Objective measurement of tissue tension in myofascial trigger point areas before and during the administration of anesthesia with complete blocking of neuromuscular transmission.

OBJECTIVES: Myofascial trigger points (MTPs) are extremely frequent in the human musculoskeletal system. Despite this, little is known about their etiology. Increased muscular tension in the trigger point area could be a major factor for the development of MTPs. To investigate the impact of muscular tension in the taut band with an MTP and thereby, the spinal excitability of associated segmental neurons, we objectively measured the tissue tension in MTPs before and during the administration of anesthesia using a transducer. METHODS: Three target muscles (m. temporalis, upper part of m. trapezius, and m. extensor carpi radialis longus) with an MTP and 1 control muscle without an MTP were examined in 62 patients scheduled for an operation. RESULTS: We found significant 2-way interactions (ANOVA, P<0.05) between the analyzed regions of the target muscles dependent on the time of measurement, that is, before and during a complete blocking of neuromuscular transmission. These effects could be demonstrated for each target muscle separately. DISCUSSION: An increased muscle tension in MTPs, and not a primary local inflammation with enhanced viscoelasticity, was the main result of our investigation. We interpret this increased muscular tension in the taut band with an MTP as increased spinal segmental excitability. In line with this, we assume a predominant, but not unique, impact of increased spinal excitability resulting in an augmented tension of segmental-associated muscle fibers for the etiology of MTP. Consequently, postisometric relaxation might be a promising therapeutic option for MTPs.

Erythropoietin as additive of HTK preservation solution in cold ischemia/reperfusion injury of steatotic livers.

BACKGROUND: Organ shortage in liver transplantation has justified usage of marginal donor livers to expand the donor organ pool. The particular susceptibility of steatotic livers to I/R injury necessitates optimal preservation conditions in order to minimize preservation-reperfusion injury for successful transplantation. METHODS: The effect of erythropoietin (EPO) as additive to HTK preservation solution was studied in a mouse model. Lean and steatotic livers were harvested, stored for 24 h in 4°C HTK solution containing either EPO or saline and reperfused for 2 h with 37°C Krebs-Henseleit buffer. Livers without cold storage served as sham controls. RESULTS: Flushing of livers upon cold storage revealed a transaminase release, which was 2- to 10-fold higher in steatotic versus lean livers. EPO was effective in reducing the enzyme release to 50% in steatotic but not in lean livers. EPO prevented cold storage-induced denudation of the endothelial lining in steatotic livers, but aggravated it in lean livers. During reperfusion, steatotic livers presented with lower oxygen consumption and higher enzyme release than lean livers. In all livers, parameters of reperfusion injury remained unaffected by EPO. Expression of UCP2 was found markedly higher in steatotic livers. After I/R, steatotic livers revealed a significant drop of UCP2, whereas expression in lean livers was only slightly affected. EPO diminished Erk phosphorylation to almost the same extent in both mouse strains. CONCLUSION: Fortification of the preservation solution by EPO ameliorates cold ischemic injury of steatotic livers and may thus be considered for use as an adjunctive agent to increase the success of transplanting steatotic livers.

Desmopressin-induced Moschcowitz-like syndrome after treatment of uterine atonic bleeding in a 28-year-old pregnant woman: case report and overview of the literature.

Here we report of a patient who developed a Moschcowitz-like syndrome following a desmopressin treatment of severe postpartum hemorrhage. The patient got an anaphylactic reaction after cervical ripening with dinoproston, leading to an emergency cesarean. A postpartum uterine atony with a blood loss more than 1500 ml resulted in a disseminated intravascular coagulation that was treated with mass transfusion of blood products, including platelets and factor VII. Desmopressin is used as rescue medication in situations of severe bleeding. It was given in this life-threatening situation and presumably triggered a Moschcowitz-like syndrome. Desmopressin exerts its haemostatic effect by releasing von Willebrand factor, which is elevated in pregnancy per se. This results in an increased risk of developing microthrombi, leading to a Moschcowitz-like syndrome. In conclusion, desmopressin should not be administered in pregnant patients owing to its potential risk of triggering the development of thrombotic-thrombocytopenic purpura.

Male germ cell expression of the PAS domain kinase PASKIN and its novel target eukaryotic translation elongation factor eEF1A1.

PASKIN links energy flux and protein synthesis in yeast, regulates glycogen synthesis in mammals, and has been implicated in glucose-stimulated insulin production in pancreatic beta-cells. Using newly generated monoclonal antibodies, PASKIN was localized in the nuclei of human testis germ cells and in the midpiece of human sperm tails. A speckle-like nuclear pattern was observed for endogenous PASKIN in HeLa cells in addition to its cytoplasmic localization. By yeast two-hybrid screening, we identified the multifunctional eukaryotic translation elongation factor eEF1A1 as a novel interaction partner of PASKIN. This interaction was mapped to the PAS A and kinase domains of PASKIN and to the C-terminus of eEF1A1 using mammalian two-hybrid and GST pull-down assays. Kinase assays, mass spectrometry and site-directed mutagenesis revealed PASKIN auto-phosphorylation as well as eEF1A1 target phosphorylation mainly but not exclusively at Thr432. Wild-type but not kinase-inactive PASKIN increased the in vitro translation of a reporter cRNA. Whereas eEF1A1 did not localize to the nucleus, it co-localizes with PASKIN to the cytoplasm of HeLa cells. The two proteins also showed a remarkably similar localization in the midpiece of the sperm tail. These data suggest regulation of eEF1A1 by PASKIN-dependent phosphorylation in somatic as well as in sperm cells.

Non-hypoxic induction of HIF-3alpha by 2-deoxy-D-glucose and insulin.

Hypoxia-inducible factors (HIFs) are key mediators of cellular adaptation to hypoxia, but also respond to non-hypoxic stimuli. To clarify involvement in metabolic disturbances, HIFs were characterised in rats subjected to insulin-induced hypoglycaemia or cellular glucoprivation provoked by 2-deoxy-D-glucose (2-DG). Using real-time qPCR, organ-specific expression of HIF-1alpha, -2alpha, -3alpha, -1beta, and of the target gene GLUT-1 was determined. Distribution of HIF-3alpha proteins was examined by immunohistochemistry. Both, insulin and 2-DG resulted in a widespread increase in HIF-3alpha mRNA. HIF-2alpha mRNA increased in lung and heart after 2-DG only, whereas other HIFs remained unaffected. A pronounced increase of protein levels in cerebral cortex was observed for HIF-3alpha. Functional significance of HIF induction was reflected in enhancement of GLUT-1 mRNA. Transcriptional up-regulation of HIF-3alpha represents a typical response to in vivo hypoglycaemia and glucoprivation. These data suggest an involvement of the HIF system in metabolic derangements as for instance caused by diabetes.

Cardiac remodeling in erythropoietin-transgenic mice.

BACKGROUND: Transgenic (tg) mice with chronic overexpression of the human erythropoietin gene are characterized by an increased hematocrit of about 0.80 in adulthood. This is accompanied by cardiac dysfunction and premature death. The aim of this study was to examine whether this cardiac dysfunction was accompanied by hypertrophy of the heart with remodeling of the extracellular matrix (ECM). METHODS: 3-months-old wild type (wt) and tg mice without cardiac hypertrophy were compared with the respective 7-months-old mice. The mRNA of brain natriuretic peptide (BNP), of the matrix metalloproteinases (MMP)-2, -8, -9, -13, of the tissue inhibitor of metalloproteinase (TIMP)-1, -2, -3, -4 and of collagen I and III was detected by ribonuclease protection assay. The activity of MMPs was measured by zymography. RESULTS: There was hypertrophy of both ventricles in 7-months-old tg mice, which was accompanied by elevated mRNA expression of BNP. MMP-2 activity was increased and MMP-9 activity was decreased in the left ventricle (LV) of 3-months-old tg mice. This was accompanied by elevated TIMP-4 expression, followed by a shift of collagen mRNA expression from type III to type I in this ventricle. CONCLUSION: The shift to collagen I in the heart of tg mice might be associated with a stiffer ventricle resulting in diastolic dysfunction. This may be responsible for a relative and intermittent LV- and right ventricle (RV)-insufficiency which was likely to have occurred as evidenced by the elevation of lung and liver weight with hemorrhage and interstitial fibrosis after 7 months.

Hypoxia-induced mitogenic factor has antiapoptotic action and is upregulated in the developing lung: coexpression with hypoxia-inducible factor-2alpha.

Hypoxia-induced mitogenic factor (HIMF), also called FIZZ1 or RELMalpha, was a newly found cytokine. Hypoxia caused robust HIMF induction in the lung, and HIMF has potent pulmonary vasoconstrictive, proliferative, and angiogenic properties. To investigate the role of HIMF in lung development, we determined its spatial and temporal expression. From embryonic day (E)16 to postnatal day (P)28, HIMF was strongly expressed in the cytoplasm of bronchial epithelial cells, type II cells, endothelial cells, and primitive mesenchymal cells. Treatment with HIMF resulted in a significant reduction of apoptosis in cultured embryonic lung, thus revealing a previously unknown function of HIMF. Because HIMF gene is upregulated by hypoxia and contains a hypoxia-inducible transcription factor (HIF) binding site, we subsequently investigated whether HIMF was coexpressed with HIF-2alpha or HIF-1alpha. HIF-1alpha expression was temporally distinct from HIMF expression. In contrast, HIF-2alpha was present in endothelial cells, bronchial epithelial cells, and type II cells from E18 to P28. Thus, HIMF and HIF-2alpha were temporally and spatially coexpressed in the developing lung. These results indicate a role for HIMF in lung development, possibly under the control of HIF-2, and suggest that HIMF regulates apoptosis and may participate in lung alveolarization and maturation.

A dominant-negative isoform of hypoxia-inducible factor-1 alpha specifically expressed in human testis.

Spermatogenesis in the seminiferous tubuli of the testis occurs under a high proliferation rate, suggesting considerable oxygen consumption. Because of the lack of blood vessels, the oxygen partial pressure in the lumen of these tubuli is very low. We previously identified a testis isoform of the hypoxia-inducible factor (HIF)-1alpha in the mouse, termed mHIF-1alphaI.1. Here, we demonstrate that expression of mHIF-1alphaI.1 increases during puberty, further demonstrating its gene induction in postmeiotic germ cells. Using 5'-rapid amplification of cDNA ends, we identified a novel HIF-1alpha isoform in the human testis, called hHIF-1alphaTe. Like mHIF-1alphaI.1, hHIF-1alphaTe mRNA is derived from an alternative promoter-first exon combination, but with a different genomic organization and a different nucleotide sequence. Reverse transcription-polymerase chain reaction analysis confirmed that hHIF-1alphaTe is exclusively expressed in the testis. As determined by immunofluorescence of ejaculated sperm cells, HIF-1alpha protein is mainly localized in the postacrosomal head and in the midpiece of spermatozoa. Though overlapping with mitochondrial localization in human and mouse spermatozoa, neither hHIF-1alphaTe nor hHIF-1alpha associated with mitochondria. In contrast with the ubiquitously expressed HIF-1alpha protein and the mouse testis-specific mHIF-1alphaI.1 isoform, the hHIF-1alphaTe mRNA sequence predicts a protein with an N-terminal truncation of the DNA-binding domain. As shown by yeast two-hybrid assays, hHIF-1alphaTe still formed heterodimeric complexes with HIF-1beta. However, hHIF-1alphaTe was incapable of forming a DNA-binding HIF-1 complex. Overexpression of exogenous hHIF-1alphaTe resulted in the inhibition of the endogenous HIF-1 transcriptional activity, demonstrating that the testis-specific hHIF-1alphaTe isoform is a dominant-negative regulator of normal HIF-1 activity.

Norepinephrine-induced acute heart failure in transgenic mice overexpressing erythropoietin.

OBJECTIVE: Overexpression of erythropoietin (Epo) in mice (Epo-tg6) leads to an increase in hematocrit and blood volume, and strongly reduces endurance upon exercise. It was the aim of this study to characterize the mechanisms underlying the reduced cardiac performance. METHODS: Left (LV) and right (RV) ventricular function was measured with and without norepinephrine (NE) stimulation in 12 anaesthetized Epo-tg6 and in 13 wild-type (WT) control mice. RESULTS: There were no differences in heart function under baseline resting conditions. Stimulation with NE (10 microl bolus injections of 1-100 ng per mouse) in WT mice led to a dose-dependent increase in heart rate (HR), LV developed pressure (LVDP) and rate of rise in LV pressure (LV dP/dt(max)), while LV end-diastolic pressure (LVEDP) was unchanged. Except for HR, these parameters increased to a lesser extent in EPO-tg6 mice. Strikingly, LVEDP strongly increased in Epo-tg6 mice after NE (up to >20 mmHg). Eleven out of 13 Epo-tg6, but none of the WT mice died or required resuscitation after high-doses of NE. In these cases severe diastolic dysfunction became overt since the relative myocardial relaxation time was significantly prolonged and the duration of diastole was shortened. Moreover, the ECG showed a marked ST segment depression as well as deep negative T-waves. The NE-induced reduction in myocardial adenosin-triphosphate (ATP) content was more pronounced in Epo-tg6 mice after 10 min of continuous NE infusion (50 ng/min per mouse). CONCLUSION: NE-induced stress in Epo-tg6 mice led to acute heart failure associated with diastolic dysfunction and myocardial ischemia.

Altered pulmonary vascular reactivity in mice with excessive erythrocytosis.

Pulmonary vascular remodeling during chronic hypoxia may be the result of either oxygen deprivation or erythrocytosis. To separate experimentally the effects of hypoxia and erythrocytosis, we analyzed transgenic mice that constitutively overexpress the human erythropoietin gene in an oxygen-independent manner. These mice are characterized by polycythemia but have normal blood pressure, heart rate, and cardiac output. In transgenic mice, pulmonary artery pressure (PAP) was increased in vivo but was reduced in blood-free perfused lungs. The thromboxane receptor agonist U46619 caused a smaller rise in PAP in isolated transgenic lungs than in lungs from wild-type mice. The transgenic pulmonary vasculature was characterized by elevated prostacyclin production, stronger endothelial nitric oxide synthase expression, and reduced pulmonary vascular smooth muscle thickness. The fact that transgenic polycythemic mice have marked pulmonary hypertension in vivo but not in vitro suggests that their pulmonary hypertension is due to the increased blood viscosity, thus supporting an independent role of polycythemia in the development of pulmonary hypertension. In addition, our findings indicate that the lungs of transgenic animals adapt to the high PAP by elevated synthesis of vasodilators and reduced vascular smooth muscle thickness that tend to reduce vascular tone and vascular responsiveness.

Targeted disruption of the mouse PAS domain serine/threonine kinase PASKIN.

PASKIN is a novel mammalian serine/threonine kinase containing two PAS (Per-Arnt-Sim) domains. PASKIN is related to the Rhizobium oxygen sensor protein FixL and to AMP-regulated kinases. Like FixL, the sensory PAS domain of PASKIN controls the kinase activity by autophosphorylation in a (unknown) ligand-dependent manner. In Saccharomyces cerevisiae, the two PASKIN orthologues PSK1 and PSK2 phosphorylate three translation factors and two enzymes involved in glycogen synthesis, thereby coordinately regulating protein synthesis and glycolytic flux. To elucidate the function of mammalian PASKIN, we inactivated the mouse Paskin gene by homologous recombination in embryonic stem cells. Paskin(-/-) mice showed normal development, growth, and reproduction. The targeted integration of a lacZ reporter gene allowed the identification of the cell types expressing mouse PASKIN. Surprisingly, PASKIN expression is strongly upregulated in postmeiotic germ cells during spermatogenesis. However, fertility and sperm production and motility were not affected by the PASKIN knockout. The Ppp1r7 gene encoding Sds22, a regulatory subunit of protein phosphatase 1, shares the promoter region with the Paskin gene, pointing towards a common transcriptional regulation. Indeed, Sds22 colocalized with the cell types expressing PASKIN in vivo, suggesting a functional role of protein phosphatase-1 in the regulation of PASKIN autophosphorylation.

Hemostasis and coagulation at a hematocrit level of 0.85: functional consequences of erythrocytosis.

We have generated a transgenic mouse line that reaches a hematocrit concentration of 0.85 due to constitutive overexpression of human erythropoietin in an oxygen-independent manner. Unexpectedly, this excessive erythrocytosis did not lead to thrombembolic complications in all investigated organs at any age. Thus, we investigated the mechanisms preventing thrombembolism in this mouse model. Blood analysis revealed an age-dependent elevation of reticulocyte numbers and a marked thrombocytopenia that matched the reduced megakaryocyte numbers in the bone marrow. However, platelet counts were not different from wild-type controls, when calculations were based on the distribution (eg, plasma) volume, thereby explaining why thrombopoietin levels did not increase in transgenic mice. Nevertheless, bleeding time was significantly increased in transgenic animals. A longitudinal investigation using computerized thromboelastography revealed that thrombus formation was reduced with increasing age from 1 to 8 months in transgenic animals. We observed that increasing erythrocyte concentrations inhibited profoundly and reversibly thrombus formation and prolonged the time of clot development, most likely due to mechanical interference of red blood cells with clot-forming platelets. Transgenic animals showed increased nitric oxide levels in the blood that could inhibit vasoconstriction and platelet activation. Finally, we observed that plasmatic coagulation activity in transgenic animals was significantly decreased. Taken together, our findings suggest that prevention of thrombembolic disease in these erythrocytotic transgenic mice was due to functional consequences inherent to increased erythrocyte concentrations and a reduction of plasmatic coagulation activity, the cause of which remains to be elucidated.

Rescue of a patient out of a grain container: the quicksand effect of grain.

Grain storage containers not only present inherent dangers to the operators, but also to the rescuers if someone falls in. Here we report the rescue of a patient from a grain container using a novel technique involving a cylinder placed around the patient. This allowed the grain to be sucked out from around the patient and enabled his rescue uninjured. The rescue action was complicated by acute chest pain in the patient while he was submerged in the grain, and a severe asthma attack in the emergency physician. The rescue and the dilemmas encountered are described together with a review of the relevant literature.

Heat induction of the unphosphorylated form of hypoxia-inducible factor-1alpha is dependent on heat shock protein-90 activity.

Hypoxia-inducible factor (HIF)-1alpha is the oxygen-sensitive subunit of HIF-1, a transcriptional master regulator of oxygen homeostasis. Oxygen-dependent prolyl hydroxylation targets HIF-1alpha for ubiquitinylation and proteasomal degradation. Unexpectedly, we found that exposing mice to elevated temperatures resulted in a strong HIF-1alpha induction in kidney, liver, and spleen. To elucidate the molecular mechanisms responsible for this effect, HepG2 hepatoma cells were exposed to different temperatures (34-42 degrees C) under normoxic (20% O(2)) or hypoxic (3% O(2)) conditions. Heat was sufficient to stabilize mainly a phosphatase-resistant, low molecular weight form of HIF-1alpha (termed HIF-1alpha(a)). Heat-induced HIF-1alpha(a) accumulated in the nucleus but neither bound to DNA nor trans-activated reporter or target gene expression, demonstrating the need for post-translational modifications for these functions. The protein banding pattern of heat-induced HIF-1alpha in immunoblot analyses was clearly distinct from the HIF-1alpha pattern after prolyl hydroxylase inhibition (by hypoxia or iron chelation/replacement) or following proteasome inhibition, suggesting that heat stabilizes HIF-1alpha by a novel mechanism. Inhibition of the ATP-dependent chaperone activity of HSP90 by novobiocin or geldanamycin prevented heat-induced as well as hypoxia-induced HIF-1alpha accumulation, indicating a common role of the HSP90 chaperone activity in HIF-1alpha stabilization by these two environmental parameters.

Physiologically low oxygen concentrations in fetal skin regulate hypoxia-inducible factor 1 and transforming growth factor-beta3.

In the first-trimester mammalian fetus, skin wounds heal with perfect reconstitution of the dermal architecture without scar formation. Understanding environmental molecular regulation in fetal wound healing may reveal scar-limiting therapeutical strategies for the prevention of postnatal scarring wound repair. Therefore, we performed studies on fetal skin oxygenation and skin and wound expression of hypoxia-inducible factor 1alpha (HIF-1alpha) in the sheep model in vivo and performed studies on the potential relevance of HIF-1alpha during wound healing in vitro. Skin oxygen partial pressure levels were hypoxic throughout normal development. In nonscarring fetal skin at gestation day (GD)60, HIF-1alpha could be detected neither in healthy nor in wounded tissue. At GD100, in wounds with minimal scar formation, HIF-1alpha was expressed in fibroblasts and was markedly up-regulated at the wound edge. In scarring fetal wounds at GD120, HIF-1alpha was predominantly expressed in inflammatory cells. Expression of transforming growth factor beta3 (TGF-beta3), a potent antiscarring cytokine, overlapped with HIF-1a expression at GD100. HIF-1alpha-deficient mouse embryonic fibroblasts showed impaired migratory capabilities and demonstrated that TGF-beta3, but not proscarring TGF-beta1, manifests hypoxia- and HIF-1alpha-dependent regulation. In conclusion, HIF-1alpha-dependent regulation of a potent antiscarring cytokine may provide new strategies for antiscarring manipulation of wound healing.

Isoform-specific expression of hypoxia-inducible factor-1alpha during the late stages of mouse spermiogenesis.

The heterodimeric hypoxia-inducible factor (HIF)-1 is a transcriptional master regulator of several genes involved in mammalian oxygen homeostasis, including erythropoietin, vascular endothelial growth factor, and factors involved in glucose transport and metabolism. The mouse Hif1a gene is expressed from two distinct promoter/first exon combinations resulting in tissue-specific (mHIF-1alphaI.1) and ubiquitous (mHIF-1alphaI.2) mRNA isoforms. By in situ hybridization, we detected mHIF-1alphaI.1 mRNA exclusively in the elongated spermatids of the testis. In vitro studies indicated that the switch from mHIF-1alphaI.2 to mHIF-1alphaI.1 mRNA expression does not occur at the premeiotic stages of mouse spermatogenesis. Exposure of mice to hypoxic conditions induced mHIF-1alphaI.2 protein in spermatocytes and probably in Sertoli cells but not in spermatogonia. In contrast, expression of the putative mHIF-1alphaI.1 protein in spermatozoa of the testis and epididymis was oxygen independent and located to the midpiece of the spermatozoal flagellum. Both the switch in transcript expression during spermiogenesis and the unexpected protein localization in mature sperm cells suggest a so far unrecognized function of HIF-1alpha.