Klaus Redmann

Oxidative DNA damage in human sperm can be detected by Raman microspectroscopy

OBJECTIVE To determine whether Raman microspectroscopy can identify different levels of oxidative sperm nDNA damage and to corroborate the findings using an established method and an alternative but complementary spectroscopic technique. DESIGN Three-way comparison of Raman profiles, Fourier transform infrared spectroscopy (FTIR) spectra, and flow-cytometric assessments of sperm nDNA damage. SETTING University-based research laboratory. PATIENT(S) Thirty-eight men attending the infertility clinic at the Centre of Reproductive Medicine and Andrology. INTERVENTION(S) Induction of oxidative damage by Fentons reaction on semen samples. MAIN OUTCOME MEASURE(S) Raman profiles, FTIR spectra, and flow-cytometric analysis of DNA fragmentation. RESULT(S) Raman and FTIR spectra contained distinctive differences between untreated and fragmented nDNA sperm that were indicative of oxidative attack. The changes in Raman profiles were similar to those previously seen and corresponded to the DNA backbone. The peak attributions were corroborated by the FTIR spectra. Principal component analysis of the entire Raman spectra distinguished samples with varying degrees of damage. After determination of a cutoff value (0.63), estimation of the percentage of sperm with nDNA damage using the intensity ratio of Raman peaks (1,050/1,095 cm(-1)) correlated linearly to the flow-cytometric assessment. CONCLUSION(S) Raman microspectroscopy still requires further validation but may potentially provide a means of assessing the nDNA status of a living sperm.

Effects of the FSH receptor gene polymorphism p.N680S on cAMP and steroid production in cultured primary human granulosa cells

The study was designed to evaluate in vitro the cellular mechanisms of the single nucleotide polymorphism (SNP) p.N680S of the FSH receptor gene (FSHR) in human granulosa cells (GC) and included patients homozygous for the FSHR SNP (NN/SS) undergoing ovarian stimulation. GC were isolated during oocyte retrieval and cultured for 1–7 days. Basal oestradiol and progesterone concentrations were measured after short-term culture. The kinetics of cAMP, oestradiol and progesterone concentrations in response to various amounts of FSH were analysed in a 6–7 day culture. Basal oestradiol, but not progesterone, concentrations on day 1 of GC culture, were significantly higher in NN compared with SS (P = 0.045), but non-responsive to FSH stimulation. Immunofluorescence microscopy demonstrated the re-appearance of FSHR expression with increasing days in culture. Upon stimulation with FSH, GC cultured for 6–7 days displayed a dose-dependent increase of cAMP, oestradiol and progesterone but no difference in the EC50 values between both variants. Primary long-term GC cultures are a suitable system to study the effects of FSH in vitro. However, the experiments suggest that factors down-stream of progesterone production or external to GC might be involved in the clinically observed differences in an FSHR variant-mediated response to FSH.

Adriamycin-induced cardiomyopathy in the dog--an appropriate model for research on partial left ventriculectomy?

OBJECTIVE To evaluate the adriamycin-induced cardiomyopathy in the dog for research on partial left ventriculectomy (PLV). METHODS An intracoronary catheter was introduced into the left main stem via the first diagonal branch in a retrograde fashion in 6 adult FBI (Foxhound Boehringer Ingelheim) dogs weighing 30 to 35 kg. The catheter was connected to a percutaneous access port that was used for weekly adriamycin administration (10 mg over a 1-hour period for 5 times). Follow-up examinations (transthoracic echocardiography, hemodynamic parameters, cardiopulmonary status, and neurohormones) were done before, 1 week after the last adriamycin administration, and 6 weeks later. After the last measurements, all dogs were euthanized with saturated potassium chloride under general anesthesia and the hearts were excised for histologic examinations. All data were calculated as mean values and standard error of the mean. Differences were calculated by the Wilcoxon signed rank test for paired and unpaired data. p values less than 0.05 were considered significant. RESULTS Central venous pressure (2.2 +/- 0.8 vs 5.2 +/- 0.4 mm Hg, p = 0.03), mean pulmonary artery pressure (8.6 +/- 1.1 vs 12.4 +/- 0.5 mm Hg, p = 0.03), pulmonary wedge pressure (2.6 +/- 0.9 vs 7.0 +/- 0 mm Hg, p = 0.03), left ventricular endsystolic diameter (2.5 +/- 0.2 vs 3.1 +/- 0.4 cm, p = 0.03), and enddiastolic (4.5 +/- 0.2 vs 4.9 +/- 0.2 cm, p = 0.03) diameter increased significantly after adriamycin administration, whereas cardiac output (4.0 +/- 0.3 vs 3.3 +/- 0.1 liter/min, p = 0.03), stroke volume index (66.0 +/- 7.4 vs 54.0 +/- 3.9 ml/beat/m(2), p = 0.03), and ejection fraction (61.1 +/- 5.1 vs 37.7 +/- 5.7%, p = 0.03) decreased markedly. These changes were accompanied by a significant decline of oxygen delivery (1130 +/- 170 vs 790 +/- 65 ml/min, p = 0.03), which led to an enhanced oxygen extraction (0.12 +/- 0.01 vs 0.24 +/- 0.01, p = 0.03). Consequently, venous oxygen saturation (82.7 +/- 4.1 vs 71.3 +/- 2.5%, p = 0.03) decreased. Troponin I (0.02 +/- 0.025 vs 1.7 +/- 0.6 ng/ml, p = 0.03) and the anti-diuretic hormone (1.9 +/- 0.9 vs 20.0 +/- 1.9 pg/ml, p = 0.03) increased significantly after adriamycin administration. Deterioration of cardiac function continued after termination of adriamycin administration, albeit slower than during adriamycin administration. All hearts had severe histologic alterations, which were characteristic of adriamycin-induced toxicity: cytoplasmic vacuolation, myocyte degeneration, and increased fibrosis. CONCLUSIONS The adriamycin-induced cardiomyopathy in the dog is similar to the dilated cardiomyopathy in humans and may be an appropriate model for PLV.

Statistical Analysis of the Angle of Intrusion of Porcine Ventricular Myocytes from Epicardium to Endocardium Using Diffusion Tensor Magnetic Resonance Imaging

Pairs of cylindrical knives were used to punch semicircular slices from the left basal, sub‐basal, equatorial, and apical ventricular wall of porcine hearts. The sections extended from the epicardium to the endocardium. Their semicircular shape compensated for the depth‐related changing orientation of the myocytes relative to the equatorial plane. The slices were analyzed by diffusion tensor magnetic resonance imaging. The primary eigenvector of the diffusion tensor was determined in each pixel to calculate the number and angle of intrusion of the long axis of the aggregated myocytes relative to the epicardial surface. Arrays of axially sectioned aggregates were found in which 53% of the approximately two million segments evaluated intruded up to ±15°, 40% exhibited an angle of intrusion between ±15° and ±45°, and 7% exceeded an angle of ±45°, the positive sign thereby denoting an epi‐ to endocardial spiral in clockwise direction seen from the apex, while a negative sign denotes an anticlockwise spiral from the epicardium to the endocardium. In the basal and apical slices, the greater number of segments intruded in positive direction, while in the sub‐basal and equatorial slices, negative angles of intrusion prevailed. The sampling of the primary eigenvectors was insensitive to postmortem decomposition of the tissue. In a previous histological study, we also documented the presence of large numbers of myocytes aggregated with their long axis intruding obliquely from the epicardial to the endocardial ventricular surfaces. We used magnetic resonance diffusion tensor imaging in this study to provide a comprehensive statistical analysis. Anat Rec, 290:1413–1423, 2007.

Reassembly of somatic cells and testicular organogenesis in vitro

Testicular organogenesis in vitro requires an environment allowing a reassembly of testicular cell types. Previous in vitro studies using male murine germ cells cultured in a defined three-dimensional environment demonstrated tubulogenesis and differentiation into spermatozoa. Combining scaffolds as artificial culture substrates with testicular cell culture, we analysed the colonization of collagen sponges by rat testicular cells focusing on cell survival and reassembly of tubule-like-structures in vitro. Isolated testicular cells obtained from juvenile Sprague Dawley and eGFP transgenic rats were cultured on collagen sponges (DMEM high glucose+Glutamax, 35°C, 5% CO2 with or without gonadotropins). Live cell imaging revealed the colonization of cells across the entire scaffold for up to 35 days. After two days, histology showed cell clusters attached to the collagen fibres and displaying signs of tubulogenesis. Clusters consisted mainly of Sertoli and peritubular cells which surrounded some undifferentiated spermatogonia. Flow cytometry confirmed lack of differentiation as no haploid cells were detected. Leydig cell activity was detected by a rise of testosterone after gonadotropin stimulation. Our approach provides a novel method which is in particular suitable to follow the somatic testicular cells in vitro an issue of growing importance for the analysis of germ line independent failure of spermatogenesis.

High-frequency oscillation in an adult porcine model

Objective: Controversy exists as to whether high‐frequency oscillatory ventilation can be used on babies and small laboratory animals only, or whether high‐frequency oscillatory ventilation can also be efficient in the adult patient and large (>65 kg body weight) laboratory animals. Moreover, controversy exists as to whether limitations in high‐frequency oscillation efficiency are caused by the size and shape of the bronchial system, by the lack of low impedant intersegmental gas flow in lung parenchyma, or by inappropriate high‐frequency ventilators and ancillary hardware. Therefore, our objective in this study using the adult pig as a model of the adult patient was to test whether the adult airway system is suited to the use of high‐frequency oscillatory ventilation or whether there are geometrical, structural, or functional limitations to efficient ventilation by high‐frequency oscillation. Design: Prospective, controlled, randomized comparison over 8 to 16 hrs of ventilatory management. Setting: Experimental thoracovascular surgery laboratory in a university hospital. Subjects: Fifteen adult, female, house swine (weight 90 to 140 kg). Interventions: We evaluated the ventilatory effect of a wide range of oscillation frequencies (10–15 to 35–45 Hz), tidal volumes (0.5 to 2.2 mL/kg), and bias flow volumes (10 to 70 L/min) at a mean airway pressure of 12 ± 1 cm H2O in anesthetized and relaxed pigs who did not have lung injury. Measurements and Main Results: Arterial blood gases are mainly dependent on tidal volume, frequency, and mean airway pressure. A threshold bias flow volume of 35 ± 5 L/min is required to prevent CO2 rebreathing. In the group of lightweight animals (65 to 99 kg), the most efficient frequency band for CO2 elimination was ∽25 Hz. The most efficient frequency band for arterial oxygenation was found to vary between individuals more than the most efficient frequency band for CO2 elimination. In the group of heavy animals (100 to 140 kg), no most efficient mean frequency could be assessed, probably because the excitation system was limited. We confirmed that tidal volume on its own had an effect on CO2 elimination (“tidal‐volume effect”), although CO2 elimination was mainly determined by the product of tidal volume and oscillation frequency (oscillated minute volume), at least up to a critical frequency. Beyond that frequency, CO2 elimination could not be enhanced. The most efficient mean airway pressure in unimpaired lungs was assessed at 12 ± 1 cm H2O. Conclusions: Adult pigs with a body weight in the range of the weight of clinical adult patients can be ventilated by high‐frequency oscillation at tidal volumes smaller than, equal to, or slightly more than anatomical deadspace. The most efficient frequency for gas exchange varied between individuals. Tidal volume had an enhancing effect on CO2 elimination. The frequency dependency of Pao2 may have been related to a frequency‐dependent structural remodeling of the airway system, which occurred even though the mean airway pressure was kept constant. These results demonstrate that failure of adequate ventilation by high‐frequency oscillation is caused by a) CO2 rebreathing, b) the avoidance of an appropriate alveolar recruitment strategy, and c) an underpowered, high‐frequency ventilatory system (oscillator) that is unable to deliver appropriate pressure oscillations. These limitations led to insufficient CO2 elimination and/or inadequate arterial oxygenation. (Crit Care Med 1994; 22:S37‐S48)

Partial left ventriculectomy in modified adriamycin-induced cardiomyopathy in the dog.

BACKGROUND The purpose of this study was to evaluate modified adriamycin-induced cardiomyopathy in the dog for research on partial left ventriculectomy (PLV). METHODS An intracoronary catheter was introduced into the left main stem via the first marginal branch in a retrograde fashion in 12 adult foxhound dogs. The catheter was connected to a percutaneous access port that was used for weekly adriamycin administration (10 mg over a 1-hour period on 5 occasions). Follow-up examinations (transthoracic echocardiography, hemodynamic parameters, cardiopulmonary status, neurohormones) were done before, 1 week after the last adriamycin administration, and then 6 weeks later. This protocol was performed in 6 dogs (control group: Group 1). The other 6 dogs underwent PLV 1 week after the last adriamycin administration (Group 2). After the last measurements, all dogs were killed with saturated potassium chloride under general anesthesia and the hearts were excised for histologic examination. All data were calculated as mean and standard error of the mean. Differences were calculated by the Wilcoxon signed-rank test for paired and unpaired data. p < 0.05 was considered statistically significant. RESULTS One dog from each group died suddenly during adriamycin administration (probably due to ventricular arrhythmia). In addition, 1 dog from Group 2 suffered from a severe systemic inflammatory response syndrome after PLV and died 36 hours after surgery. Thus, 5 dogs from Group 1 and 4 from Group 2 underwent the entire study protocol. Adriamycin administration resulted in a severe dilated cardiomyopathy that was comparable in both groups (significant increase of central venous pressure, mean pulmonary artery pressure, pulmonary wedge pressure, left ventricular end-systolic and end-diastolic diameters, oxygen extraction, troponin I and anti-diuretic hormone, whereas cardiac output, ejection fraction and venous oxygen saturation decreased significantly). Deterioration of cardiac function continued after termination of adriamycin administration in Group 1 dogs, albeit not as progressively as during adriamycin administration. In contrast, cardiac function improved in Group 2 dogs after PLV, but did not reach baseline values. Cardiac index increased and oxygen extraction (p = 0.03) decreased, resulting in an enhanced venous oxygen saturation (p = 0.02). In particular, the distance of the papillary muscles at end diastole (p = 0.02) and at end systole (p = 0.02) at the mid-papillary level decreased significantly after PLV, resulting in reduced left ventricular diameter and volume (statistically significant for left ventricular end-systolic diameter and volume). All hearts had severe histologic alterations characteristic of adriamycin-induced toxicity, including cytoplasmic vacuolation, myocyte degeneration and increased fibrosis. CONCLUSION Modified adriamycin-induced cardiomyopathy in the dog may be suitable for research on PLV.

Immediate Effects of Partial Left Ventriculectomy on Left Ventricular Function

Abstract Background: Attempts to prolong life or to improve the quality of life by partial left ventriculectomy in patients suffering from dilated cardiomyopathy have yielded strikingly variable results in leading surgical centers. Hypothesis: The outcome of patients after partiat left ventriculectomy depends on intraoperative myocardial protection together with appropriate long‐term pharmacotherapy. We further assume that partial removal of the fibrotic ventricular wall may lead to a particularly inhomogeneous pattern of wall stress, giving rise to the potential of a paradoxical increase in wall stress and the creation of arrhythmogenic foci. Methods: During surgery in 24 patients, local mesh tension was measured using needle‐force probes in up to five sites within the left ventricular wall before and after resection of the interpapillary mural segment. The data were used to calculate regional peak developed force and to identify any differences in the timing of local mechanical activity between the measured regions. Results: Mean decrease in regional wall stress was 42% (76 sites of measurement). However, we discovered a paradoxicat increase of 42% in 18 sites of measurement. The time delay in the onset of force development between the measured regions prior to surgery was 0 msec in 10 patients, up to 30 msec in 7 patients, and beyond 80 msec in 7 patients. After resection, the time delay increased considerably in incidence and duration. Conclusion: Ventriculectomy is an effective means of reducing wall stress. The unexpectedly high incidence of inhomogeneities in wall stress after asymmetrical surgical ventricular remodeling, currently typical for the classical Batista procedure, together with the asynchronous regional ventricular function that we found to increase after partial left ventriculectomy, needs further elucidation by electrophysiological investigations.

Late ventricular structure after partial left ventriculectomy

Nine months after partial ventriculectomy, a 53-year-old man died of progressive heart failure. His heart was examined to determine the alignment of the muscle fibers around the ventricular scar, which was 11 cm long, 1.3 cm thick and 4 cm wide. The scar reached 2 to 12 mm beyond the surgical suture line. The fibers in the middle and subendocardial layers were malaligned, resulting in convergence, compression and regional necrosis.

Preoperative immunoglobulin treatment in patients with Werlhof’s disease undergoing cardiac operation

BACKGROUND Patients with Werlhofs disease and undergoing a cardiac surgical procedure with cardiopulmonary bypass are at increased risk for bleeding complications. We report the usefulness of preoperative immunoglobulin treatment in selected patients. METHODS Between May 1995 and July 1998, 10 patients with Werlhofs disease underwent a cardiac surgical procedure with cardiopulmonary bypass in our department. Five patients with mean platelet counts of less than 80x10(9)/L received immunoglobulin therapy preoperatively (group 1). The other 5 patients with mean platelet counts higher than 80x10(9)/L were not so treated (group 2). RESULTS In group 1, mean platelet count increased from 54x10(9)/L 5 days before operation to 112x10(9)/L after immunoglobulin treatment (p = 0.018) and did not fall to less than 60x10(9)/L postoperatively. Patients in group 1 received 16 units of packed red blood cells and 5 units of platelet concentrate. Patients in group 2 required 24 units of packed red blood cells, 5 units of platelet concentrate, and 23 units of fresh frozen plasma. Only 1 patient (group 2) had a surgical bleeding complication that required reexploration. Mean drainage loss was 1,100 mL in group 1 and 1,210 mL in group 2. CONCLUSIONS Our data demonstrate that immunoglobulin treatment of patients with Werlhofs disease and mean platelet counts of less than 80x10(9)/L significantly augments platelet counts preoperatively. It may be useful in selected patients.