The differences in the anatomy of the thoracolumbar and sacral autonomic outflow are quantitative.

PURPOSE: We have re-evaluated the anatomical arguments that underlie the division of the spinal visceral outflow into sympathetic and parasympathetic divisions. METHODOLOGY: Using a systematic literature search, we mapped the location of catecholaminergic neurons throughout the mammalian peripheral nervous system. Subsequently, a narrative method was employed to characterize segment-dependent differences in the location of preganglionic cell bodies and the composition of white and gray rami communicantes. RESULTS AND CONCLUSION: One hundred seventy studies were included in the systematic review, providing information on 389 anatomical structures. Catecholaminergic nerve fibers are present in most spinal and all cranial nerves and ganglia, including those that are known for their parasympathetic function. Along the entire spinal autonomic outflow pathways, proximal and distal catecholaminergic cell bodies are common in the head, thoracic, and abdominal and pelvic region, which invalidates the "short-versus-long preganglionic neuron" argument. Contrary to the classically confined outflow levels T1-L2 and S2-S4, preganglionic neurons have been found in the resulting lumbar gap. Preganglionic cell bodies that are located in the intermediolateral zone of the thoracolumbar spinal cord gradually nest more ventrally within the ventral motor nuclei at the lumbar and sacral levels, and their fibers bypass the white ramus communicans and sympathetic trunk to emerge directly from the spinal roots. Bypassing the sympathetic trunk, therefore, is not exclusive for the sacral outflow. We conclude that the autonomic outflow displays a conserved architecture along the entire spinal axis, and that the perceived differences in the anatomy of the autonomic thoracolumbar and sacral outflow are quantitative.

Are live lectures a discontinued model? A survey on the influence of synchronous online lecturing on the perception of teaching and assessment outcome.

Objectives: In the early phase of their studies, students are confronted with a number of teaching and learning methods they are usually not familiar with. Beyond, learning in a university environment requires a high degree of self-organization. Thus, the transition from learning in a school environment to university can be challenging for students and associated with adjustment difficulties. We hypothesized that synchronous online lecturing might be able to serve as a thematic superstructure and a curricular guide that can positively influence course perception, motivation and exam outcome. Methods: We investigated this hypothesis in a retrospective approach by comparing results from histology exams (2020 n=411, 2021 n= 423) and questionnaires for course evaluation received from medical and dentistry second semester students of the RWTH Aachen University, Germany, in 2020 (n=113 questionnaire participants) and 2021 (n=106 questionnaire participants). While in 2020, due to the Corona Pandemic, no synchronous online lectures were held, these were reintroduced in 2021. Results: Our results show several differences in between the two study cohorts. Most important findings include a significantly (p<0.001) lower number of students that failed to pass or withdrew from the exam in 2021, an increased motivation to deal with the learning content (p<0.001) and a higher perceived quality of the study materials (p<0.001) in 2021. Conclusion: Our study indicates that synchronous online lectures can be an important tool to help students to accustom to new learning environments and to structure private study. Further studies will now have to show whether live (online) lectures can have the same significance during clinical training.

Efficient 3D light-sheet imaging of very large-scale optically cleared human brain and prostate tissue samples.

The ability to image human tissue samples in 3D, with both cellular resolution and a large field of view (FOV), can improve fundamental and clinical investigations. Here, we demonstrate the feasibility of light-sheet imaging of ~5 cm3 sized formalin fixed human brain and up to ~7 cm3 sized formalin fixed paraffin embedded (FFPE) prostate cancer samples, processed with the FFPE-MASH protocol. We present a light-sheet microscopy prototype, the cleared-tissue dual view Selective Plane Illumination Microscope (ct-dSPIM), capable of fast 3D high-resolution acquisitions of cm3 scale cleared tissue. We used mosaic scans for fast 3D overviews of entire tissue samples or higher resolution overviews of large ROIs with various speeds: (a) Mosaic 16 (16.4 µm isotropic resolution, ~1.7 h/cm3), (b) Mosaic 4 (4.1 µm isotropic resolution, ~ 5 h/cm3) and (c) Mosaic 0.5 (0.5 µm near isotropic resolution, ~15.8 h/cm3). We could visualise cortical layers and neurons around the border of human brain areas V1&V2, and could demonstrate suitable imaging quality for Gleason score grading in thick prostate cancer samples. We show that ct-dSPIM imaging is an excellent technique to quantitatively assess entire MASH prepared large-scale human tissue samples in 3D, with considerable future clinical potential.

The innervation of the cervical facet joints-an anatomical and histological approach.

Cervical facet joint pain syndrome accounts for a great amount of cervical pain worldwide. This study aims to provide updated knowledge of cervical facet joint innervation with new anatomical findings. Twenty-seven cervical facet joints and their innervating structures were dissected from five halves of three human neck specimens. Histologic staining was used to confirm that the samples were nervous tissues, and all samples were documented with photography. Histology: Thirty-six assumed facet joint branch samples were obtained and stained. Twenty-two of these were confirmed to be nervous tissue. Therefore, 61% of the samples were identified as facet joint branches. Of all samples, 28% were not nerves. Dissection: At least one medial branch was clearly identified at each dissected cervical level. At some cervical levels, more than one medial branch was found. Anatomical differences, such as a plexus-like innervation in the high cervical region, were observed. Direct facet joint branches were also discovered. These branches originate directly from the dorsal root of the spinal nerve and were independent from medial branches during their direct pathway toward the facet joint. Direct cervical facet joint branches were identified and a more diverse innervation pattern than previously described of the cervical facet joints was found.

A unified 3D map of microscopic architecture and MRI of the human brain.

We present the first three-dimensional (3D) concordance maps of cyto- and fiber architecture of the human brain, combining histology, immunohistochemistry, and 7-T quantitative magnetic resonance imaging (MRI), in two individual specimens. These 3D maps each integrate data from approximately 800 microscopy sections per brain, showing neuronal and glial cell bodies, nerve fibers, and interneuronal populations, as well as ultrahigh-field quantitative MRI, all coaligned at the 200-µm scale to the stacked blockface images obtained during sectioning. These unprecedented 3D multimodal datasets are shared without any restrictions and provide a unique resource for the joint study of cell and fiber architecture of the brain, detailed anatomical atlasing, or modeling of the microscopic underpinnings of MRI contrasts.

Nerve fiber density differences in the temporal dura mater: An explanation for headache after temporal lobectomy? An anatomical study.

OBJECTIVE: Patients who undergo a temporal lobectomy for drug-resistant epilepsy more frequently complain about postoperative headache compared to patients who undergo a craniotomy in any other region. The pathophysiological mechanism is not well understood. It is hypothesized that a relatively high density of sensory nerve fibers in the temporomesial dura underlies a higher sensitivity to pain upon stimulation. The objective of this study was to address this hypothesis by comparing the nerve fiber density in the temporomesial dura to that in the temporolateral dura. METHODS: Temporomesial (n = 6) and temporolateral (n = 6) dura mater samples (2.5 × 2 cm) were dissected from the middle cranial fossa of 5 formalin fixed human cadavers. Paraffin embedded specimens were cut in a sagittal direction into 5 µm sections (temporomesial group n = 106, temporolateral group n = 113), and immunohistochemically stained for S100 as a marker of myelinated nerve fibers. The number of S100-immunoreactive nerve fiber bundles was counted in an anterior-posterior direction by a blinded observer, expressed as mean ± standard error of the mean per cm for each group, and statistically analyzed by a linear mixed-effects model. To assess potential observer bias, a randomized subset of the sections (n = 28) was evaluated by a second blinded observer and statistically analyzed by intraclass correlation coefficient (ICC). RESULTS: The temporomesial dura expressed 4.1 ± 2.1 and the temporolateral dura displayed 1.0 ± 0.7 nerve fiber bundles per cm (ß = 3.2, SE= 0.30, 95% CI [2.6, 3.8], p < 0.001). There is a significant decrease in nerve fiber bundle density in the mesial to lateral direction (mean difference -0.1, SE= 0.0, 95% CI [-0.1, -0.2], p < 0.001). The ICC was 0.69. CONCLUSIONS: The density of myelinated nerve fiber bundles is about 4 times higher in the temporomesial dura, than in the temporolateral dura. Assuming that dural innervation primarily consists of sensory trigeminal fibers, this observation suggests that a summation of stimuli to surpass the threshold to convey pain is reached sooner in the temporomesial than in the temporolateral dura mater.

7 Tesla MRI Followed by Histological 3D Reconstructions in Whole-Brain Specimens.

Post mortem magnetic resonance imaging (MRI) studies on the human brain are of great interest for the validation of in vivo MRI. It facilitates a link between functional and anatomical information available from MRI in vivo and neuroanatomical knowledge available from histology/immunocytochemistry. However, linking in vivo and post mortem MRI to microscopy techniques poses substantial challenges. Fixation artifacts and tissue deformation of extracted brains, as well as co registration of 2D histology to 3D MRI volumes complicate direct comparison between modalities. Moreover, post mortem brain tissue does not have the same physical properties as in vivo tissue, and therefore MRI approaches need to be adjusted accordingly. Here, we present a pipeline in which whole-brain human post mortem in situ MRI is combined with subsequent tissue processing of the whole human brain, providing a 3-dimensional reconstruction via blockface imaging. To this end, we adapted tissue processing procedures to allow both post mortem MRI and subsequent histological and immunocytochemical processing. For MRI, tissue was packed in a susceptibility matched solution, tailored to fit the dimensions of the MRI coil. Additionally, MRI sequence parameters were adjusted to accommodate T1 and T2∗ shortening, and scan time was extended, thereby benefiting the signal-to-noise-ratio that can be achieved using extensive averaging without motion artifacts. After MRI, the brain was extracted from the skull and subsequently cut while performing optimized blockface imaging, thereby allowing three-dimensional reconstructions. Tissues were processed for Nissl and silver staining, and co-registered with the blockface images. The combination of these techniques allows direct comparisons across modalities.

Scalable Labeling for Cytoarchitectonic Characterization of Large Optically Cleared Human Neocortex Samples.

Optical clearing techniques and light sheet microscopy have transformed fluorescent imaging of rodent brains, and have provided a crucial alternative to traditional confocal or bright field techniques for thin sections. However, clearing and labeling human brain tissue through all cortical layers and significant portions of a cortical area, has so far remained extremely challenging, especially for formalin fixed adult cortical tissue. Here, we present MASH (Multiscale Architectonic Staining of Human cortex): a simple, fast and low-cost cytoarchitectonic labeling approach for optically cleared human cortex samples, which can be applied to large (up to 5 mm thick) formalin fixed adult brain samples. A suite of small-molecule fluorescent nuclear and cytoplasmic dye protocols in combination with new refractive index matching solutions allows deep volume imaging. This greatly reduces time and cost of imaging cytoarchitecture in thick samples and enables classification of cytoarchitectonic layers over the full cortical depth. We demonstrate application of MASH to large archival samples of human visual areas, characterizing cortical architecture in 3D from the scale of cortical areas to that of single cells. In combination with scalable light sheet imaging and data analysis, MASH could open the door to investigation of large human cortical systems at cellular resolution and in the context of their complex 3-dimensional geometry.

Innervation of the human spleen: A complete hilum-embedding approach.

INTRODUCTION: The spleen is hypothesized to play a role in the autonomic nervous system (ANS)-mediated control of host defence, but the neuroanatomical evidence for this assumption rests on a sparse number of studies, which mutually disagree with respect to the existence of cholinergic or vagal innervation. METHODS: We conducted an immuno- and enzyme-histochemical study of the innervation of the human spleen using a complete hilum-embedding approach to ensure that only nerves that entered or left the spleen were studied, and that all splenic nerves were included in the sampled area. Furthermore, a complete embedded spleen was serially sectioned to prepare a 3D reconstruction of the hilar nerve plexus. RESULTS: All detected nerves entering the spleen arise from the nerve plexus that surrounds branches of the splenic artery and are catecholaminergic. Inside the spleen these nerves continue within the adventitia of the white pulpal central arteries and red pulpal arterioles. Staining for either choline acetyltransferase or acetylcholinesterase did not reveal any evidence for cholinergic innervation of the human spleen, irrespective of the type of fixation (regularly fixed, fresh-frozen post-fixed or fresh-frozen cryoslides). Furthermore, no positive VIP staining was observed (VIP is often co-expressed in postganglionic parasympathetic nerves). CONCLUSION: Our comprehensive approach did not produce any evidence for a direct cholinergic (or VIP-ergic) innervation of the spleen. This finding does not rule out (indirect) vagal innervation via postganglionic non-cholinergic periarterial fibres.

The human phrenic nerve serves as a morphological conduit for autonomic nerves and innervates the caval body of the diaphragm.

Communicating fibres between the phrenic nerve and sympathetic nervous system may exist, but have not been characterized histologically and immunohistochemically, even though increased sympathetic activity due to phrenic nerve stimulation for central sleep apnoea may entail morbidity and mortality. We, therefore, conducted a histological study of the phrenic nerve to establish the presence of catecholaminergic fibres throughout their course. The entire phrenic nerves of 35 formalin-fixed human cadavers were analysed morphometrically and immunohistochemically. Furthermore, the right abdominal phrenic nerve was serially sectioned and reconstructed. The phrenic nerve contained 3 ± 2 fascicles in the neck that merged to form a single fascicle in the thorax and split again into 3 ± 3 fascicles above the diaphragm. All phrenic nerves contained catecholaminergic fibres, which were distributed homogenously or present as distinct areas within a fascicle or as separate fascicles. The phrenicoabdominal branch of the right phrenic nerve is a branch of the celiac plexus and, therefore, better termed the "phrenic branch of the celiac plexus". The wall of the inferior caval vein in the diaphragm contained longitudinal strands of myocardium and atrial natriuretic peptide-positive paraganglia ("caval bodies") that where innervated by the right phrenic nerve.

Patient-Specific Topographic Anatomy of the Deep Circumflex Iliac Artery Flap: Comparing Standard and Modified Computed Tomographic Angiography.

PURPOSE: Computed tomographic angiography (CTA) is reported to give insight into patient-specific anatomy of the flap pedicle preoperatively. We compared information available from standard CTA (s-CTA) with that gained by modifying the conventional CTA technique (modified CTA [m-CTA]). Dissected cadavers served as the control group. MATERIALS AND METHODS: We evaluated 16 s-CTA scans (32 deep circumflex iliac arteries [DCIAs]) and 12 m-CTA scans (17 DCIAs) using 3-dimensional software (Vesalius; ps-medtech, Amsterdam, The Netherlands). We dissected 17 cadavers (n = 34 DCIAs) to serve as the control group. The positions of 4 landmarks (anterior superior iliac spine, origin of DCIA, origin of ascending branch, and crossing of horizontal branch and iliac crest) were defined in a 3-dimensional coordinate system. RESULTS: We found significant differences concerning the distances from the origin of the DCIA to the femoral bifurcation (P < .05) and the anterior superior iliac spine to the crossing point of the horizontal branch with the iliac crest (P < .05) between CTA scans and cadaveric studies. The imaging quality of the m-CTA scans was shown to be more consistent than and superior to that of the s-CTA scans. The visible length of the DCIA was longer on m-CTA scans (mean, 134.32 mm) than on s-CTA scans (mean, 73.62 mm). We could evaluate the branching off of perforators and the relation of the pedicle to the surrounding bone and soft tissue in more detail on m-CTA scans. Standard CTA allowed the bilateral evaluation of the pedicle, whereas m-CTA allowed the evaluation of the injected side only. CONCLUSIONS: The quality and quantity of information available from CTA could be improved by modifying the s-CTA examination by injection as close as possible to the target vessel. Standard CTA delivered information about both sides, whereas m-CTA may need an additional injection for contralateral-side imaging.

Ultra-High Field MRI Post Mortem Structural Connectivity of the Human Subthalamic Nucleus, Substantia Nigra, and Globus Pallidus.

INTRODUCTION: The subthalamic nucleus, substantia nigra, and globus pallidus, three nuclei of the human basal ganglia, play an important role in motor, associative, and limbic processing. The network of the basal ganglia is generally characterized by a direct, indirect, and hyperdirect pathway. This study aims to investigate the mesoscopic nature of these connections between the subthalamic nucleus, substantia nigra, and globus pallidus and their surrounding structures. METHODS: A human post mortem brain specimen including the substantia nigra, subthalamic nucleus, and globus pallidus was scanned on a 7 T MRI scanner. High resolution diffusion weighted images were used to reconstruct the fibers intersecting the substantia nigra, subthalamic nucleus, and globus pallidus. The course and density of these tracks was analyzed. RESULTS: Most of the commonly established projections of the subthalamic nucleus, substantia nigra, and globus pallidus were successfully reconstructed. However, some of the reconstructed fiber tracks such as the connections of the substantia nigra pars compacta to the other included nuclei and the connections with the anterior commissure have not been shown previously. In addition, the quantitative tractography approach showed a typical degree of connectivity previously not documented. An example is the relatively larger projections of the subthalamic nucleus to the substantia nigra pars reticulata when compared to the projections to the globus pallidus internus. DISCUSSION: This study shows that ultra-high field post mortem tractography allows for detailed 3D reconstruction of the projections of deep brain structures in humans. Although the results should be interpreted carefully, the newly identified connections contribute to our understanding of the basal ganglia.

Students' perceptions of anatomy across the undergraduate problem-based learning medical curriculum: a phenomenographical study.

BACKGROUND: To get insight in how theoretical knowledge is transformed into clinical skills, important information may arise from mapping the development of anatomical knowledge during the undergraduate medical curriculum. If we want to gain a better understanding of teaching and learning in anatomy, it may be pertinent to move beyond the question of how and consider also the what, why and when of anatomy education. METHODS: A purposive sample of 78 medical students from the 2nd, 3rd, 4th and 6th year of a PBL curriculum participated in 4 focus groups. Each group came together twice, and all meetings were recorded and transcribed verbatim. Data were analysed with template analysis using a phenomenographical approach. RESULTS: Five major topics emerged and are described covering the students' perceptions on their anatomy education and anatomical knowledge: 1) motivation to study anatomy, 2) the relevance of anatomical knowledge, 3) assessment of anatomical knowledge, 4) students' (in)security about their anatomical knowledge and 5) the use of anatomical knowledge in clinical practice. CONCLUSIONS: Results indicated that a PBL approach in itself was not enough to ensure adequate learning of anatomy, and support the hypothesis that educational principles like time-on-task and repetition, have a stronger impact on students' perceived and actual anatomical knowledge than the educational approach underpinning a curriculum. For example, students state that repetitive studying of the subject increases retention of knowledge to a greater extent than stricter assessment, and teaching in context enhances motivation and transfer. Innovations in teaching and assessment, like spiral curriculum, teaching in context, teaching for transfer and assessment for learning (rewarding understanding and higher order cognitive skills), are required to improve anatomy education.

Zona pellucida birefringence correlates with developmental capacity of bovine oocytes classified by maturational environment, COC morphology and G6PDH activity.

In the present study we aimed to analyse structural changes during in vitro maturation of the bovine zona pellucida (ZP) by scanning electron microscopy (SEM) ands zona pellucida birefringence (ZPB). Here we show that alterations during in vitro maturation invasively analysed by SEM are reflected in ZPB. In vivo-matured oocytes displayed significantly lower birefringence parameters and significantly higher blastocyst rates compared with in vitro-derived oocytes (39.1% vs 21.6%). The same was observed for in vitro-matured oocytes with cumulus-oocyte complex (COC) Quality 1 (Q1) compared with Q3-COCs with respect to zona birefringence and developmental capacity. Immature oocytes with Q1-COCs displayed higher ZPB values and a higher developmental capacity to the blastocyst stage (27.7% vs 16.9%) compared with immature Q3-COCs. Considering in vitro-matured oocytes, only those with Q1-COC showed a trend for ZPB similar to in vivo-matured oocytes. Therefore, a decreasing trend for ZPB during in vitro maturation seems to be typical for high-quality oocytes and successful cytoplasmic maturation. In accordance, fully-grown immature oocytes reached significantly higher blastocyst rates (32.0% vs 11.5%) and lower ZPB values compared with still-growing ones. In conclusion, we successfully evaluated the applicability of zona imaging to bovine oocytes: alterations during in vitro maturation invasively analysed by scanning electron microscopy were reflected in the birefringence of the zona pellucida of bovine oocytes affecting developmental capacity at the same value. Therefore ZPB measurement by live zona imaging has potential to become a new tool to assess correctness of in vitro maturation and to predict developmental competence.

Pulsed radiofrequency treatment adjacent to the lumbar dorsal root ganglion for the management of lumbosacral radicular syndrome: a clinical audit.

INTRODUCTION: Lumbosacral radicular syndrome (LRS) is probably the most frequent neuropathic pain syndrome. Three months to 1 year after onset, 30% of the patients still experience ongoing pain. The management of those patients is complex, and treatment success rates are rather low. The beneficial effect of pulsed radiofrequency (PRF) therapy has been described for the treatment of LRS in case reports and in retrospective and prospective studies. Up until now, no neurological complications have been reported after PRF treatment. The current clinical audit has been performed to assess the amount of pain relief after a single PRF treatment. METHODS: Sixty consecutive patients who received a PRF treatment adjacent to the lumbar dorsal root ganglion for the management of LRS in the period 2007-2009 were included. The main study objective was to measure the reduction of pain after the pulsed radiofrequency treatment by using the global perceived effect. The primary end point was defined as at least 50% pain relief for a period of 2 months or longer. RESULTS: The primary end point was achieved in 29.5% of all the PRF interventions. After 6 months, 50% pain relief was still present in 22.9% of the cases and after 12 months in 13.1% of the cases. The need for pain medication was significantly lower after pulsed radiofrequency treatment in the success group compared with the nonsuccess group. CONCLUSIONS: PRF treatment can be considered for the management of LRS patients. These results need to be confirmed in a randomized clinical trial.

Recommendations for reviewing a manuscript for the GMS Zeitschrift für Medizinische Ausbildung.

The aim of this paper, written by the committee of educational research methodology of the "Society for Medical Education" of the German-speaking countries, will give recommendations for the review process of scientific papers in medical education. The recommendations are based on the results of a workshop in 2007 and on a survey among reviewers of the journal GMS Z Med Ausbild. It reflects on international standards and research in medical education in Germany. The paper describes reviewer's function concerning the journal of GMS Z Med Ausbild and specifies criteria for the review process with regard to the editors and reviewers; it also gives proposals for a feedback to the author. The catalogue of criteria for the reviewers is pictured in a checklist. The present recommendations shall help to increase the quality of the review process and to improve the national and international acceptance of the journal GMS Z Med Ausbild. Additionally, transparency of the review processes will support authors to submit a scientific article of high quality.

Blended learning positively affects students' satisfaction and the role of the tutor in the problem-based learning process: results of a mixed-method evaluation.

Problem-based learning (PBL) is an established didactic approach in medical education worldwide. The impact of PBL depends on the tutors' quality and the students' motivation. To enhance students' motivation and satisfaction and to overcome the problems with the changing quality of tutors, online learning and face-to-face classes were systematically combined resulting in a blended learning scenario (blended problem-based learning--bPBL). The study aims at determining whether bPBL increases the students' motivation and supports the learning process with respect to the students' cooperation, their orientation, and more reliable tutoring. The blended PBL was developed in a cooperation of students and teachers. The well-established Seven Jump-scheme of PBL was carefully complemented by eLearning modules. On the first training day of bPBL the students start to work together with the online program, but without a tutor, on the final training day the tutor participates in the meeting for additional help and feedback. The program was evaluated by a mixed-method study. The traditional PBL course was compared with the blended PBL by means of qualitative and quantitative questionnaires, standardized group interviews, and students' test results. In addition log-files were analyzed. A total of 185 third-year students and 14 tutors took part in the study. Motivation, subjective learning gains and satisfaction achieved significantly higher ratings by the bPBL students compared with the students learning by traditional PBL. The tutors' opinion and the test results showed no differences between the groups. Working with the web-based learning environment was assessed as very good by the students. According to the log-file analysis, the web-based learning module was frequently used and improved the cooperation during the self-directed learning.

Cryopreservation of spermatozoa in alginic acid capsules.

OBJECTIVE: To develop a method of freezing small amounts of spermatozoa in polymerized alginic acid drops, which can be liquified after thawing for recovery of the spermatozoa. DESIGN: Prospective clinical study. SETTING: Medical School, RWTH Aachen, Aachen Germany. PATIENT(S): None. INTERVENTION(S): Validation of the encapsulation method with bovine sperm; cryopreservation of human spermatozoa in alginic capsules. MAIN OUTCOME MEASURE(S): We optimized the cryopreservation method by testing different parameters influencing the freezing procedure, such as concentration of alginic acid, size of drops, time of polymerization, and culture media. RESULT(S): The final protocol was as follows: encapsulation by 7.3 mg/mL alginic acid forming 10-muL drops polymerized for 30 seconds and liquefied for 2.5 minutes in sodium citrate. Cryopreservation of human spermatozoa by this protocol resulted in a decreased motility of 18.3% compared with standard protocols but a 19.9% higher vitality of the immotile spermatozoa. CONCLUSION(S): No difference in viability of spermatozoa after both sperm-freezing procedures could be observed. Further investigation will be undertaken to reduce the amount of immotile but viable sperm after microencapsulation in alginic acid.

Regulation of taurocholate transport in freshly isolated proximal tubular cells of the rat kidney by protein kinases.

BACKGROUND/AIMS: The bile acids filtered through the glomeruli nearly completely escape urinary excretion due to an efficient tubular reabsorption process. Reabsorption is mediated mainly by the sodium-dependent bile acid transporter (ASBT) which is located in the brush border membranes of proximal tubular cells. The present study addresses the question whether this transporter is subject to short-term regulation by protein kinases. METHODS: The effects of specific activators or inhibitors of eight different protein kinases (PKs) on 3H-taurocholate uptake of proximal tubular cells were investigated. The cells were freshly isolated from rat kidneys by nycodenz density gradient centrifugation. RESULTS: Activation of the cAMP/PKA system by forskolin, 8-Br-cAMP, or the cAMP phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine significantly diminished cellular 3H-taurocholate uptake whereas 8-Br-cGMP had no effect. Also the MEK1/2 inhibitors PD98059 and U0126, and the p38 mitogen-activated protein (MAP) kinase inhibitor SB203580 decreased 3H-taurocholate uptake. Phorbol myristate acetate and dioctanolglycerol, activators of PKC, and chelerythrine, a selective inhibitor of PKC, did not affect 3H-taurocholate uptake. Likewise the phosphatidylinositol-3 kinase inhibitor wortmannin and the tyrosine kinase inhibitor genistein induced no significant change of cellular 3H-taurocholate uptake. In a sodium-free medium forskolin and PD98059 did not affect 3H-taurocholate uptake but SB203580 significantly decreased it. CONCLUSION: It is concluded that PKA and MAP kinases are involved in the regulation of the ASBT-mediated taurocholate uptake into proximal tubular cells. p38 MAP kinase may have an additional effect on a sodium-independent tubular taurocholate transporter.