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The main complication of aortocoronary reconstruction with vein grafts is restenosis in the course of time. The aim was to assess the effect of a periadventitial polyester system releasing sirolimus on intimal hyperplasia of autologous grafts. The controlled-release system comprises a polyester mesh coated with a sirolimus-eluting copolymer of L lactic acid and ε-caprolactone system designed to be wrapped around an autologous venous graft during its implantation. In vitro sirolimus release and its effects on smooth muscle and endothelial cells were assessed. In vitro, the copolymer-coated polyester mesh released sirolimus over a period of 6 weeks. Mesh-eluted sirolimus inhibited the growth of smooth muscle and endothelial cells in seven-day in vitro experiments. After seven days of sirolimus release from the mesh, smooth muscle and endothelial cell counts decreased by 29% and 75%, respectively, with the cells maintaining high viability. We implanted v. jugularis ext. into a. carotis communis in rabbits. The vein graft was either intact, or was wrapped with a pure polyester mesh, or with a sirolimus-releasing mesh. Three and six weeks after surgery, the veins were subjected to standard histological staining and the thicknesses of the tunica intima, the media and the intima-media complex were...

The main complication of aortocoronary reconstruction with vein grafts is restenosis in the course of time. The aim was to assess the effect of a periadventitial polyester system releasing sirolimus on intimal hyperplasia of autologous grafts. The controlled-release system comprises a polyester mesh coated with a sirolimus-eluting copolymer of L lactic acid and ε-caprolactone system designed to be wrapped around an autologous venous graft during its implantation. In vitro sirolimus release and its effects on smooth muscle and endothelial cells were assessed. In vitro, the copolymer-coated polyester mesh released sirolimus over a period of 6 weeks. Mesh-eluted sirolimus inhibited the growth of smooth muscle and endothelial cells in seven-day in vitro experiments. After seven days of sirolimus release from the mesh, smooth muscle and endothelial cell counts decreased by 29% and 75%, respectively, with the cells maintaining high viability. We implanted v. jugularis ext. into a. carotis communis in rabbits. The vein graft was either intact, or was wrapped with a pure polyester mesh, or with a sirolimus-releasing mesh. Three and six weeks after surgery, the veins were subjected to standard histological staining and the thicknesses of the tunica intima, the media and the intima-media complex were...

Grundlage der vorgelegten Arbeit sind die bei der Alzheimerschen Erkrankung beobachtbaren pathologischen Merkmale, wie die progressive Akkumulation von β-Amyloid-Plaques, cholinerger Dysfunktion und zerebrovaskuläre Abnormalitäten. Die in englischer Sprache verfasste Dissertation ist eine tierexperimentelle Studie, die versucht, den Zusammenhang von β-Amyloid, cholinerger Neurotransmission und zerebralem Gefäßsystem bei der Alzheimerschen Erkrankung näher zu charakterisieren. An Hirnmaterial aus der transgenen Maus Tg2576, die die schwedische Mutation des humanen Amyloidpräkursorproteins als Transgen trägt und ab dem 10. Lebensmonat durch humane β-Amyloid-Plaqueablagerungen in der Hirnrinde imponiert, wurden im Altersverlauf (4 bis 18 Monate) immunhistochemische Untersuchungen zur morphologischen Integrität der zerebralen Mikrogefäße, der kortikalen cholinergen Nervterminalen und der intrazerebralen cholinergen neurovaskulären Innervation durchgeführt. Am somatosensorischen Kortex werden beispielhaft die Expression des Glukosetransporters 1 oder Solanum tuberosum Lektin als Kapillarmarker und des vesikulären Acetylcholintransporters als Marker für cholinerge Fasern mittels Immunfluoreszenz und Laser-Scanning Mikroskopie erfasst, einer semiquantitativen Computer-gestützten Bildanalytischen Auswertung unterzogen und mit dem Ausmaß der kortikalen Plaquebeladung korreliert. So konnte gezeigt werden, dass die Dichte der Blutgefäße und cholinergen Fasern im somatosensorischen Kortex von transgenen Tieren mit dem Alter im Vergleich zu nichttransgenen Kontrolltieren abnimmt, was mit einer Reduktion der perivaskulären cholinergen Innervation einhergeht. Die erhobenen Befunde stützen die von J.C. de la Torre und T. Mussivand schon im Jahre 1993 formulierte „vaskuläre Hypothese“, wonach bei der sporadischen Form der Alzheimerschen Erkrankung alters- und Lebensstil-bedingte Schädigungen des zerebralen Gefäßsystems eine zentrale Rolle bei der Manifestierung der Erkrankung spielen.

Grundlage der vorgelegten Arbeit sind die bei der Alzheimerschen Erkrankung beobachtbaren pathologischen Merkmale, wie die progressive Akkumulation von β-Amyloid-Plaques, cholinerger Dysfunktion und zerebrovaskuläre Abnormalitäten. Die in englischer Sprache verfasste Dissertation ist eine tierexperimentelle Studie, die versucht, den Zusammenhang von β-Amyloid, cholinerger Neurotransmission und zerebralem Gefäßsystem bei der Alzheimerschen Erkrankung näher zu charakterisieren. An Hirnmaterial aus der transgenen Maus Tg2576, die die schwedische Mutation des humanen Amyloidpräkursorproteins als Transgen trägt und ab dem 10. Lebensmonat durch humane β-Amyloid-Plaqueablagerungen in der Hirnrinde imponiert, wurden im Altersverlauf (4 bis 18 Monate) immunhistochemische Untersuchungen zur morphologischen Integrität der zerebralen Mikrogefäße, der kortikalen cholinergen Nervterminalen und der intrazerebralen cholinergen neurovaskulären Innervation durchgeführt. Am somatosensorischen Kortex werden beispielhaft die Expression des Glukosetransporters 1 oder Solanum tuberosum Lektin als Kapillarmarker und des vesikulären Acetylcholintransporters als Marker für cholinerge Fasern mittels Immunfluoreszenz und Laser-Scanning Mikroskopie erfasst, einer semiquantitativen Computer-gestützten Bildanalytischen Auswertung unterzogen und mit dem Ausmaß der kortikalen Plaquebeladung korreliert. So konnte gezeigt werden, dass die Dichte der Blutgefäße und cholinergen Fasern im somatosensorischen Kortex von transgenen Tieren mit dem Alter im Vergleich zu nichttransgenen Kontrolltieren abnimmt, was mit einer Reduktion der perivaskulären cholinergen Innervation einhergeht. Die erhobenen Befunde stützen die von J.C. de la Torre und T. Mussivand schon im Jahre 1993 formulierte „vaskuläre Hypothese“, wonach bei der sporadischen Form der Alzheimerschen Erkrankung alters- und Lebensstil-bedingte Schädigungen des zerebralen Gefäßsystems eine zentrale Rolle bei der Manifestierung der Erkrankung spielen.:CHAPTER 1: INTRODUCTION 1.1 Alzheimer’s disease 1 1.2 APP processing and β-amyloid production 2 1.3 Cholinergic dysfunction in Alzheimer’s disease 5 1.4 Cerebrovascular abnormalities in Alzheimer’s disease 8 1.5 Cholinergic innervation of intracortical cerebral microvessels 9 1.6 Transgenic Tg2576 mouse model of Alzheimer’s disease 11 1.7 Aim of study 14 CHAPTER 2: MATERIALS AND METHODS 2.1 Materials 15 2.1.1 Chemical reagents used 15 2.1.2 Biological reagents used 15 2.1.3 Preparation of solutions and buffers 15 2.1.4 Antibodies and reagents used for immunohistochemistry 17 2.1.5 Transgenic animals 19 2.2 Methods 20 2.2.1 Tissue preparation and sampling of sections 20 2.2.2 Immunohistochemistry 20 2.2.2.1 Protocol of immunofluorescent labeling 20 2.2.2.2 Protocol of immunoperoxidase labeling (ABC technique) 21 2.2.2.3 Combination of primary and secondary antibodies 22 2.2.2.4 Protocol of β–amyloid immunolabeling (Formic acid epitope retrieval method) 23 2.2.3 Histochemistry 23 2.2.3.1 Thioflavin S staining 23 2.2.3.2 Nissl staining 23 2.2.3.3 Solanum Tuberosum Lectin (STL) staining 24 2.2.4 Double and triple-coloured immuno-/ histochemical staining of brain sections 24 2.2.5 Microscopy and digital image processing 25 2.2.6 Morphological and morphometric analyses 25 2.2.6.1 Cortical microvessels 25 2.2.6.2 Cortical cholinergic innervation 27 2.2.6.2.1 Total density of VAChT-immunoreactivity 27 2.2.6.2.2 Estimation of the density of varicosities on cholinergic fibres 29 2.2.6.3 Estimation of cholinergic perivascular innervation of cortical microvessels 29 2.2.6.4 Three-dimensional-imaging of vessels innervation 30 2.2.7 Statistical analysis 30 CHAPTER 3: RESULTS 3.1 Developmental and amyloid plaque-related changes in cerebral cortical capillaries in transgenic Tg2576 Alzheimer mice 31 3.1.1 Morphological distribution of brain vessels in the cerebral cortex of wild type mice 31 3.1.2 Microvessel density under plaque burden 33 3.2 Developmental and amyloid plaque-related changes in cholinergic neurotransmission in cholinoceptive target regions of transgenic Tg2576 mice 39 3.2.1 Visualisation of cholinergic nerve terminals in mouse brain 39 3.2.2 VAChT-Expression in wild type and transgenic Tg2576 mice 40 3.3 Role of cholinergic system in β-amyloid-related changes in the cerebrovascular system of transgenic Tg2576 mice 46 3.3.1 Solanum tuberosum lectin (STL) histochemistry in visualisation of brain vessels, β-amyloid, and microglia 46 3.3.1.1 Solanum tuberosum lectin and brain vessels 46 3.3.1.2 Solanum tuberosum lectin and β-amyloid plaques 47 3.3.1.3 Solanum tuberosum lectin staining to visualize glial cells 48 3.3.2 Cholinergic perivascular innervation of cerebral cortical microvessels in transgenic Tg2576 and wild type mice 50 CHAPTER 4: DISCUSSION 4.1 β-Amyloid and brain vascular system: the vascular hypothesis of Alzheimer’s disease 55 4.1.1 Evidences of a role of vascular mechanisms in Alzheimer’s disease 55 4.1.2 Effect of β-amyloid on brain vascular system 57 4.1.3 Effect of ischemia and hypoperfusion on APP processing 59 4.1.4 Effect of β-amyloid on cholinergic function in brain vascular system 59 4.2 Aim of study and main results obtained 61 4.3 Age-related changes in cerebral cortical microvessels in the presence and absence of β-amyloid plaque load 62 4.4 Age-related changes of cholinergic terminals in cholinoceptive target regions in the presence and absence of β-amyloid plaque load 64 4.4.1 VAChT – a reliable marker for detection of cholinergic terminals in cerebral cortex 64 4.4.2 The barrel field of the somatosensory cortex 1 (S1BF) as a model region to reveal age-related changes in cholinergic innervation 65 4.4.3 VAChT expression: morphological and morphometric studies 66 4.5 Age-related changes in cholinergic innervation of cerebral cortical microvessels in the presence and absence of β-amyloid plaque load 69 4.5.1 STL – a mono-marker for detection of cortical vessels, senile amyloid plaques and activated microglia in cerebral cortex 69 4.5.2 Cholinergic perivascular innervation of cerebral cortical microvessels in transgenic Tg2576 mice 70 4.5.3 Quantitation of cholinergic input on cerebral microvessels of mouse brain 71 4.6 Summary and conclusions 75 REFERENCES 77

Indiana University-Purdue University Indianapolis (IUPUI) / Obesity increases cardiovascular disease risk and is associated with factors of the “metabolic syndrome” (MetS), a disorder including hypertension, hypercholesterolemia and/or impaired glucose tolerance. Expanding adipose and subsequent inflammation is implicated in vascular dysfunction in MetS. Perivascular adipose tissue (PVAT) surrounds virtually every artery and is capable of releasing factors that influence vascular reactivity, but the effects of PVAT in the coronary circulation are unknown. Accordingly, the goal of this investigation was to delineate mechanisms by which lean vs. MetS coronary PVAT influences vasomotor tone and the coronary PVAT proteome. We tested the hypothesis that MetS alters the functional expression and vascular contractile effects of coronary PVAT in an Ossabaw swine model of the MetS. Utilizing isometric tension measurements of coronary arteries in the absence and presence of PVAT, we revealed the vascular effects of PVAT vary according to anatomical location as coronary and mesenteric, but not subcutaneous adipose tissue augmented coronary artery contractions to KCl. Factors released from coronary PVAT increase baseline tension and potentiate constriction of isolated coronary arteries relative to the amount of adipose tissue present. The effects of coronary PVAT are elevated in the setting of MetS and occur independent of endothelial function. MetS is also associated with substantial alterations in the coronary PVAT proteome and underlying increases in vascular smooth muscle Ca2+ handling via CaV1.2 channels, H2O2-sensitive K+ channels and/or upstream mediators of these ion channels. Rho-kinase signaling participates in the increase in coronary artery contractions to PVAT in lean, but not MetS swine. These data provide novel evidence that the vascular effects of PVAT vary according to anatomic location and are influenced by the MetS phenotype.