[Value of stem cell therapy for the treatment of stress incontinence. Current status and perspectives]. / Stellenwert der Stammzelltherapie für die Behandlung der Belastungsinkontinenz. Aktueller Stand und Ausblick.

Parallel to a fundamental change in the therapeutic approach to managing stress incontinence, an increasing number of patients ask for reconstruction of the outer, striated urethral sphincter as therapy for urinary stress incontinence. Regenerative medicine is starting to offer solutions using stem cells as a part of oncological therapy or in reconstructive surgery. In addition to the many auspicious experimental approaches, one published study reports the effective therapeutic use of myogenic stem cells in urinary stress incontinent patients. Before this procedure is adopted into general clinical practice, further studies with validated evaluations and a sound legal basis are needed.

[Tissue engineering and stem cell research in urology for a reconstructive or regenerative treatment approach]. / "Tissue engineering" und Stammzellforschung in der Urologie für den rekonstruktiven bzw. regenerativen Therapieansatz.

With the involvement of clinical reconstructive urology in the field of tissue engineering, outstanding results have been achieved in basic research as well as in some clinics. Stem cell research has even opened up possibilities for regenerative aspects. In close cooperation with various disciplines, the Department of Urology at the University of Tübingen investigates different clinical aspects with regard to reconstructive and regenerative urology. The regeneration of the external urethral sphincter requires functionally integrated muscle cells. In addition stricture reconstruction with multilayer urothelium should become less invasive and the re-stricture rate reduced. After the application of differentiating stem cells was proven, the clinical setting needed to be set for legal issues. In addition to the specification of culture media and verification in the animal model, the possibility to harvest omnipotent stem cells out of human testis and to differentiate those into the three germ layers was demonstrated. With the reduced invasiveness of harvesting the urothelium cells by a bladder wash using specific culture fluids, the cell culture was significantly improved enabling successful creation of urothelium by stratification. In addition urothelial cells in a matrix are further improved for endoscopic application. The close cooperation of different disciplines shortens the time to develop therapeutic approaches with a close clinical relationship in reconstructive and regenerative urology.

New molecules for hippocampal development.

Pathfinding by developing axons towards their proper targets is an essential step in establishing appropriate neuronal connections. Recent work involving cell culture assays and molecular biology strategies, including knockout animals, strongly indicates that a complex network of guidance signals regulates the formation of hippocampal connections during development. Outgrowing axons are routed towards the hippocampal formation by specific expression of long-range cues, which include secreted class 3 semaphorins, netrin 1 and Slit proteins. Local membrane- or substrate-anchored molecules, such as ligands of the ephrin A subclass, provide layer-specific positional information. Understanding the molecular mechanisms that underlie axonal guidance during hippocampal development might be of importance in making therapeutic use of sprouting fibers, which are produced following the loss of afferents in CNS lesion.

A role for the Eph ligand ephrin-A3 in entorhino-hippocampal axon targeting.

Neurons of layers II and III of the entorhinal cortex constitute the major afferent connection of the hippocampus. The molecular mechanisms that target the entorhinal axons to specific layers in the hippocampus are not known. EphA5, a member of the Eph receptor family, which has been shown to play critical roles in axon guidance, is expressed in the entorhinal cortex, the origin of the perforant pathway. In addition, ligands that interact with EphA5 are expressed in distinct hippocampal regions during development of the entorhino-hippocampal projection. Of these ligands, ephrin-A3 mRNA is localized both in the granular cell layer of the dentate gyrus and in the pyramidal cell layer of the cornu ammonis, whereas ephrin-A5 mRNA is only expressed in the pyramidal cell layer of the cornu ammonis. In the dentate gyrus, the ligand protein is not present in the termination zone of the entorhinal efferents (the outer molecular layer of the dentate gyrus) but is concentrated in the inner molecular layer into which entorhinal efferents do not grow. We used outgrowth and stripe assays to test the effects of ephrin-A3 and ephrin-A5 on the outgrowth behavior of entorhinal axons. This functional analysis revealed that entorhinal neurites were repelled by ephrin-A3 but not by ephrin-A5. These observations suggest that ephrin-A3 plays an important role in the layer-specific termination of the perforant pathway and that this ligand may interact with the EphA5 receptor to restrict entorhinal axon terminals in the outer molecular layer of the dentate gyrus.

Glycogen synthase kinase 3beta links neuroprotection by 17beta-estradiol to key Alzheimer processes.

Estrogen exerts many of its receptor-mediated neuroprotective functions through the activation of various intracellular signal transduction pathways including the mitogen activating protein kinase (MAPK), phospho inositol-3 kinase and protein kinase C pathways. Here we have used a hippocampal slice culture model of kainic acid-induced neurotoxic cell death to show that estrogen can protect against oxidative cell death. We have previously shown that MAPK and glycogen synthase kinase-3beta (GSK-3beta) are involved in the cell death/cell survival induced by kainic acid. In this model and other cellular and in vivo models we have shown that estrogen can also cause the phosphorylation and hence inactivation of GSK-3beta, a known mediator of neuronal cell death. The effect of estrogen on GSK-3beta activity is estrogen receptor mediated. Further, this estrogen/GSK-3beta interaction may have functional consequences in cellular models of some key pathogenic pathways associated with Alzheimer's disease. More specifically, estrogen affects the basal levels of tau phosphorylation at a site known to be phosphorylated by GSK-3beta. Taken together, these data indicate a novel molecular and functional link between estrogen and GSK-3beta and may have implications for estrogen receptor modulation as a target for the prevention of neurodegenerative disorders.

Glucocorticoids enhance oxidative stress-induced cell death in hippocampal neurons in vitro.

In patients with Alzheimer's disease, hippocampal cells are among the first neuronal cells of the brain to degenerate. Both rat primary hippocampal neurons and cells of the clonal mouse hippocampal cell line HT22 express endogenous functional glucocorticoid receptors (GRs), as shown by transient transfection of cells with a luciferase reporter plasmid containing GR-responsive elements. The influence of activated GRs on oxidative stress-induced neuronal cell death in vitro was investigated employing these hippocampal model systems. Two oxidative stressors were investigated, the free radical-inducing Alzheimer's disease-associated amyloid beta-protein, which is toxic to hippocampal neurons, and the excitatory amino acid glutamate, which induces oxidative cell death in HT22 cells via an increase in intracellular peroxides. Cellular viability was assessed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide test and trypan exclusion staining, followed by microscopical cell counting. Glucocorticoids strongly increased the vulnerability of the hippocampal cells to amyloid beta-protein and glutamate. This increase could be blocked by the specific GR antagonist RU486. Our data suggest that changes in hippocampal GR homeostasis and regulation may render hippocampal neurons more vulnerable to oxidative stress-induced neuronal degeneration.

Corticotropin-releasing hormone receptor (type I) antisense targeting reduces anxiety.

Two brain-derived corticotropin-releasing hormone receptors have been cloned, termed corticotropin-releasing hormone receptors type I and type 2. Antisense oligodeoxynucleotides targeted to the cloned rat and mouse corticotropin-releasing hormone receptors type I messenger RNA reduced the binding of the natural ligand of the corticotropin-releasing hormone receptors type I and also the release of adenocorticotrophic hormone in primary rat anterior pituitary cells and in clonal mouse pituitary cells (AtT-20) by up to 60% in an application time-dependent manner. Studies on intracellular uptake of fluorescence-labelled oligodeoxynucleotides indicated a cytoplasmic accumulation starting within two to four hours after application of oligodeoxynucleotides in vitro. In vivo, antisense oligodeoxynucleotides infused intra-cerebroventricularly reduced binding of radiolabelled corticotropin-releasing hormone receptors in central sites of the rat brain. Anxiety induced by i.c.v. administration of corticotropin-releasing hormone was attenuated by corticotropin-releasing hormone receptors type I antisense treatment as determined in the elevated plus maze and in the novel open field test. The corticotropin-releasing hormone-induced behavioural changes were absent in corticotropin-releasing hormone receptors type I antisense-pretreated animals. These results show that the selected antisense probes used were able to suppress corticotropin-releasing hormone receptors type I function in vitro as well as in vivo and suggest that the development of drugs blocking this specific receptor might lead to a novel class of anxiolytics.

Localization of tyrosine hydroxylase mRNA in the axons of the hypothalamo-neurohypophysial system.

With in situ hybridization we examined the localization of mRNA coding for tyrosine hydroxylase (TH) in the rat hypothalamo-neurohypophysial system (HNS) under conditions of acute osmotic stress. Fifteen min after salt loading, hybridization signal of TH mRNA could be located in the magnocellular hypothalamic nuclei and in the median eminence (ME). In untreated animals, TH mRNA was detected only in the ME. In osmotically challenged animals that had been pretreated with colchicine, signals for TH mRNA remained confined to the ME, while pretreatment of salt loaded rats with a polymerase II transcription inhibitor resulted in labelling of the magnocellular perikarya but a decrease of the hybridization signal in the ME. Our results suggest that also TH mRNA is among the RNAs which are axonally transported in the HNS. TH mRNA can probably be stored in axons of the hypothalamo-neurohypophysial tract, to be transported retrogradely and translated upon certain stimuli.

Molecular and cellular analysis of rP1.B in the rat hypothalamus: in situ hybridization and immunohistochemistry of a new P-domain neuropeptide.

P-domain peptides, a new family of secretory polypeptides, have been identified mainly in the gastroenteropancreatic tract of humans, rodents, and amphibians as well as in amphibian skin. In the present study, with PCR and RNA analysis a transcript has been discovered in rat brain termed rP1.B. The deduced polypeptide consists of a single P-domain and its amino acid sequence matches that of rat intestinal trefoil factor (rITF). Thus far, rP1.B is the only P-domain peptide expressed in neuronal cells of the CNS. Immunostained magnocellular perikarya were visible in the paraventricular, supraoptic and periventricular nuclei. Parvocellular rP1.B neurons were found in the arcuate nucleus. Additionally, specific hybridization signals with radiolabeled transcripts were observed in the same regions. rP1.B in the rat hypothalamus may be involved in the control of hypothalamo-hypophysial functions.

Vasopressin antisense oligonucleotide induces temporary diabetes insipidus in rats.

The purpose of this study was to downregulate the transcriptional message of arginine vasopressin (AVP) by antisense treatment. A complete phosphorothioate antisense oligodesoxynucleotide corresponding to the beginning of the coding region of rat AVP mRNA was constructed and injected into the lateral ventricle of rats. Within 3-6 h animals exhibited a temporary diabetes insipidus, which lasted up to 9 h. Accordingly, vasopressin immunoreactivity in the hypothalamic nuclei was reduced. Our results demonstrate that a specific and reversible inhibition of neuropeptide expression can be accomplished in the intact hypothalamo-neurohypophysial system by antisense treatment, thus providing a novel tool for studies on stimulus-secretion coupling in vivo.

Haloperidol-induced cell death--mechanism and protection with vitamin E in vitro.

Haloperidol, a dopamine receptor antagonist and sigma-receptor-active neuroleptic drug, is cytotoxic to primary hippocampal neurones, C6 glioma cells and NCB20 cells. A 24 h challenge of these cells with haloperidol resulted in reduced cell viability and ultimately cell lysis. The most dramatic changes in cellular morphology were the retraction of cellular extensions, development of membrane blebs, and finally cell detachment from the culture dish. DNA isolated from haloperidol-treated cells was randomly degraded, indicating a necrotic rather than an apoptotic pathway of cell death. Vitamin E (alpha-tocopherol), a lipophilic free radical scavenger, prevented haloperidol-induced DNA fragmentation and ultimately cell death. These findings suggest that haloperidol induces necrotic cell death in which free radicals play a major role.

Melatonin prevents oxidative stress-induced cell death in hippocampal cells.

The pineal hormone melatonin has been suggested to be a very effective antioxidant in the brain. Oxidative stress may play a role in certain neuropathological conditions and the hippocampus is an early target of neurodegeneration. Using cells of the clonal hippocampal cell line HT22 and organotypic hippocampal rat brain slice cultures, we investigated a possible protective role of melatonin against oxidative stress-induced hippocampal cell death. Glutamate alone induced oxidative apoptotic cell death in HT22 cells as detected with Hoechst staining and DNA fragmentation analysis. Preincubation with 1 mM melatonin protected HT22 cells against glutamate-induced cell death and protected organotypic hippocampal slices against H2O2-induced cell death. These findings suggest that this neurohormone may be useful in the prevention of neurodegenerative diseases.

Corticotropin-releasing hormone (CRH) antisense oligodeoxynucleotide induces anxiolytic effects in rat.

Antisense oligodeoxynucleotide complementary to the start coding region of rat corticotropin releasing hormone (CRH) mRNA was intracerebroventricularly infused into rats three times at 12-h intervals. In the shuttle-box avoidance procedure antisense-treated rats showed, within 6 h, significant acceleration and increase in the total number of discriminative avoidance responses compared with controls, treated with the corresponding sense probe or vehicle alone. Following the shuttle-box experiment hypothalamic CRH hybridization signals and immunoreactivity were reduced, while CRH immunoreactivity in the median eminence remained unchanged. Plasma ACTH and corticosterone were decreased in antisense-treated animals. It is likely that in addition to a selective blockade of CRH translation, antisense treatment may also interrupt secretion of CRH. Antisense targeting of the hypothalamo-hypophysial-adrenal axis may provide new strategies for the neuropharmacology of affective disorders.

Activation of c-Fos contributes to amyloid beta-peptide-induced neurotoxicity.

Amyloid beta peptide, a major component of Alzheimer's disease plaques, is directly toxic to various neuronal cell lines and primary neurons in culture. The mechanism underlying A beta neurotoxicity may include an increase in intracellular calcium and reactive oxygen species. In the present study, exposure of a mouse hippocampal cell line (HT-22) to the 25-35 peptide fragment of A beta (10 microM) caused a rapid and sustained increase in nuclear c-Fos immunoreactivity. Inhibition of A beta-mediated c-Fos activation by c-fos antisense oligodeoxynucleotides (5 microM) significantly protected against A beta toxicity as assessed by MTT assay. The signal transduction pathway for c-fos induction remains speculative, however, there seems to be a causal relationship between c-Fos transcription factor and A beta toxicity.

Entorhinal cortex lesion studied with the novel dye fluoro-jade.

We used the fluorescent dye Fluoro-Jade, capable of selectively staining degenerating neurons and their processes, in order to analyze degenerative effects of transecting the hippocampus from its main input, the entorhinal cortex in vivo and in organotypical hippocampal slice culture. Degenerating fibers stained with Fluoro-Jade were present as early as 1 day postlesion in the outer molecular layer of the dentate gyrus and could be detected up to 30 days postlesion. However, the intensity of the Fluoro-Jade staining in the outer molecular layer faded from postlesional day 20 onward. Punctate staining, various cells and neural processes became visible in this area suggesting that degenerating processes were phagocytosed by microglial cells or astrocytes. We conclude that Fluoro-Jade is an early and sensitive marker for studying degenerating neurites in the hippocampal system.

Centrally administered oligodeoxynucleotides in rats: occurrence of non-specific effects.

We studied the effects of various intracerebroventricularly administered oligodeoxynucleotides on body temperature, locomotor activity, food intake and water consumption in rats during a 24 h period with a radio-telemetric system. Both complete phosphorothioate oligodeoxynucleotides and end-inverted oligodeoxynucleotides dose-dependently elevated body temperature, suppressed food and fluid intake and inhibited nighttime activity. Apparently these effects do not depend on the nucleotide sequence because antisense and sense arginine vasopressin and oxytocin oligodeoxynucleotides, as well as a missense oligodeoxynucleotide produced comparable changes in the autonomous and behavioral parameters. In control experiments neither contaminants from the chemical synthesis nor endotoxins produced such effects, whereas native DNA from salmon sperm did. Fever and sickness-like behavior in response to missense phosphorothioate oligodeoxynucleotides were accompanied by elevated concentrations of circulating corticosterone and by a marked increase in interleukin 6 mRNA in brain and spleen, indicating that centrally administered oligodeoxynucleotides stimulate the production of pyrogenic inflammatory mediators in both central nervous system and peripheral tissues. Our results indicate that centrally administered oligodeoxynucleotides produce beside their intended sequence-specific effects also transient and sequence-independent effects due to their nucleic acid structure.

Ventral tegmental area (VTA) injections of tyrosine hydroxylase phosphorothioate antisense oligonucleotide suppress operant behavior in rats.

Dopaminergic A 10 neurons are known to be the essential part of the brain reinforcement system. An antisense oligonucleotide corresponding to the start coding region of rat tyrosine hydroxylase (TH) mRNA, the transcriptional message of the rate limiting enzyme in the metabolic pathway leading to catecholamine synthesis, was constructed and injected into the ventral tegmental area (VTA). 36 h after injection operant behavior was markedly reduced, and this suppression was fully reversed within 5 days following the antisense injection. Accordingly, TH immunoreactivity in the VTA was reduced in comparison to control experiments using mixed bases oligonucleotides. Our results demonstrate that highly specific inhibition of TH expression can be accomplished in the intact mesolimbic system by antisense treatment, thus providing a novel tool for studies on motivational processes in vivo.

Antisense oligodeoxynucleotide complementary to oxytocin mRNA blocks lactation in rats.

The posterior lobe peptide oxytocin (OT) is known to control lactation and parturition, as well as maternal and sexual behavior. An antisense oligodeoxynucleotide (ODN) directed against the mRNA of OT was injected intracerebroventricularly 6 times in 12 hour intervals to manipulate the transcriptional message of OT in lactating rats. OT immunoreactivity in magnocellular hypothalamic nuclei and in the posterior lobe of the pituitary was reduced in antisense treated animals in comparison to ODN with scrambled base composition and vehicle controls. This decline in OT levels was associated with a decrease of pup weight. Our results demonstrate that central infusions of antisense ODN significantly reduce OT expression in vivo.