Introduction: La circulation extracorporelle (CEC) peut entraîner une dysfonction endothéliale pulmonaire et l’hypertension pulmonaire. Le SN50 agit au niveau de la signalisation cellulaire pour prévenir ces réactions à la CEC et pourrait renverser la dysfonction endothéliale pulmonaire post-CEC sans effets néfastes sur l’hémodynamie. Méthodes: Quatre groups de porcs ont reçu un parmi quatre traîtements avant de subir 90 minutes de CEC et 60 minutes de reperfusion: (1) milrinone nébulisé; (2) sildenafil nébulisé; (3) placebo nébulisé; et (4) SN-50 intraveineux. Un monitoring hémodynamique invasif a été utilisé. La réactivité vasculaire des artères pulmonaires de deuxième ordre a été évaluée face à l’acétylcholine et la bradykinine. Résultats: Le sildénafil produit une augmentation significative de la pression de l’artère pulmonaire (PAP) moyenne à 60 minutes de reperfusion par rapport au début de la chirurgie. Les relaxations dépendantes de l’endothélium face à la bradykinine étaient meilleurs dans les groupes milrinone et SN-50 et surtout dans le groupe sildénafil par rapport au groupe placébo. Le SN-50 produisait de moins bonnes relaxations dépendantes de l’endothélium face à l’acétylcholine que les autres traitements incluant placébo. Conclusion: Le sildénafil prévient mieux la dysfonction endothéliale pulmonaire que les autres traitements. Les bénéfices du SN-50 sont possiblement sous-estimés vu que la dose n’a pas pu être ajustée à la durée de CEC. Le sildenafil inhalé mérite une étude plus importante chez l’humain et le SN-50 dans un model de CEC animal. / Background: Cardiopulmonary bypass (CPB) can lead to pulmonary endothelial dysfunction and consequent pulmonary hypertension. The novel agent SN-50 acts at the level of the transduction pathway to prevent these responses and may limit or reverse post-CPB pulmonary endothelial dysfunction and pulmonary hypertension without the untoward effects on hemodynamics seen with other known therapies. Methods: Four groups of Landrace-Yorkshire swine that received one of four treatments before undergoing 90 minutes of normothermic CPB and 60 minutes of reperfusion were compared: (1) Nebulized milrinone; (2) nebulized sildenafil; (3) placebo consisting of nebulized NaCl solution; and (4) intravenous SN-50. Invasive hemodynamic monitoring was used throughout all experiments. Vascular reactivity of second-degree pulmonary arteries was evaluated in response to acetylcholine and bradykinin. Results: Sildenafil produced a significant increase in mean pulmonary artery pressure (PAP) at 60 minutes after CPB compared to baseline. Both the sildenafil and milrinone groups had increased mean PAP/MAP ratio at 60 minutes after CPB compared to baseline, however this ratio was not different between the groups. Endothelial-dependent relaxations to bradykinin were improved in the SN-50 and milrinone groups and especially the sildenafil group as compared to placebo. SN-50 produced worse endothelium-dependent relaxations in response to acetylcholine compared to the other groups including placebo. Conclusion: Sildenafil better prevented pulmonary endothelial dysfunction than all other treatments. The improvements seen with SN-50 may be suboptimal as dose could not be titrated to length of CPB. Inhaled sildenafil and SN-50 both merit further study in human trials and animal models, respectively.

Os pacientes que usam lítio (Li) para tratamento do transtorno bipolar frequentemente apresentam poliúria e deficiência de concentração urinária, sintomas do Diabetes Insipidus Nefrogênico (DIN). Animais tratados com Li apresentam baixos níveis de produção de adenosina monofosfato cíclico (AMPc) em resposta ao hormônio antidiurético (HAD). O Sildenafil (Sil), um inibidor da fosfodiesterase 5 (PDE5), eleva os níveis intracelulares de guanosina monofosfato cíclico (GMPc), levando a inserção de aquaporina 2 (AQP2) na membrana plasmática das células do ducto coletor. Portanto, inibidores de PDE podem promover a inserção de AQP2 na membrana plasmática mesmo sem a ativação do receptor de HAD, indicando a participação de uma via alternativa mediada pelo GMPc. Nós investigamos as vias de ação do Sil no tratamento da DIN induzida pelo Li. Ratos Wistar foram divididos nos seguintes grupos: grupo controle, recebendo dieta alimentar normal durante quatro semanas; grupo Li, recebendo dieta alimentar normal com 40 mmol Li por quilo de dieta durante quatro semanas; grupo Li + Sil, recebendo dieta alimentar normal com 40 mmol Li por quilo de dieta durante quatro semanas e 200 mg por quilo de dieta de Sil a partir da segunda semana; grupo Sil, recebendo dieta alimentar normal durante a primeira semana e a partir da segunda semana recebendo dieta normal com 200 mg de Sil por quilo de dieta. Os animais do grupo Li desenvolveram poliúria, diminuição da osmolalidade urinária e diminuição da expressão da AQP2 tanto na fração citoplasmática como de membrana celular e o Sil reverteu essas alterações. Demonstramos ainda que a concentração de GMPc intracelular estava aumentada nos túbulos papilares tratados com Sil. Observamos que a provável via de fosforilação da AQP2 induzida pelo GMPc é pela PKA. Além disso, o tratamento com Sil aumenta a expressão de pCreb, fator de transcrição para ativação do gene da AQP2. Observamos ainda que o Li diminui a expressão de eNOS e o tratamento com Sil normaliza essa diminuição. Assim, concluímos que o tratamento com Sil em ratos com DIN melhora a poliúria aumentando a produção e a inserção de AQP2. O tratamento com Sil pode ser benéfico para pacientes que sofrem com DIN induzido pelo Li / Patients taking lithium to treat bipolar disorder often present polyuria and urinary concentrating defect. In addition, lithium-treated animals present lower cyclic adenosine monophosphate production in response to vasopressin. Sildenafil (Sil), a phosphodiesterase 5 (PDE5) inhibitor, elevates intracellular cyclic guanosine monophosphate (cGMP) levels, leading to plasma membrane accumulation of aquaporin 2 (AQP2). Therefore, PDE inhibitors might induce AQP2 membrane insertion even without vasopressin receptor activation by activating a parallel cGMP-mediated signal transduction pathway. We investigated Sil pathways of action in rats with lithium-induced nephrogenic Diabetes Insipidus (NDI). Wistar rats received lithium (40 mmol/kg food) or not for 4 weeks (Li or control), some rats also receiving sildenafil (200 mg/kg food) in weeks 2-4, with or without lithium (Li+Sil orSil). Animals in Li group developed polyuria, decreased urinary osmolality and decreased expression of AQP2 in both the cytoplasmic fraction and the cell membrane and Sil reversed these changes. We also demonstrated that intracellular cGMP concentration was increased in papillary tubules treated with Sil. We found that PKA may be involved in the pathway of cGMP induced AQP2 phosphorylation. In addition, Sil treatment increases Creb phosphorylation. Creb phosphorylation, acts as AQP2 gene transcription factor. We also observed that Li decreases eNOS expression and treatment with Sil normalizes this alteration. We conclude that Sil treatment improves polyuria by increasing production and insertion of AQP2. Sil treatment may be beneficial to patients suffering from induced DIN Li

The development of new therapeutical approaches, devised to foster the regeneration of neuronal circuits after injury and/or in neurodegenerative diseases, is of great importance. The impairment of dopaminergic projections is especially severe, because these projections are involved in crucial brain functions such as motor control, reward and cognition. In the work presented here, organotypic brain slice co-cultures of (a) the mesostriatal and (b) the mesocortical dopaminergic projection systems consisting of tissue sections of the ventral tegmental area/substantia nigra (VTA/SN), in combination with the target regions of (a) the striatum (STR) or (b) the prefrontal cortex (PFC), respectively, were used to evaluate different approaches to stimulate neurite outgrowth: (i) inhibition of cAMP/cGMP turnover with 3’,5’ cyclic nucleotide phosphodiesterase inhibitors (PDE-Is), (ii) blockade of calcium currents with nimodipine, and (iii) the co-cultivation with bone marrow-derived mesenchymal stromal/stem cells (BM-MSCs). The neurite growth-promoting properties of the tested substances and cell populations were analyzed by neurite density quantification in the border region between the two brain slices, using biocytin tracing or tyrosine hydroxylase labeling and automated image processing procedures. In addition, toxicological tests and gene expression analyses were conducted. (i) PDE-Is were applied to VTA/SN+STR rat co-cultures. The quantification of neurite density after both biocytin tracing and tyrosine hydroxylase labeling revealed a growth promoting effect of the PDE2A-Is BAY60-7550 and ND7001. The application of the PDE10-I MP-10 did not alter neurite density in comparison to the vehicle control. (ii) The effects of nimodipine were evaluated in VTA/SN+PFC rat co-cultures. A neurite growth-promoting effect of 0.1 µM and 1 µM nimodipine was demonstrated in a projection system of the CNS. In contrast, the application of 10 µM nimodipine did not alter neurite density, compared to the vehicle control, but induced the activation of the apoptosis marker caspase 3. The expression levels of the investigated genes, including Ca2+ binding proteins (Pvalb, S100b), immediate early genes (Arc, Egr1, Egr2, Egr4, Fos and JunB), glial fibrillary acidic protein, and myelin components (Mal, Mog, Plp1) were not significantly changed (with the exception of Egr4) by the treatment with 0.1 µM and 1 µM nimodipine. (iii) Bulk BM-MSCs that were classically isolated by plastic adhesion were compared to the subpopulation Sca-1+Lin-CD45--derived MSCs (SL45-MSCs). The neurite growth-promoting properties of both MSC populations were quantified in VTA/SN+PFC mouse co-cultures. For this purpose, the MSCs were seeded on glass slides that were placed underneath the co-cultures. A significantly enhanced neurite density within the co-cultures was induced by both bulk BM-MSCs and SL45-MSCs. SL45-MSCs increased neurite density to a higher degree. The characterization of both MSC populations revealed that the frequency of fibroblast colony forming units (CFU-f ) is 105-fold higher in SL45-MSCs. SL45-MSCs were morphologically more homogeneous and expressed higher levels of nestin, BDNF and FGF2 compared to bulk BM-MSCs. Thus, this work emphasizes the vast potential for molecular targeting with respect to the development of therapeutic strategies in the enhancement of neurite regrowth.

L’hypertension systolique isolée (HSI) est le résultat de changements au niveau de la paroi vasculaire qui ont pour conséquence d’augmenter la rigidité artérielle. Ces modifications surviennent surtout au niveau des grosses artères comme l’aorte et sont associées au vieillissement. La fragmentation des fibres élastiques, leur calcification (élastocalcinose) et la fibrose font partie des changements majeurs observés avec l’âge. En plus de ces changements, le vieillissement vasculaire provoque des modifications au niveau des cellules qui composent la paroi. Les cellules endothéliales sécrètent moins de monoxyde d’azote (NO) provoquant une dysfonction endothéliale et les cellules musculaires lisses vasculaires (CMLVs) synthétisent maintenant des protéines matricielles et osseuses. Situé entre le sang et les CMLVs, l’endothélium contrôle le tonus vasculaire par la sécrétion de plusieurs substances vasoactives qui interagissent entre elles afin de maintenir l’homéostasie du système vasculaire. Parmi celles-ci, on note l’endothéline (ET), un puissant vasoconstricteur et le NO, un gaz vasorelaxants. Ce dernier est aussi reconnu pour bloquer la production d’ET par un mécanisme dépendant du guanosine monophosphate cyclique (GMPc). Comme il y a une interaction entre le NO et l’ET, et que cette dernière est impliquée dans la calcification artérielle, le NO pourrait être impliqué dans la modulation de l’élastocalcinose et de la rigidité artérielle par l’inhibition de l’ET et la modification de la composition de la paroi. Cet effet, qui se produirait au delà des effets vasorelaxants du NO, offre un potentiel thérapeutique intéressant pour l’HSI. Afin d’évaluer l’implication du NO dans la calcification vasculaire et la rigidité artérielle, un modèle animal d’HSI a été utilisé (modèle warfarine vitamine K, WVK). Ce modèle d’élastocalcinose est basé sur l’inhibition de la maturation d’une protéine anti-calcifiante, la matrix Gla protein (MGP), par la warfarine. Afin de déterminer l’implication physiologique du NO dans l’initiation et la progression de l’élastocalcinose, sa production a été inhibée par un analogue de la L-arginine, le L-NG-nitroarginine methyl ester (L-NAME). Lors des processus d’initiation de la calcification, le L-NAME a prévenu l’élastocalcinose sans toutefois modifier la vitesse de l’onde de pouls (PWV). Suite au traitement L-NAME, l’expression de la NO synthase inductible (iNOS) a été diminuée alors qu’elle a été augmentée lors du traitement WVK. Elle pourrait donc être impliquée dans les processus de calcification vasculaire. De plus, la NO synthase endothéliale (eNOS) semble également impliquée puisqu’elle a été augmentée dans le modèle WVK. Cette hausse pourrait être bénéfique pour limiter l’élastocalcinose alors que l’expression de la iNOS serait délétère. Lors de la progression de la calcification, le L-NAME a augmenté l’élastocalcinose et le PWV. Dans ce contexte, l’ET serait impliquée dans l’amplification de la calcification vasculaire entrainant une hausse de la rigidité artérielle. Comme le NO endogène limite la progression de la calcification et conséquemment la rigidité artérielle, il semble être protecteur. L’efficacité d’une modulation de la voie du NO dans le modèle WVK a été étudiée par l’administration d’un donneur de NO, le sinitrodil, ou d’un inhibiteur de la phosphosdiestérase 5 (PDE5), le tadalafil. La modulation de la voie du NO semble être bénéfique sur la rigidité artérielle, mais seulement de façon aiguë. En effet, le sinitrodil a modifié de transitoirement la rigidité au niveau de l’aorte possiblement par la modulation du tonus vasculaire sans toutefois avoir des effets sur la composition de la paroi. Comme le modèle WVK n’affecte pas la fonction endothéliale, les concentrations endogènes de NO semblent être optimales puisque le sinitrodil provoque une augmentation de l’élastocalcinose possiblement par le développement d’une tolérance. Tout comme le sinitrodil, le tadalafil a modulé de manière aiguë la rigidité artérielle sans modifier la composition de la paroi. Globalement, ces travaux ont permis de mettre en évidence les effets bénéfiques du NO endogène pour limiter le développement de l’HSI, suggérant qu’une dysfonction endothéliale, tel qu’observé lors du vieillissement, a un impact négatif sur la maladie. / Isolated systolic hypertension (ISH) is the result of complex changes in the vascular wall and consequently the increase of arterial stiffness. These modifications occur mainly in conductance arteries, like the aorta, and are associated with aging. The fragmentation of elastic fibers, calcification (elastocalcinosis), and fibrosis are major changes with age. In addition to these changes in the extracellular matrix, vascular aging also induces vascular cell wall modifications. These include decreased production of nitric oxide (NO) by endothelial cells, which induces endothelial dysfunction, and the production of matrix and bone proteins by vascular smooth muscle cells (VSMCs). Located between the blood and VSMCs, the endothelium controls vascular tone by secreting various vasoactive factors. These factors interact with each other to maintain the hemodynamic of the vascular system. Among these factors, the vasoconstrictor endothelin (ET) and the vasodilator NO. The latter has been shown to block ET production via a cyclic guanosine monophosphates-(cGMP) dependent mechanism, whereas ET has been implicated in arterial calcification. Therefore, NO might be involved in the modulation of elastocalcinosis and arterial stiffness by inhibiting ET and modifying the vascular wall composition. This effect of NO could offer interesting therapeutic potential for ISH. To evaluate the implication of NO in the vascular calcification and arterial stiffness, an animal model of ISH was used. This model of elastocalcinosis is based on the inhibition of the maturation of the anti-calcific protein, matrix Gla protein (MGP), by warfarin (WVK model). To gain insight into the physiological role of endogenous NO in the initiation and progression of elastocalcinosis, its production was inhibited by the administration of L-NAME. Interestingly, elastocalcinosis was prevented by L-NG-nitroarginine methyl ester (L-NAME) administration without any modifications of the pulse wave velocity (PWV) during the initiation of the calcification processes. After the L-NAME treatment, the expression of inducible NO synthase (iNOS) was decreased, whereas upon treatment with warfarin alone the expression of iNOS was increased, which could be implicated in vascular calcification and arterial stiffness. In addition, endothelial NO synthase (eNOS) seems to be implicated in this process as its expression was also increased upon WVK treatment. This increase could be beneficial to limit elastocalcinosis, whereas the increase in iNOS expression could be harmful. L-NAME administration during the progression of calcification increased elastocalcinosis and PWV. In an endothelial dysfunction context, ET has been shown to be involved in the amplification process of vascular calcification causing an increase in arterial stiffness. As NO limits the progression of calcification and consequently arterial stiffness, endogenous NO seems to be protective in the aorta. The efficacy of exogenous modulation of the NO pathway in the WVK model was studied upon administration of the NO donor, sinitrodil, or the phosphodiesterase type 5 inhibitor (PDE5), tadalafil. The exogenous modulation of the NO pathway seemed to be beneficial for arterial stiffness, but only in an acute manner. Indeed, sinitrodil modified the acute stiffness in the aorta potentially by vascular tone modulation, without having any effect on vascular wall composition. Since endothelial function was not affected upon WVK model, endogenous NO concentrations seem to be optimal. Thus, exogenous NO potentially caused an increase of elastocalcinosis by inducing tolerance to NO. As well as sinitrodil, tadalafil modulated the arterial stiffness in an acute manner without modifying the composition of the vascular wall. Broadly, these studies provide evidence that endogenous NO can limit ISH development, suggesting that endothelial dysfunction, as observed in aging, has a negative impact on this pathology.

Os pacientes que usam lítio (Li) para tratamento do transtorno bipolar frequentemente apresentam poliúria e deficiência de concentração urinária, sintomas do Diabetes Insipidus Nefrogênico (DIN). Animais tratados com Li apresentam baixos níveis de produção de adenosina monofosfato cíclico (AMPc) em resposta ao hormônio antidiurético (HAD). O Sildenafil (Sil), um inibidor da fosfodiesterase 5 (PDE5), eleva os níveis intracelulares de guanosina monofosfato cíclico (GMPc), levando a inserção de aquaporina 2 (AQP2) na membrana plasmática das células do ducto coletor. Portanto, inibidores de PDE podem promover a inserção de AQP2 na membrana plasmática mesmo sem a ativação do receptor de HAD, indicando a participação de uma via alternativa mediada pelo GMPc. Nós investigamos as vias de ação do Sil no tratamento da DIN induzida pelo Li. Ratos Wistar foram divididos nos seguintes grupos: grupo controle, recebendo dieta alimentar normal durante quatro semanas; grupo Li, recebendo dieta alimentar normal com 40 mmol Li por quilo de dieta durante quatro semanas; grupo Li + Sil, recebendo dieta alimentar normal com 40 mmol Li por quilo de dieta durante quatro semanas e 200 mg por quilo de dieta de Sil a partir da segunda semana; grupo Sil, recebendo dieta alimentar normal durante a primeira semana e a partir da segunda semana recebendo dieta normal com 200 mg de Sil por quilo de dieta. Os animais do grupo Li desenvolveram poliúria, diminuição da osmolalidade urinária e diminuição da expressão da AQP2 tanto na fração citoplasmática como de membrana celular e o Sil reverteu essas alterações. Demonstramos ainda que a concentração de GMPc intracelular estava aumentada nos túbulos papilares tratados com Sil. Observamos que a provável via de fosforilação da AQP2 induzida pelo GMPc é pela PKA. Além disso, o tratamento com Sil aumenta a expressão de pCreb, fator de transcrição para ativação do gene da AQP2. Observamos ainda que o Li diminui a expressão de eNOS e o tratamento com Sil normaliza essa diminuição. Assim, concluímos que o tratamento com Sil em ratos com DIN melhora a poliúria aumentando a produção e a inserção de AQP2. O tratamento com Sil pode ser benéfico para pacientes que sofrem com DIN induzido pelo Li / Patients taking lithium to treat bipolar disorder often present polyuria and urinary concentrating defect. In addition, lithium-treated animals present lower cyclic adenosine monophosphate production in response to vasopressin. Sildenafil (Sil), a phosphodiesterase 5 (PDE5) inhibitor, elevates intracellular cyclic guanosine monophosphate (cGMP) levels, leading to plasma membrane accumulation of aquaporin 2 (AQP2). Therefore, PDE inhibitors might induce AQP2 membrane insertion even without vasopressin receptor activation by activating a parallel cGMP-mediated signal transduction pathway. We investigated Sil pathways of action in rats with lithium-induced nephrogenic Diabetes Insipidus (NDI). Wistar rats received lithium (40 mmol/kg food) or not for 4 weeks (Li or control), some rats also receiving sildenafil (200 mg/kg food) in weeks 2-4, with or without lithium (Li+Sil orSil). Animals in Li group developed polyuria, decreased urinary osmolality and decreased expression of AQP2 in both the cytoplasmic fraction and the cell membrane and Sil reversed these changes. We also demonstrated that intracellular cGMP concentration was increased in papillary tubules treated with Sil. We found that PKA may be involved in the pathway of cGMP induced AQP2 phosphorylation. In addition, Sil treatment increases Creb phosphorylation. Creb phosphorylation, acts as AQP2 gene transcription factor. We also observed that Li decreases eNOS expression and treatment with Sil normalizes this alteration. We conclude that Sil treatment improves polyuria by increasing production and insertion of AQP2. Sil treatment may be beneficial to patients suffering from induced DIN Li

The development of new therapeutical approaches, devised to foster the regeneration of neuronal circuits after injury and/or in neurodegenerative diseases, is of great importance. The impairment of dopaminergic projections is especially severe, because these projections are involved in crucial brain functions such as motor control, reward and cognition. In the work presented here, organotypic brain slice co-cultures of (a) the mesostriatal and (b) the mesocortical dopaminergic projection systems consisting of tissue sections of the ventral tegmental area/substantia nigra (VTA/SN), in combination with the target regions of (a) the striatum (STR) or (b) the prefrontal cortex (PFC), respectively, were used to evaluate different approaches to stimulate neurite outgrowth: (i) inhibition of cAMP/cGMP turnover with 3’,5’ cyclic nucleotide phosphodiesterase inhibitors (PDE-Is), (ii) blockade of calcium currents with nimodipine, and (iii) the co-cultivation with bone marrow-derived mesenchymal stromal/stem cells (BM-MSCs). The neurite growth-promoting properties of the tested substances and cell populations were analyzed by neurite density quantification in the border region between the two brain slices, using biocytin tracing or tyrosine hydroxylase labeling and automated image processing procedures. In addition, toxicological tests and gene expression analyses were conducted. (i) PDE-Is were applied to VTA/SN+STR rat co-cultures. The quantification of neurite density after both biocytin tracing and tyrosine hydroxylase labeling revealed a growth promoting effect of the PDE2A-Is BAY60-7550 and ND7001. The application of the PDE10-I MP-10 did not alter neurite density in comparison to the vehicle control. (ii) The effects of nimodipine were evaluated in VTA/SN+PFC rat co-cultures. A neurite growth-promoting effect of 0.1 µM and 1 µM nimodipine was demonstrated in a projection system of the CNS. In contrast, the application of 10 µM nimodipine did not alter neurite density, compared to the vehicle control, but induced the activation of the apoptosis marker caspase 3. The expression levels of the investigated genes, including Ca2+ binding proteins (Pvalb, S100b), immediate early genes (Arc, Egr1, Egr2, Egr4, Fos and JunB), glial fibrillary acidic protein, and myelin components (Mal, Mog, Plp1) were not significantly changed (with the exception of Egr4) by the treatment with 0.1 µM and 1 µM nimodipine. (iii) Bulk BM-MSCs that were classically isolated by plastic adhesion were compared to the subpopulation Sca-1+Lin-CD45--derived MSCs (SL45-MSCs). The neurite growth-promoting properties of both MSC populations were quantified in VTA/SN+PFC mouse co-cultures. For this purpose, the MSCs were seeded on glass slides that were placed underneath the co-cultures. A significantly enhanced neurite density within the co-cultures was induced by both bulk BM-MSCs and SL45-MSCs. SL45-MSCs increased neurite density to a higher degree. The characterization of both MSC populations revealed that the frequency of fibroblast colony forming units (CFU-f ) is 105-fold higher in SL45-MSCs. SL45-MSCs were morphologically more homogeneous and expressed higher levels of nestin, BDNF and FGF2 compared to bulk BM-MSCs. Thus, this work emphasizes the vast potential for molecular targeting with respect to the development of therapeutic strategies in the enhancement of neurite regrowth.:Table of contents Abbreviations 1 1. Introduction 2 1.1 The dopaminergic system 2 1.2 Neurite regeneration following mechanical lesions of the CNS 7 1.3 Organotypic brain slice co-cultures 8 1.4 Promising substances and cells to enhance neuroregeneration 10 1.5 The aim of the thesis 14 2. The original research articles 16 2.1 Phosphodiesterase 2 inhibitors promote axonal outgrowth in organotypic slice co-cultures 17 2.2 Nimodipine enhances neurite outgrowth in dopaminergic brain slice co-cultures 35 2.3 Mesenchymal stem cells support neuronal fiber growth in an organotypic brain slice co-culture model 50 3. References 66 Appendices 73 Summary 73 Zusammenfassung 78 Curriculum Vitae 84 Track Record 85 Selbständigkeitserklärung 87 Acknowledgments 88

This diploma thesis is focused on the determination of phosphodiesterase 5 selective inhibitors in communal waste waters. In this study phosphodiesterase inhibitors levels in municipal waste waters from sewage treatment plants with different numbers of equivalent inhabitants were analyzed. Namely it was sewage treatment plants in Brno – Modřice, Luhačovice and Hodonín. In each of the above mentioned facilities 24 - hour cumulated samples of the influent and effluent waste water were collected. On the Brno - Modřice sewage treatment plant also weekly monitoring of the concentration of phosphodiesterase inhibitors was realized.