Due to the high metabolic demand and low capacity for energy storage of the brain, neurons are vitally reliant on a constant oxygen supply. Under chronic mild hypoxic conditions (10% oxygen), angiogenesis is induced in the brain in an attempt to restore tissue oxygen tension to normal levels. In brain hypoxia, vascular endothelial growth factor (VEGF) plays a critical role in angiogenesis; however, the role of its homolog placental growth factor (PlGF) is unknown. Using PlGF knockout (PlGF-/-) mice exposed to whole body hypoxia (10% oxygen) for 7, 14 and 21-days, we show that PlGF-/- animals exhibit a delay in the angiogenic response of the brain to hypoxia. PlGF-/- microvessels had a significant increase in fibrinogen accumulation and extravasation, which correlated with disruption of the tight-junction protein claudin-5. These vessels displayed large lumens, were surrounded by reactive astrocytes, lacked mural cell coverage and endothelial VEGF expression, and regressed after 21 days of hypoxia. The lack of PlGF, in combination with reduced VEGF expression levels observed in the brain of PlGF-/- animals during the first 5 days of hypoxia, is likely the cause of the delayed angiogenic response and the prothrombotic phenotype of these mice. In vitro studies conducted to analyze mechanisms involved in the impaired angiogenic phenotype and enhanced astrocytic reactivity to hypoxia of PlGF-/- animals indicated that: i) PlGF-/- mouse brain endothelial cells exhibit alterations in intracellular signaling pathways associated with sprouting (ERK1/2) and vessel branching morphogenesis (GSK-3β) and ii) PlGF-/- astrocytes overexpress VEGF receptor-2 (VEGFR-2) which through activation of the ERK1/2 signaling pathway leads to a more proliferative astrocytic phenotype. These astrocytes were more resistant to oxygen and glucose deprivation (OGD) than PlGF+/+ astrocytes, a characteristic that was shown to be independent of the classical antiapoptotic VEGFR-2-dependent PI3K/Akt pathway. The findings presented in this thesis demonstrated a critical role of PlGF in vascular remodeling in the hypoxic brain.

Due to the high metabolic demand and low capacity for energy storage of the brain, neurons are vitally reliant on a constant oxygen supply. Under chronic mild hypoxic conditions (10% oxygen), angiogenesis is induced in the brain in an attempt to restore tissue oxygen tension to normal levels. In brain hypoxia, vascular endothelial growth factor (VEGF) plays a critical role in angiogenesis; however, the role of its homolog placental growth factor (PlGF) is unknown. Using PlGF knockout (PlGF-/-) mice exposed to whole body hypoxia (10% oxygen) for 7, 14 and 21-days, we show that PlGF-/- animals exhibit a delay in the angiogenic response of the brain to hypoxia. PlGF-/- microvessels had a significant increase in fibrinogen accumulation and extravasation, which correlated with disruption of the tight-junction protein claudin-5. These vessels displayed large lumens, were surrounded by reactive astrocytes, lacked mural cell coverage and endothelial VEGF expression, and regressed after 21 days of hypoxia. The lack of PlGF, in combination with reduced VEGF expression levels observed in the brain of PlGF-/- animals during the first 5 days of hypoxia, is likely the cause of the delayed angiogenic response and the prothrombotic phenotype of these mice. In vitro studies conducted to analyze mechanisms involved in the impaired angiogenic phenotype and enhanced astrocytic reactivity to hypoxia of PlGF-/- animals indicated that: i) PlGF-/- mouse brain endothelial cells exhibit alterations in intracellular signaling pathways associated with sprouting (ERK1/2) and vessel branching morphogenesis (GSK-3β) and ii) PlGF-/- astrocytes overexpress VEGF receptor-2 (VEGFR-2) which through activation of the ERK1/2 signaling pathway leads to a more proliferative astrocytic phenotype. These astrocytes were more resistant to oxygen and glucose deprivation (OGD) than PlGF+/+ astrocytes, a characteristic that was shown to be independent of the classical antiapoptotic VEGFR-2-dependent PI3K/Akt pathway. The findings presented in this thesis demonstrated a critical role of PlGF in vascular remodeling in the hypoxic brain.

Cleaved fragments of the extracellular matrix protein perlecan have been shown to promote neuroprotection and repair after ischemic stroke. The cysteine proteases cathepsin B and L as well as the metalloprotease bone morphogenic protein 1 (BMP-1) are capable of releasing the biologically active C-terminal laminin-like globular domain (LG3) of perlecan. Exercise, a known method of reducing stroke risk and severity, has been shown to increase the expression of some proteases associated with perlecan processing. Using a transient distal middle cerebral artery occlusion (MCAo) model for focal ischemic stroke we show that while 7 days of running only slightly decreased infarct volume, BMP1 and perlecan (HSPG2) RNA expression in skeletal muscle was significantly increased in 3-month-old male wild type C57/BL6 mice. Moreover, elevated levels of BMP1 RNA were still detectable after 3 days of detraining, suggesting a prolonged effect of exercise on BMP1 expression. Levels of LG3 in the blood were below the limit of detection in the current study, however it is likely that a more sensitive method would enable analysis of serum. These preliminary findings suggest that LG3 could be a molecular mediator of neuroprotection afforded by exercise though further studies are required.

Due to the high metabolic demand and low capacity for energy storage of the brain, neurons are vitally reliant on a constant oxygen supply. Under chronic mild hypoxic conditions (10% oxygen), angiogenesis is induced in the brain in an attempt to restore tissue oxygen tension to normal levels. In brain hypoxia, vascular endothelial growth factor (VEGF) plays a critical role in angiogenesis; however, the role of its homolog placental growth factor (PlGF) is unknown. Using PlGF knockout (PlGF-/-) mice exposed to whole body hypoxia (10% oxygen) for 7, 14 and 21-days, we show that PlGF-/- animals exhibit a delay in the angiogenic response of the brain to hypoxia. PlGF-/- microvessels had a significant increase in fibrinogen accumulation and extravasation, which correlated with disruption of the tight-junction protein claudin-5. These vessels displayed large lumens, were surrounded by reactive astrocytes, lacked mural cell coverage and endothelial VEGF expression, and regressed after 21 days of hypoxia. The lack of PlGF, in combination with reduced VEGF expression levels observed in the brain of PlGF-/- animals during the first 5 days of hypoxia, is likely the cause of the delayed angiogenic response and the prothrombotic phenotype of these mice. In vitro studies conducted to analyze mechanisms involved in the impaired angiogenic phenotype and enhanced astrocytic reactivity to hypoxia of PlGF-/- animals indicated that: i) PlGF-/- mouse brain endothelial cells exhibit alterations in intracellular signaling pathways associated with sprouting (ERK1/2) and vessel branching morphogenesis (GSK-3β) and ii) PlGF-/- astrocytes overexpress VEGF receptor-2 (VEGFR-2) which through activation of the ERK1/2 signaling pathway leads to a more proliferative astrocytic phenotype. These astrocytes were more resistant to oxygen and glucose deprivation (OGD) than PlGF+/+ astrocytes, a characteristic that was shown to be independent of the classical antiapoptotic VEGFR-2-dependent PI3K/Akt pathway. The findings presented in this thesis demonstrated a critical role of PlGF in vascular remodeling in the hypoxic brain.

No description available.

The surfactant protein-G (SP-G) has recently been discovered in the brain and linked to fluid balance regulations. Stroke is characterized by impaired vessel integrity, promoting water influx and edema formation. The neurovascular unit concept (NVU) has been generated to cover not only ischemic affections of neurons or vessels but also other regionally associated cells. This study provides the first spatio-temporal characterization of SP-G and NVU elements after experimental stroke. Immunofluorescence labeling was applied to explore SP-G, vascular and cellular markers in mice (4, 24, and 72 h of ischemia), rats (24 h of ischemia), and sheep (two weeks of ischemia). Extravasated albumin indicated vascular damage within ischemic areas. Quantifications revealed decreasing SP-G signals in the ischemia-affected neocortex and subcortex. Inverse immunosignals of SP-G and vascular elements existed throughout all models. Despite local associations between SP-G and the vasculature, a definite co-localization was not seen. Along with a decreased SP- G-immunoreactivity in ischemic areas, signals originating from neurons, glial elements, and the extracellular matrix exhibited morphological alterations or changed intensities. Collectively, this study revealed regional alterations of SP-G, vascular, and non-vascular NVU elements after ischemia, and may thus stimulate the discussion about the role of SP-G during stroke.

Title: Nitric oxide and its neurophysiologic role Objectives: The main aim of my diploma work is to evaluate the role of nitric oxide and its effects during epileptic seizure in rats. I summarize the present knowledge about nitric oxide and its effects on human organism in the theoretical section of this paper and I will suppose the positive effect of nitric oxide inhibitor in epileptic seizures in rats by the realization of my experiment. Methods: The experiment will be performed in the laboratory of Academy of Science of Czech Republic. Implementation will come through examining a set of ten rats. The first six will be given v (L-NPA) in an amount of 0,5mg to one side, which will take place by applying bilaterally to coele. Then after ten minutes 0,5µl of 4-aminopyridine (4-AP) will be applied bilaterally to coele. The control group, consisting of four rats will be given saline instead of L-NPA. The experiment will run for two days. Results: The course of epileptic seizure with application of L-NPA to examined group was compared with the control group. The experiment confirmed the positive effect of L-NPA in terms of delay and reduction of seizure. Keywords: nitric oxide, Nω -propyl-l-arginine, neurovascular coupling, 4-anopyridine, epilepsy

Zwischen neuronaler und glialer Aktivierung, dem Energiemetabolismus und dem zerebralen Gefäßbett besteht eine enge Beziehung - als Phänomen der neurometabolischen und neurovaskulären Kopplung bekannt. Diese Korrelation von elektrischer und metabolischer Aktivität sowie dem regionalen zerebralen Blutfluß besteht räumliche und zeitliche fokussiert und ist charakteristisch für das Gehirn. Das Verständnis um die Mechanismen und Mediatoren der neurovaskulären Kopplung ist von grundlegender Bedeutung für die korrekte Interpretation moderner bildgebender Verfahren im klinischen Einsatz. Ein Schwerpunkt der Untersuchungen lag in der Überprüfung der Hypothese eines frühen Sauerstoff-oder Glukose-Mangels im Gewebe unter funktioneller Stimulation als mögliches primäres Signal für eine nachfolgende vaskuläre Antwort. Anhand der Befunde kann jedoch ausgeschlossen werden, daß ein möglicherweise kurzzeitig auftretender Substratmangel im Gewebe registriert wird, und auf diesem Wege die Blutflußantwort auf funktionelle Stimulation initiiert wird. Die Frage nach dem eigentlichen Signal für die Blutflußantwort und dem primären Ort der Regulation (Arteriole, Kapillare, Venole) bleibt weiterhin ungeklärt. Die qualitative wie auch quantitative Charakterisierung des Verlaufs der Blutfluß- und Blutoxygenierungsveränderungen unter somatosensorischer Stimulation bildet die Grundlage für die Untersuchung von Veränderungen dieses Musters unter pathophyiologischen Bedingungen als sogenannter Fingerabdruck spezifischer zerebraler Schädigungen. Im Rahmen des zweiten Schwerpunkts beschäftigt sich vorliegende Arbeit mit der Rolle des Bioradikals Stickstoffmonoxid (NO) bei der neurovaskulären Kopplung. Ein im kortikalen Gewebe physiologisch vorhandener basaler NO Spiegel moduliert über die Bereitstellung einer basalen cyclischen Guanosin-3',5'-Monophosphat (cGMP) Konzentration vornehmlich in glatten Gefäßmuskelzellen die Reaktivtät zerebraler Gefäße sowohl auf globale vasodilatatorische Stimuli wie die Hyperkapnie als auch auf funktionelle Aktivität wie die Whisker-Stimulation der Ratte. Da die Bereitstellung des basalen NO / cGMP Spiegels im zerebralen Gewebe für physiologische vaskuläre Antworten von grundlegender Bedeutung ist, ist zu erwarten, daß eine Störung desselben durch pathophysiologische Vorgänge weitreichende Folgen für die adequate Versorgung aktivierter Hirnareale haben dürfte. / A tight relation exists between neuronal and glial cell activation, cerebral energy metabolism, and cerebral vasculature - a phenomenon known as neurometabolic and neurovascular coupling. The correlation of electrical and metabolic activity, and regional cerebral blood flow occurs at high temporal and spatial resolution, and is characteristic for the brain. We need to understand the physiology of neurometabolic and neurovascular coupling to fully exploit the potential of modern functional brain imaging, which utilizes vascular responses to map brain activity. Therefore, understanding the signalling cascade of regional vasodilation due to functional activation is of great importance. In the present work, we have shown that there is no evidence neither for an early deoxygenation nor for an early decrease in glucose concentration in the tissue at the onset of increased neuronal activity. The first event inducing regional cerebral blood flow increase as well as the vascular compartment, at which vasodilation starts (arteriole, capillary, venule), is still not known so far and has to be further investigated. In disease neurovascular coupling may be disturbed, while this disturbance may itself further contribute to tissue damage. Therefore a thorough elucidation of the physiology and pathophysiology of neurometabolic and neurovascular coupling may contribute to the development of treatment strategies in acute and chronic CNS disorders. Beside the mechanisms, little is known concerning the mediators of neurovascular coupling. The involvement of the highly diffusible vasodilator bioradical nitric oxide (NO) in the regulation of regional cerebral blood flow is widely accepted. In the present work, it has been shown that NO acts as a modulator rather than a mediator of vascular relaxation due to functional activation or systemic hypercapnia in the cerebral cortex, permitting vasodilation mediated by other agents. This modulation mainly occurs via a basal cyclic guanosin-3',5'-monophosphate (cGMP) production within the vascular smooth muscle cell. The basal modulatory concentration of NO / cyclic GMP may be disturbed during cerebrovascular disease, leading to a mismatch of regional cerebral blood flow and metabolic demand.

Die Arbeit stellt die optische Methode der Nahinfraotspektroskopie (NIRS) in der Anwendung zur Messung von Oxygenierungsänderungen am Kopf des Erwachsenen dar. Dabei werden zunächst methodische Aspekte behandelt. Dies schließt (i) die Frage ein ob Änderungen des Redox-Status der Cytochromoxidase mit der NIRS bestimmt werden können; (ii) diskutiert die Ergebnisse zur Darstellbarkeit schneller optischer Signale, wie sie in invasiven Studien als Korrelat der elektrophysiologisch messbaren neuronalen Prozesse bekannt sind und (iii) richtet sich auf die bessere Definition des Messvolumens, sowohl im Sinne einer zweidimensionalen Bildgebung der Oxygenierungs-änderungen an der Hirnoberfläche als auch die Möglichkeiten zu einer Tiefenauflösung. Bezüglich der grundlegenden physiologischen Fragestellungen thematisiert die Arbeit im zweiten Teil Fragestellungen der neuro-vaskulären Kopplung. Dies ist die Frage, wie sich die neuronale, elektrophysiologisch meßbare Aktivität von Neuronen in eine hämodynamische Antwort übersetzt. Hier sind die Einordnung der NIRS-Befunde in ein Modell der vaskulären Antwort aber auch gleichzeitige Messungen mit elektrophysiologischen Methoden und weiteren vaskulär-basierten Methoden, insbesondere der funktionellen Kernspintomographie, Fokus. Schließlich stellt die Arbeit die klinischen Perspektiven in der Neurologie dar, wie sie sich besonders für die vaskulären Erkrankungen des Gehirns aber auch für Fragestellungen bei Epilepsie und Migräne ergeben. / We here present a optical method, near-infrared spectroscopy (NIRS), and its application for non-invasive measurement of oxygenation changes in the adult head. The first part deals with methodological aspects of NIRS. This includes (i) the question whether changes of cytochrome-oxidase redox state can be monitored by NIRS; (ii) the discussion of study results investigating the non-invasive detectability of fast optical signals , known to reflect electrophysiological changes in invasive animal preparations; (iii) and finally the issue how the sampling volume can be better defined, and with respect to a two-dimensional imaging approach of cortical oxygenation changes and the methodological options to achieve a depth resolution. In a second chapter we then focus on the underlying physiological issues mainly raising questions concerning neuro-vascular coupling. Neuro-vascular coupling means the translation of neuronal activity, as can be measured by electrophysiological methods, into the haemodynamic response. Here the focus is on how the results of functional NIRS studies can be interpreted in the light of a model of the vascular response. Also the simultaneous assessment of electrophysiological parameters or the parameters of other methods based on the vascular response, especially fMRI, are dealt with in this chapter. Finally the here presented work gives perspectives of potential clinical applications of the method. This primarily means the application in stroke and other vascularly diseases in neurology, but also includes questions in the clinical fields of epilepsy and migraine.

Introduction : Le système nerveux autonome (SNA) est en situation supralévatorienne, le système somatique en situation infra lévatorienne. Le sympathique assurerait les fonctions de sécrétion et le parasympahtique, les fonctions dʼérection. Le rectum est en rapport étroit avec ces éléments nerveux. La protectomie avec exérèse totale du mésorectum pour cancer est associée à des séquelles sexuelles par lésion iatrogène des nerfs pelviens.Objectifs : Étudier la physiologie et lʼanatomie topographique et structurelle de l'innervation pelvipérinéale Matériels et méthodes : Cinq pelvis de foetus et dix pelvis dʼadultes féminins ont été prélevés pour études macroscopiques, microscopiques etimmunohistochimiques des nerfs pelviens. Les coupes ont été colorées puis immunomarquées pour détecter les fibres nerveuses (PS-100), somatiques(PMP22), autonomes adrénergiques (TH), cholinergiques (VAChT), sensitives(CGRP) et les fibres pro-érectiles (nNOS). Les lames ont été numérisées et reconstruites en 3D.Résultats : Les fibres nerveuses du SNA, richement interconnectées,véhiculent de façon mixte lʼinflux sympathique et parasympathique.Celles issues du plexus hypogastrique inférieur contrôlant les fonctions sexuelles sont regroupées avec le pédicule vaginal long et forment la bandelette neuro-vasculaire (BNV) sur la face antérolatérale du rectum à « 2et 10h ». Cette BNV est en avant de lʼexpansion postérolatérale du septum recto vaginal (SRV) qui la protège et qui est lʼéquivalent chez lʼhomme du fascia de Denonvilliers. Cette BNV concentre lʼensemble des fibres nerveusespro-érectiles destinées au périnée. Une lésion tronculaire de cette bandelette pourrait conduire à un trouble de lʼérection clitoridienne et de la lubrification vulvaire car, à ce niveau, les efférences sympathiques et parasympathiques coexistent.Conclusion: Un modèle anatomo-physiologique et pédagogiquedʼinnervation pelvipérinéale féminine a été développé. Ces travaux offrent des perspectives dʼétudes cliniques afin de mieux évaluer les dysfonctions sexuelles postopératoires. / Introduction: The nerve supply of the autonomic nervous system (ANS) to the pelvis is located above the levator ani muscle, and the somatic nerve supply to the pelvis is situated below levator ani. Sympathetic innervation assures secretory functions and parasympathetic innervation allows erection. The rectum is anatomically closely associated with these nerves. Protectomy with total excision of the mesorectum for cancer is associated with sexual sequellae due to iatrogenic damage to the pelvic nerves.Objectives: To study the physiology and topographic and structural anatomy of the innervation of the pelvis.Materials and methods: Five fœtal pelvises and ten adult female pelvises were collected for macroscopic, microscopic, and immunohistochemical studies of pelvic nerves. Sections were stained and then immunostained to reveal nerve fibres (PS-100), somatic nerves (PMP22), adrenergic autonomic nerves (TH), cholinergic autonomic nerves (VAChT), sensory nerves (CGRP) and pro-erectile nerves (nNOS). Sections were numbered and reconstructed in 3D.Results: ANS nerve fibres, densely interconnected, carry a combination of sympathetic and parasympathetic fibres.Nerve fibres controlling sexual function from the inferior hypogastric plexus are clustered along the vaginal pedicle and form the neurovascular bundle (NVB) on the anterolateral face of the rectum between “2 and 10 o’clock”. This NVB is in front of the expansion of the rectovaginal septum (RVS) which protects it. In males, the equivalent structure is the rectoprostratic fascia. This NVB contains all of the pro-erectile nerves supplying the perineum. A truncal lesion to this bundle could result in erectile dysfunction of the clitoris as well as difficulties in vulvar lubrication because sympathetic and parasympathetic efferent fibres are both present at this site.Conclusion: An educational anatomical and physiological model of the innervation of the female pelvis and perineum has been developed. This work offers perspectives for clinical studies to facilitate better evaluation of cases of post-operative sexual dysfunction.