7 research outputs found
Klinik und Pathologie von Scrapie im Mausmodell bei unterschiedlichen Inokulationsrouten
No full textIn der vorliegenden Arbeit wurden die Zusammenhänge zwischen dem Verlauf von Scrapie und der Ausbildung Scrapie-typischer Symptome sowie zwischen der Ausbildung TSE-spezifischer Vakuolen, einer Astrozytose und der Akkumulation von PrPsc im Mausmodell charakterisiert. Dazu wurden Mäuse zweier Wildtyp-Linien (CD-1 und C57/Bl6) und Tiere des transgenen tga20-Mausstammes mit einem 1%igen bzw. 10%igen Gehirnhomogenat des maus-adaptierten Prionen-Stammes Rocky Mountain Laboratory (RML) intrazerebral (ic) intraperitoneal (ip) und oral inokuliert. Tga20-Mäuse überexprimieren das zelluläre Prion-Protein um das ca. Zehnfache im Gehirn verglichen mit Wildtyp-Mäusen und weisen daher kürzere Inkubationszeiten auf. Es wurden der Verlauf der Erkrankung sowie die klinischen Symptome dokumentiert und die Gehirne der Tiere in sowohl präklinischen Krankheitsstadien als auch mit einsetzenden Symptomen und im späten Stadium der Erkrankung untersucht. Neben der ic-Inokulation wurden erstmals auch zwei periphere Inokulationswege detailliert für die tga20-Mäuse analysiert.
Die Beurteilung der Vakuolisierung erfolgte am histologischen Schnitt nach HE-Färbung. Die Ausbildung einer Astrozytose wurde anhand der Astrozytenzahl durch Immunhistochemie und anhand der GFAP-Genexpression durch RT-PCR und Northern Blot Analyse nachgewiesen. Die Akkumulation von PrPsc wurde am histologischen Schnitt der Gehirne nach Immunhistochemie und durch Western Blot Analyse ausgewertet.
Tga20-Mäuse wiesen nach allen drei Inokulationswegen verkürzte Inkubationszeiten auf verglichen mit den Wildtyp-Mäusen.
Bei der klinischen Beurteilung der Tiere fielen Unterschiede in der Symptomatik zwischen den drei Mausstämmen auf. Wichtigste Symptome der beiden Wildtyp-Mausstämme waren eine fortschreitende Ataxie, Abmagerung und Lethargie, die Ausbildung eines stumpfen und struppigen Felles sowie eine Kyphose. C57/Bl6-Mäuse wiesen außerdem in unregelmäßigen Abständen auftretende Zuckungen der Hautmuskulatur im Bereich des Rückens sowie eine Parese der Hintergliedmaßen mit lateraler Abspreizung der Hinterfüße auf. Nach ic-Inokulation waren die Symptome der tga20-Mäuse ähnlich denen der CD-1-Mäuse. Dagegen zeigten tga20-Mäuse nach den beiden peripheren Inokulationswegen eine extrem langgestreckte Körperhaltung ohne Ausbildung einer Kyphose. Es konnte somit erstmals gezeigt werden, dass die Inokulation eines bestimmten Prionen-Stammes zu unterschiedlichen klinischen Ausprägungen der Krankheit innerhalb der tga20-Mauslinie führt, dass also die klinischen Symptome abhängig von der Inokulationsroute waren.
Die Untersuchung der neuropathologischen Veränderungen in den Gehirnen der Tiere zeigte, dass die tga20-Mäuse im späten Stadium der Erkrankung eine geringere Vakuolisierung und eine schwächere astrozytäre Gliose ausbilden sowie weniger PrPsc akkumulieren verglichen mit den Wildtyp-Mäusen im vergleichbaren Krankheitsstadium. Dabei war dieser Unterschied nach den beiden peripheren Inokulationsrouten deutlicher als nach intrazerebraler Inokulation. Zudem konnte bereits in präklinischen Krankheitsstadien in den Gehirnen der Tiere beider Wildtyp-Mausstämme regelmäßig eine deutliche Ausbildung TSE-spezifischer Vakuolen sowie eine Astrozytose und PrPsc-Akkumulation festgestellt werden. Dies war in den Gehirnen der tga20-Mäuse nicht der Fall. Die präklinischen Veränderungen bei den Wildtyp-Mäusen waren meist stärker ausgeprägt als bei klinisch kranken tga20-Mäusen. Demnach scheint die Ausbildung von spongiformen Läsionen sowie eine Astrozytenaktivierung und die Akkumulation von PrPsc weder notwendig noch ausreichend zu sein für die Ausbildung von klinischen Symptomen bis hin zum späten Krankheitsstadium.
In der vorliegenden Studie konnte außerdem gezeigt werden, dass PrPsc präklinisch häufig vor dem Auftreten von TSE-spezifischen Vakuolen nachweisbar ist und bei den tga20-Mäusen auch im späten Krankheitsstadium nicht immer mit dem Nachweis von Vakuolen korreliert.The present study characterized the relationships between the course of scrapie and the development of typical scrapie symptoms as well as between the development of TSE-specific vacuoles, astrocytosis, and the accumulation of PrPsc in the mouse model. Mice of two wild-type lines (CD-1 and C57/Bl6) and mice of the transgenic tga20 strain were inoculated intracerebrally (ic), intraperitoneally (ip), and orally with a 1% and 10% brain homogenate of the mouse-adapted Rocky Mountain Laboratory (RML) prion strain. Tga20 mice overexpress cellular prion protein in the brain by a factor of ten in comparison to wild-type mice and therefore exhibit shorter incubation times. The course of the disease and the clinical symptoms were documented, and the animals’ brains were examined during the preclinical stages, after the onset of symptoms, and in the late stage of the disease. In addition to ic-inoculation, two peripheral inoculation routes were investigated for the first time in detail in tga20 mice.
Vacuolization was evaluated in HE-stained histologic sections. The development of astrocytosis was ascertained on the basis of the number of astrocytes as determined by immunohistochemical staining and on the basis of GFAP gene expression as determined by RT-PCR and northern blot analysis. PrPsc accumulation was assessed in histologic brain sections by immunohistochemical staining and western blotting.
After all three inoculation routes the tga20 mice exhibited shortened inoculation times in comparison to their wild-type counterparts. Clinical evaluation of the animals revealed differences between the symptoms in the three mouse strains. The chief symptoms of the two wild-type mouse strains were progressive ataxia, emaciation, lethargy, kyphosis and dull, rough fur. The C57/Bl6 mice also showed convulsions of the skin muscles on the back at irregular intervals as well as paresis of the hind legs with lateral spreading of the hind feet. Following ic-inoculation the symptoms in the tga20 mice were similar to those in the CD-1 mice. By contrast, after the two peripheral inoculation methods, the tga20 mice exhibited an extremely elongated posture without development of kyphosis. Thus, it was shown for the first time that inoculation of a specific prion strain leads to different clinical manifestations of the disease within the tga20 mouse strain, with the clinical symptoms depending on the inoculation route.
Examination of the neuropathologic changes in the animals’ brains showed that in the late stage of the disease the tga20 mice had less pronounced vacuolization, astrocytosis, and PrPsc accumulation than the wild-type mice in a comparable disease stage. This difference was more pronounced after the two peripheral inoculation routes than after ic-inoculation. Moreover, marked development of TSE-specific vacuoles, astrocytosis, and PrPsc accumulation were observed in the brains of both wild-type strains even in the preclinical disease stages. This was not the case in the tga20 mice. The preclinical changes were generally more pronounced in the wild-type mice than in the clinically affected tga20 mice. Hence, spongiform lesion development, astrocyte activation, and PrPsc accumulation do not appear to be necessary or sufficient for the development of clinical symptoms up to the late disease stage.
The present study also showed that PrPsc is often detectable preclinically before the appearance of TSE-specific vacuoles and in the case of tga20 mice does not always correlate with the observation of vacuoles even in the late stage of the disease
Klinik und Pathologie von Scrapie im Mausmodell bei unterschiedlichen Inokulationsrouten
No full textIn der vorliegenden Arbeit wurden die Zusammenhänge zwischen dem Verlauf von Scrapie und der Ausbildung Scrapie-typischer Symptome sowie zwischen der Ausbildung TSE-spezifischer Vakuolen, einer Astrozytose und der Akkumulation von PrPsc im Mausmodell charakterisiert. Dazu wurden Mäuse zweier Wildtyp-Linien (CD-1 und C57/Bl6) und Tiere des transgenen tga20-Mausstammes mit einem 1%igen bzw. 10%igen Gehirnhomogenat des maus-adaptierten Prionen-Stammes Rocky Mountain Laboratory (RML) intrazerebral (ic) intraperitoneal (ip) und oral inokuliert. Tga20-Mäuse überexprimieren das zelluläre Prion-Protein um das ca. Zehnfache im Gehirn verglichen mit Wildtyp-Mäusen und weisen daher kürzere Inkubationszeiten auf. Es wurden der Verlauf der Erkrankung sowie die klinischen Symptome dokumentiert und die Gehirne der Tiere in sowohl präklinischen Krankheitsstadien als auch mit einsetzenden Symptomen und im späten Stadium der Erkrankung untersucht. Neben der ic-Inokulation wurden erstmals auch zwei periphere Inokulationswege detailliert für die tga20-Mäuse analysiert.Die Beurteilung der Vakuolisierung erfolgte am histologischen Schnitt nach HE-Färbung. Die Ausbildung einer Astrozytose wurde anhand der Astrozytenzahl durch Immunhistochemie und anhand der GFAP-Genexpression durch RT-PCR und Northern Blot Analyse nachgewiesen. Die Akkumulation von PrPsc wurde am histologischen Schnitt der Gehirne nach Immunhistochemie und durch Western Blot Analyse ausgewertet.Tga20-Mäuse wiesen nach allen drei Inokulationswegen verkürzte Inkubationszeiten auf verglichen mit den Wildtyp-Mäusen. Bei der klinischen Beurteilung der Tiere fielen Unterschiede in der Symptomatik zwischen den drei Mausstämmen auf. Wichtigste Symptome der beiden Wildtyp-Mausstämme waren eine fortschreitende Ataxie, Abmagerung und Lethargie, die Ausbildung eines stumpfen und struppigen Felles sowie eine Kyphose. C57/Bl6-Mäuse wiesen außerdem in unregelmäßigen Abständen auftretende Zuckungen der Hautmuskulatur im Bereich des Rückens sowie eine Parese der Hintergliedmaßen mit lateraler Abspreizung der Hinterfüße auf. Nach ic-Inokulation waren die Symptome der tga20-Mäuse ähnlich denen der CD-1-Mäuse. Dagegen zeigten tga20-Mäuse nach den beiden peripheren Inokulationswegen eine extrem langgestreckte Körperhaltung ohne Ausbildung einer Kyphose. Es konnte somit erstmals gezeigt werden, dass die Inokulation eines bestimmten Prionen-Stammes zu unterschiedlichen klinischen Ausprägungen der Krankheit innerhalb der tga20-Mauslinie führt, dass also die klinischen Symptome abhängig von der Inokulationsroute waren. Die Untersuchung der neuropathologischen Veränderungen in den Gehirnen der Tiere zeigte, dass die tga20-Mäuse im späten Stadium der Erkrankung eine geringere Vakuolisierung und eine schwächere astrozytäre Gliose ausbilden sowie weniger PrPsc akkumulieren verglichen mit den Wildtyp-Mäusen im vergleichbaren Krankheitsstadium. Dabei war dieser Unterschied nach den beiden peripheren Inokulationsrouten deutlicher als nach intrazerebraler Inokulation. Zudem konnte bereits in präklinischen Krankheitsstadien in den Gehirnen der Tiere beider Wildtyp-Mausstämme regelmäßig eine deutliche Ausbildung TSE-spezifischer Vakuolen sowie eine Astrozytose und PrPsc-Akkumulation festgestellt werden. Dies war in den Gehirnen der tga20-Mäuse nicht der Fall. Die präklinischen Veränderungen bei den Wildtyp-Mäusen waren meist stärker ausgeprägt als bei klinisch kranken tga20-Mäusen. Demnach scheint die Ausbildung von spongiformen Läsionen sowie eine Astrozytenaktivierung und die Akkumulation von PrPsc weder notwendig noch ausreichend zu sein für die Ausbildung von klinischen Symptomen bis hin zum späten Krankheitsstadium. In der vorliegenden Studie konnte außerdem gezeigt werden, dass PrPsc präklinisch häufig vor dem Auftreten von TSE-spezifischen Vakuolen nachweisbar ist und bei den tga20-Mäusen auch im späten Krankheitsstadium nicht immer mit dem Nachweis von Vakuolen korreliert.The present study characterized the relationships between the course of scrapie and the development of typical scrapie symptoms as well as between the development of TSE-specific vacuoles, astrocytosis, and the accumulation of PrPsc in the mouse model. Mice of two wild-type lines (CD-1 and C57/Bl6) and mice of the transgenic tga20 strain were inoculated intracerebrally (ic), intraperitoneally (ip), and orally with a 1% and 10% brain homogenate of the mouse-adapted Rocky Mountain Laboratory (RML) prion strain. Tga20 mice overexpress cellular prion protein in the brain by a factor of ten in comparison to wild-type mice and therefore exhibit shorter incubation times. The course of the disease and the clinical symptoms were documented, and the animals brains were examined during the preclinical stages, after the onset of symptoms, and in the late stage of the disease. In addition to ic-inoculation, two peripheral inoculation routes were investigated for the first time in detail in tga20 mice.Vacuolization was evaluated in HE-stained histologic sections. The development of astrocytosis was ascertained on the basis of the number of astrocytes as determined by immunohistochemical staining and on the basis of GFAP gene expression as determined by RT-PCR and northern blot analysis. PrPsc accumulation was assessed in histologic brain sections by immunohistochemical staining and western blotting.After all three inoculation routes the tga20 mice exhibited shortened inoculation times in comparison to their wild-type counterparts. Clinical evaluation of the animals revealed differences between the symptoms in the three mouse strains. The chief symptoms of the two wild-type mouse strains were progressive ataxia, emaciation, lethargy, kyphosis and dull, rough fur. The C57/Bl6 mice also showed convulsions of the skin muscles on the back at irregular intervals as well as paresis of the hind legs with lateral spreading of the hind feet. Following ic-inoculation the symptoms in the tga20 mice were similar to those in the CD-1 mice. By contrast, after the two peripheral inoculation methods, the tga20 mice exhibited an extremely elongated posture without development of kyphosis. Thus, it was shown for the first time that inoculation of a specific prion strain leads to different clinical manifestations of the disease within the tga20 mouse strain, with the clinical symptoms depending on the inoculation route.Examination of the neuropathologic changes in the animals brains showed that in the late stage of the disease the tga20 mice had less pronounced vacuolization, astrocytosis, and PrPsc accumulation than the wild-type mice in a comparable disease stage. This difference was more pronounced after the two peripheral inoculation routes than after ic-inoculation. Moreover, marked development of TSE-specific vacuoles, astrocytosis, and PrPsc accumulation were observed in the brains of both wild-type strains even in the preclinical disease stages. This was not the case in the tga20 mice. The preclinical changes were generally more pronounced in the wild-type mice than in the clinically affected tga20 mice. Hence, spongiform lesion development, astrocyte activation, and PrPsc accumulation do not appear to be necessary or sufficient for the development of clinical symptoms up to the late disease stage. The present study also showed that PrPsc is often detectable preclinically before the appearance of TSE-specific vacuoles and in the case of tga20 mice does not always correlate with the observation of vacuoles even in the late stage of the disease
Mutation of the cell cycle regulator p27kip1 drives pseudohypoxic pheochromocytoma development.
No full textBACKGROUND: Pseudohypoxic tumors activate pro-oncogenic pathways typically associated with severe hypoxia even when sufficient oxygen is present, leading to highly aggressive tumors. Prime examples are pseudohypoxic pheochromocytomas and paragangliomas (p-PPGLs), neuroendendocrine tumors currently lacking effective therapy. Previous attempts to generate mouse models for p-PPGLs all failed. Here, we describe that the rat MENX line, carrying a Cdkn1b (p27) frameshift-mutation, spontaneously develops pseudohypoxic pheochromocytoma (p-PCC). METHODS: We compared rat p-PCCs with their cognate human tumors at different levels: histology, immunohistochemistry, catecholamine profiling, electron microscopy, transcriptome and metabolome. The vessel architecture and angiogenic potential of pheochromocytomas (PCCs) was analyzed by light-sheet fluorescence microscopy ex vivo and multi-spectral optoacoustic tomography (MSOT) in vivo. RESULTS: The analysis of tissues at various stages, from hyperplasia to advanced grades, allowed us to correlate tumor characteristics with progression. Pathological changes affecting the mitochrondrial ultrastructure where present already in hyperplasias. Rat PCCs secreted high levels of norepinephrine and dopamine. Transcriptomic and metabolomic analysis revealed changes in oxidative phosphorylation that aggravated over time, leading to an accumulation of the oncometabolite 2-hydroxyglutarate, and to hypermethylation, evident by the loss of the epigenetic mark 5-hmC. While rat PCC xenografts showed high oxygenation, induced by massive neoangiogenesis, rat primary PCC transcriptomes possessed a pseudohypoxic signature of high Hif2a, Vegfa, and low Pnmt expression, thereby clustering with human p-PPGL. CONCLUSION: Endogenous rat PCCs recapitulate key phenotypic features of human p-PPGLs. Thus, MENX rats emerge as the best available animal model of these aggressive tumors. Our study provides evidence of a link between cell cycle dysregulation and pseudohypoxia
Correlation of in vivo imaging to morphomolecular pathology in translational research: challenge accepted
No full textAbstract Correlation of in vivo imaging to histomorphological pathology in animal models requires comparative interdisciplinary expertise of different fields of medicine. From the morphological point of view, there is an urgent need to improve histopathological evaluation in animal model-based research to expedite translation into clinical applications. While different other fields of translational science were standardized over the last years, little was done to improve the pipeline of experimental pathology to ensure reproducibility based on pathological expertise in experimental animal models with respect to defined guidelines and classifications. Additionally, longitudinal analyses of preclinical models often use a variety of imaging methods and much more attention should be drawn to enable for proper co-registration of in vivo imaging methods with the ex vivo morphological read-outs. Here we present the development of the Comparative Experimental Pathology (CEP) unit embedded in the Institute of Pathology of the Technical University of Munich during the Collaborative Research Center 824 (CRC824) funding period together with selected approaches of histomorphological techniques for correlation of in vivo imaging to morphomolecular pathology
Imaging pheochromocytoma in small animals: Preclinical models to improve diagnosis and treatment.
No full textPheochromocytomas (PCCs) and paragangliomas (PGLs), together referred to as PPGLs, are rare chromaffin cell-derived tumors. They require timely diagnosis as this is the only way to achieve a cure through surgery and because of the potentially serious cardiovascular complications and sometimes life-threatening comorbidities that can occur if left untreated. The biochemical diagnosis of PPGLs has improved over the last decades, and the knowledge of the underlying genetics has dramatically increased. In addition to conventional anatomical imaging by CT and MRI for PPGL detection, new functional imaging modalities have emerged as very useful for patient surveillance and stratification for therapy. The availability of validated and predictive animal models of cancer is essential for translating molecular, imaging and therapy response findings from the bench to the bedside. This is especially true for rare tumors, such as PPGLs, for which access to large cohorts of patients is limited. There are few animal models of PPGLs that have been instrumental in refining imaging modalities for early tumor detection, as well as in identifying and evaluating novel imaging tracers holding promise for the detection and/or treatment of human PPGLs. The in vivo PPGL models mainly include xenografts/allografts generated by engrafting rat or mouse cell lines, as no representative human cell line is available. In addition, there is a model of endogenous PCCs (i.e., MENX rats) that was characterized in our laboratory. In this review, we will summarize the contribution that various representative models of PPGL have given to the visualization of these tumors in vivo and we present an example of a tracer first evaluated in MENX rats, and then translated to the detection of these tumors in human patients. In addition, we will illustrate briefly the potential of ex vivo biological imaging of intact adrenal glands in MENX rats
Combining [177Lu]Lu-DOTA-TOC PRRT with PARP inhibitors to enhance treatment efficacy in small cell lung cancer
No full text<jats:title>Abstract</jats:title><jats:sec>
<jats:title>Purpose</jats:title>
<jats:p>Small cell lung cancer (SCLC) is a highly aggressive tumor with neuroendocrine origin. Although SCLC frequently express somatostatin receptor type 2 (SSTR2), a significant clinical benefit of SSTR2-targeted radionuclide therapies of SCLC was not observed so far. We hypothesize that combination treatment with a PARP inhibitor (PARPi) could lead to radiosensitization and increase the effectiveness of SSTR2-targeted therapy in SCLC.</jats:p>
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<jats:title>Methods</jats:title>
<jats:p>SSTR2-ligand uptake of the SCLC cell lines H69 and H446 was evaluated in vitro using flow cytometry, and in vivo using SPECT imaging and cut-and-count biodistribution. Single-agent (Olaparib, Rucaparib, [<jats:sup>177</jats:sup>Lu]Lu-DOTA-TOC) and combination treatment responses were determined in vitro via cell viability, clonogenic survival and γH2AX DNA damage assays. In vivo, we treated athymic nude mice bearing H69 or H446 xenografts with Olaparib, Rucaparib, or [<jats:sup>177</jats:sup>Lu]Lu-DOTA-TOC alone or with combination treatment regimens to assess the impact on tumor growth and survival of the treated mice.</jats:p>
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<jats:title>Results</jats:title>
<jats:p>H446 and H69 cells exhibited low SSTR2 expression, i.e. 60 to 90% lower uptake of SSTR2-ligands compared to AR42J cells. In vitro, combination treatment of [<jats:sup>177</jats:sup>Lu]Lu-DOTA-TOC with PARPi resulted in 2.9- to 67-fold increased potency relative to [<jats:sup>177</jats:sup>Lu]Lu-DOTA-TOC alone. We observed decreased clonogenic survival and higher amounts of persistent DNA damage compared to single-agent treatment for both Olaparib and Rucaparib. In vivo, tumor doubling times increased to 1.6-fold (H446) and 2.2-fold (H69) under combination treatment, and 1.0 to 1.1-fold (H446) and 1.1 to 1.7-fold (H69) in monotherapies compared to untreated animals. Concurrently, median survival was higher in the combination treatment groups in both models compared to monotherapy and untreated mice. Fractionating the PRRT dose did not lead to further improvement of therapeutic outcome.</jats:p>
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<jats:title>Conclusion</jats:title>
<jats:p>The addition of PARPi can markedly improve the potency of SSTR2-targeted PRRT in SCLC models in SSTR2 low-expressing tumors. Further evaluation in humans seems justified based on the results as novel treatment options for SCLC are urgently needed.</jats:p>
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<jats:title>Graphical Abstract</jats:title>
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3D histopathology of human tumours by fast clearing and ultramicroscopy
No full textHere, we describe a novel approach that allows pathologists to three-dimensionally analyse malignant tissues, including the tumour-host tissue interface. Our visualization technique utilizes a combination of ultrafast chemical tissue clearing and light-sheet microscopy to obtain virtual slices and 3D reconstructions of up to multiple centimetre sized tumour resectates. For the clearing of tumours we propose a preparation technique comprising three steps: (a) Fixation and enhancement of tissue autofluorescence with formalin/5-sulfosalicylic acid. (b) Ultrafast active chemical dehydration with 2,2-dimethoxypropane and (c) refractive index matching with dibenzyl ether at up to 56 °C. After clearing, the tumour resectates are imaged. The images are computationally post-processed for contrast enhancement and artefact removal and then 3D reconstructed. Importantly, the sequence a–c is fully reversible, allowing the morphological correlation of one and the same histological structures, once visualized with our novel technique and once visualized by standard H&E- and IHC-staining. After reverting the clearing procedure followed by standard H&E processing, the hallmarks of ductal carcinoma in situ (DCIS) found in the cleared samples could be successfully correlated with the corresponding structures present in H&E and IHC staining. Since the imaging of several thousands of optical sections is a fast process, it is possible to analyse a larger part of the tumour than by mechanical slicing. As this also adds further information about the 3D structure of malignancies, we expect that our technology will become a valuable addition for histological diagnosis in clinical pathology
