Impact of the hypoxic microenvironment on spermatogonial stem cells in culture.

The stem cell niche plays a crucial role in the decision to either self-renew or differentiate. Recent observations lead to the hypothesis that O2 supply by blood and local O2 tension could be key components of the testicular niche of spermatogonial stem cells (SSCs). In this study, we investigated the impact of different hypoxic conditions (3.5%, 1%, and 0.1% O2 tension) on murine and human SSCs in culture. We observed a deleterious effect of severe hypoxia (1% O2 and 0.1% O2) on the capacity of murine SSCs to form germ cell clusters when plated at low density. Severe effects on SSCs proliferation occur at an O2 tension ≤1% and hypoxia was shown to induce a slight differentiation bias under 1% and 0.1% O2 conditions. Exposure to hypoxia did not appear to change the mitochondrial mass and the potential of membrane of mitochondria in SSCs, but induced the generation of mitochondrial ROS at 3.5% and 1% O2. In 3.5% O2 conditions, the capacity of SSCs to form colonies was maintained at the level of 21% O2 at low cell density, but it was impossible to amplify and maintain stem cell number in high cell density culture. In addition, we observed that 3.5% hypoxia did not improve the maintenance and propagation of human SSCs. Finally, our data tend to show that the transcription factors HIF-1α and HIF-2α are not involved in the SSCs cell autonomous response to hypoxia.

Expression of the short chain fatty acid receptor GPR41/FFAR3 in autonomic and somatic sensory ganglia.

G-protein-coupled receptor 41 (GPR41) also called free fatty acid receptor 3 (FFAR3) is a Gαi-coupled receptor activated by short-chain fatty acids (SCFAs) mainly produced from dietary complex carbohydrate fibers in the large intestine as products of fermentation by microbiota. FFAR3 is expressed in enteroendocrine cells, but has recently also been shown to be present in sympathetic neurons of the superior cervical ganglion. The aim of this study was to investigate whether the FFAR3 is present in other autonomic and sensory ganglia possibly influencing gut physiology. Cryostat sections were cut of autonomic and sensory ganglia of a transgenic reporter mouse expressing the monomeric red fluorescent protein (mRFP) gene under the control of the FFAR3 promoter. Control for specific expression was also done by immunohistochemistry with an antibody against the reporter protein. mRFP expression was as expected found not only in neurons of the superior cervical ganglion, but also in sympathetic ganglia of the thoracic and lumbar sympathetic trunk. Further, neurons in prevertebral ganglia expressed the mRFP reporter. FFAR3-mRFP-expressing neurons were also present in both autonomic and sensory ganglia such as the vagal ganglion, the spinal dorsal root ganglion and the trigeminal ganglion. No expression was observed in the brain or spinal cord. By use of radioactive-labeled antisense DNA probes, mRNA encoding the FFAR3 was found to be present in cells of the same ganglia. Further, the expression of the FFAR3 in the ganglia of the transgenic mice was confirmed by immunohistochemistry using an antibody directed against the receptor protein, and double labeling colocalized mRFP and the FFAR3-protein in the same neurons. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) on extracts from the ganglia supported the presence mRNA encoding the FFAR3 in most of the investigated tissues. These data indicate that FFAR3 is expressed on postganglionic sympathetic and sensory neurons in both the autonomic and somatic peripheral nervous system and that SCFAs act not only through the enteroendocrine system but also directly by modifying physiological reflexes integrating the peripheral nervous system and the gastro-intestinal tract.

[Transillumination technique in difficult intubations in heart surgery]. / Die Transilluminationstechnik bei der schwierigen Intubation in der Herzanästhesie.

OBJECTIVE: The avoidance of myocardial depression still remains the goal of the management for cardiosurgical patients, also when unexpected difficulties in intubation face the anaesthetist. Therefore the difficult intubation should be managed by a short and easy procedure which provides safe results. To analyse the use of transillumination technique with a lightwand device (Trachlight(R), Laerdale, USA) in case of unexpected difficult intubation is the aim of our report. Methods All cardiosurgical patients (NYHA II - IV) were included from Jan 1998 - Dec 2001. After failure of the first intubation attempt by means of direct laryngoscopy (with non-adjustable vocal cord level) this intubation was qualified as an difficult intubation. In all these cases a lightwand device (Trachlight(R), Laerdale, USA) was applicated. Success, duration of the procedure, blood pressure, heart rate were recorded. Results 195 patients (out of total 7406) who could not be directly intubated by laryngoscopy (vocal cord level and arytenoid cartilage not visible), were classified as a difficult intubation. During the first year 1998 the light guided intubation (LGI) was successful as secondary procedure in 94 %, 3 cases, in which LGI failed the intubation was performed by fiberoptic method or McCoy blade. From 1999 to 2001 all difficult intubation could be managed by light guided intubation. In all cases of unexpected difficult intubation the procedure of the light guided intubation took as less than 3 min. The directly measured arterial blood pressure elevated by 14 % in comparison with the pressure prior to the passage through the larynx. Discussion and Conclusions 1998 after sufficient familiarisation of staff with the light wand device the transillumination technique was introduced as an alternative of using the McCoy blade or of using the fiberoptic method in the case of difficult intubation. Short neck or obesity, which occur as main reasons for intubation problems are surprisingly easy to control by light wand device. Therefore the light guided intubation could be an alternative procedure for unexpected difficult intubation in the setting of adult cardiac anesthesia.

[Intraoperative blood requirements and allogeneic blood transfusion in cardioanesthesia. Data analysis of 7729 patients in 12 cardiac surgical clinics]. / Intraoperativer Fremdblutverbrauch und Eigenbluttransfusion in der Kardioanästhesie. Datenanalyse von 7729 Patienten aus 12 herzchirurgischen Kliniken.

UNLABELLED: Allogeneic blood requirements in cardiac surgery shows a wide variation even for comparable procedures. The aim of the present study was to compare the intraoperative allogeneic blood requirement in defined cardiac operations among 12 cardiac centers in Germany. METHOD: A data set with 25 variables concerning the intraoperative course in adult cardiac patients with myocardial revascularization, valve replacement (aortic or/and mitral valve) or combined procedures was distributed to the participating centers. The data of all patients between January 1th 1998 and June 30th 1998 were included. Besides demographic data, the intraoperative transfusion of allogeneic and autologous blood, fresh frozen plasma and the concomitant hematocrit values were registered. Data were analyzed for all centers and separated for each center. RESULTS: The data of 7,729 patients were analyzed. The intraoperative allogeneic blood requirement was 0.6 +/- 1.3 units for all patients. It varied among the centers from 0.25 +/- 0.6 units to 0.97 +/- 1.6 units (P < 0.05). The percentage of patients receiving allogeneic blood was 27% and differed among the centers from 17% to 35%. Female patients were transfused in 53% (36-39%) compared to male patients with 16% (9-20%) (P < 0.05). The rate of autologous blood predonation varied from 0.5% to 23%. Patients without autologous predonation were transfused in 28% compared to 4% in patients with predonation (P < 0.05). In patients with autologous predonation the intraoperative transfusion of allogeneic blood was significantly reduced (0.1 +/- 0.39 vs 0.6 +/- 1.4 units, P < 0.05). However, some centers with a high percentage of autologous predonation also demonstrated a high rate of perioperative allogeneic transfusion. CONCLUSION: The incidence of allogeneic blood transfusion in cardiac surgery depends on the institution and not on the surgical procedure. A common threshold value of hemoglobin for the transfusion of blood trigger even for comparable procedures could not be detected among the centers. Especially in female patients, there was a wide variation in allogeneic blood transfusion. Autologous blood predonation reduces blood requirement significantly, however, it is practiced with variing intensity. The data set did not include information about transfusion regimen in the postoperative period, thus, these data do not allow to draw conclusions for the whole perioperative period.

Bipolar clamping improves the sensitivity of mutation detection by temperature gradient gel electrophoresis.

Temperature gradient gel electrophoresis (TGGE) is a rapid and sensitive screening method for point mutations and other small DNA alterations. Usually a polymerase chain reaction (PCR)-product of 150 to 500 bp that has been clamped at one end by a psoralen molecule or a "GC-clamp" is tested for abnormal melting characteristics by electrophoresis in a temperature gradient. Under optimal conditions, a heterozygous mutation within the fragment is detected through the presence of three additional bands in the TGGE gel, the mutant homoduplex and two heteroduplex bands. However, the ideal pattern of four sharp bands is not always found due to inconsistencies in melting behavior along the sequence of the DNA fragment under study. Some of these fragments show fuzzy bands that may impede or even prevent the detection of a mutation. Here, we describe a method to overcome this problem by utilizing one psoralen clamp at each end of the PCR product. Using TGGE assays established for exons 16, 17, and 18 of the NF1 gene and for exon 14 of the FBN1 gene as examples, we show that bipolar clamping may transform blurred bands into sharp ones and may visualize mutations that could not be detected by conventional single-sided clamping.

[The effect of substitution with AT III- and PPSB-concentrates in patients with terminal liver insufficiency]. / Substitutionseffekt von AT III-und PPSB-Konzentraten bei Patienten mit terminaler Leberinsuffizienz.

UNLABELLED: Patients with end-stage liver disease frequently develop combined coagulopathies due to increased procoagulant and fibrinolytic turnover as well as thrombocytopenia. The onset of clinical symptoms of a haemorrhagic diathesis requires balanced substitution of coagulation factors, since fresh frozen plasma alone does not always maintain a sufficient haemostatic potential in these patients. This substitution commonly follows standard rules based on the assumption that 0.5-1 IU of a coagulation factor or inhibitor concentrate given per kg body weight will increase its endogenous activity by 1%. We set out to investigate the validity of this standard regime in patients with end-stage liver disease scheduled for orthotopic liver transplantation. PATIENTS AND METHODS: Fifty-one adult patients were prospectively studied. In 37 patients with antithrombin III (AT III) activity < 70%, an AT III preparation (Kybernin, Behring, Marburg, Germany) was given preoperatively (mean dose 2616 +/- 207 IU) following standard calculations in order to increase endogenous activity to 80%. Twenty-seven of the patients had chronic hepatic failure (CHF group) with histologically proven cirrhosis and 10 had acute hepatic failure (AHF group). Blood samples were drawn prior to a 15-min infusion of AT III concentrate and 30 min thereafter. In 14 patients with prothrombin time (PT) < 40%, AT III levels had been corrected earlier during the clinical course to achieve activities > 70%. Prothrombin complex (PPSB, Beriplex, Behring, Marburg, Germany) was substituted (mean dose 2304 +/- 289 IU) to increase procoagulant activity to PT > 60%. Blood samples were drawn in the same fashion as in the AT III group. The amounts of AT III and PPSB concentrates (delta AT III, [IU/kg]; delta PPSB [IU/kg]) required to increase AT III activity and PT, respectively, by 1% were calculated. RESULTS: Standard calculations for AT III substitution indicated AT III recovery in all 37 patients who received AT III concentrate. However, there was a statistically significant difference between patients with CLF and ALF. In patients with CLF, delta AT III was found to be 0.8 IU/kg (+/- 0.1 SEM) and in those with ALF it was 1.5 IU/kg (+/- 0.1 SEM) (P < 0.05, t-test). In patients treated with PPSB concentrate, delta PPSB was 1.6 U/kg (+/- 0.2 SEM) for both CLF and ALF groups. CONCLUSIONS: In patients with end-stage liver disease standard rules for substitution with AT III-concentrate are adequate only for patients with CLF. In patients with ALF higher AT III doses are required to achieve the expected effect on endogenous AT III activity. Procoagulant activity, as reflected by PT, can be increased by 1% when 1.6 IU/kg PPSB concentrate is given. However, this study shows the effects of coagulation concentrates only 30 min after administration. An increased volume of distribution and increased turnover may explain the poor recovery of AT III activity in the ALF group, indicating that the dose of coagulation concentrate should be estimated against the background of the patient's clinical symptoms and diagnosis.