficiency of the PCR. Different RNA extraction/reverse transcription efficiencies are normalized by the addition of an unrelated cRNA. Quantitation is achieved by parallel amplifications of reference samples containing known amounts of PCR products. Precision was achieved by multiple measurements of several samples. Our approach allows for the detection of less than twofold differences (27-88%, depending on the RNA species studied) among samples. A comparison of biopsies from highly trained endurance runners with biopsies from untrained subjects showed that the increased mitochondrial density in the runners' samples is accompanied by a proportional increase in the concentration of the mRNA of cytochrome c oxidase subunit IV.

The use of 33P-labelled riboprobes for in situ hybridizations: localization of myosin alkali light-chain mRNAs in adult human skeletal muscle.

33P-labelled probes were used to localize the mRNAs coding for the myosin alkali light-chain isoforms MLC 1f/3f and MLC 1sb in adult human muscles, which are distributed in characteristic fibre type specific patterns. In situ hybridizations of 33P-labelled probes were compared with probes carrying 35S or digoxigenin labels. Signals of equal strength were obtained with each of the three labels. The preferentially peripheral localization of these mRNAs in the muscle fibres could be clearly seen with all three probes, with digoxigenin probes providing the best resolution. 33P can serve as a viable alternative in this type of experiment. These experiments with adult human muscles also showed that the post mortem stability of RNA in human muscle is better than generally assumed. We could detect no signs of degradation in RNA prepared from heart ventricle as well as skeletal muscle up to 24 hours post mortem. In situ hybridizations worked equally well in biopsy material as in post mortem samples.

Fast myosin light chain expression in chicken muscles studied by in situ hybridization.

We have studied the fiber type-specific expression of the fast myosin light chain isoforms LC 1f, LC 2f, and LC 3f in adult chicken muscles using in situ hybridization and two-dimensional gel electrophoresis. Type II (fast) fibers contain all three fast myosin light chain mRNAs; Types I and III (slow) fibers lack them. The myosin light chain patterns of two-dimensional gels from microdissected single fibers match their mRNA signals in the in situ hybridizations. The results confirm and extend previous studies on the fiber type-specific distribution of myosin light chains in chicken muscles which used specific antibodies. The quantitative ratios between protein and mRNA content were not the same for all three fast myosin light chains, however. In bulk muscle samples, as well as in single fibers, there was proportionally less LC 3f accumulated for a given mRNA concentration than LC 1f or LC 2f. Moreover, the ratio between LC 3f mRNA and protein was different in samples from muscles, indicating that LC 3f is regulated somewhat differently than LC 1f and LC 2f. In contrast to other in situ hybridization studies on the fiber type-specific localization of muscle protein mRNAs, which reported the RNAs to be located preferentially at the periphery of the fibers, we found all three fast myosin light chain mRNAs quite evenly distributed within the fiber's cross-sections, and also in the few rare fibers which showed hybridization signals several-fold higher than their surrounding counterparts. This could indicate principal differences in the intracellular localization among the mRNAs coding for various myofibrillar protein families.