26 research outputs found
Concise review : increasing the validity of cerebrovascular disease models and experimental methods for translational stem cell research
Interspecies differences, anatomical and physiological aspects, as wells as simplified study designs contribute to an overestimation of treatment effects and limit the transferability of experimental results into clinical applications. Confounders of cell therapies for cerebrovascular disorders (CVD) include common CVD comorbidities, frequent medications potentially affecting endogenous and transplanted stem cells, as well as ageâ and immuneâsystemârelated effects. All those can contribute to a substantial modeling bias, ultimately limiting the prospective quality of preclinical research programs regarding the clinical value of a particular cell therapy. In this review, we discuss the nature and impact of most relevant confounders. We provide suggestions on how they can be considered to enhance the validity of CVD models in stem cell research. Acknowledging substantial and sometimes surprising effects of housing conditions, chronobiology, and intersex differences will further augment the translational value of animal models. We finally discuss options for the implementation of highâquality functional and imaging readout protocols. Altogether, this might help to gain a more holistic picture about the therapeutic impact of a particular cell therapy for CVD, but also on potential side and offâsite effects of the intervention
Frameless Stereotaxy in Sheep - Neurosurgical and Imaging Techniques for Translational Stroke Research
Bone marrow-derived mononuclear cells do not exert acute neuroprotection after stroke in spontaneously hypertensive rats
Bone marrow-derived mononuclear cells (BM-MNCs) were shown to improve the outcome in animal stroke models and clinical pilot studies on BM-MNCs for stroke patients were already conducted. However, relevant aspects of pre-clinical evaluation, such as the use of animals with comorbidities and dose-response studies, were not thoroughly addressed so far. We therefore investigated different BM-MNC doses in the clinical meaningful stroke model of spontaneously hypertensive (SH) rats. Three hours after the onset of transient middle cerebral artery occlusion (MCAO) animals received either one of three syngeneic BM-MNC doses or placebo intravenously. The primary endpoint was the infarct size. Secondary endpoints included functional outcome, mortality, inflammatory processes, and the dose-response relationship. In contrast to previous studies which used healthy animals no beneficial effect of BM-MNCs was found. Infarct volumes, mortality, behavioral outcomes, and the extent of the inflammatory response to cerebral ischemia were comparable in all groups. In conclusion, we could not demonstrate that early BM-MNC treatment improves the outcome after stroke in SH rats. Whether BM-MNCs improve neurological recovery after delayed treatment initiation was not investigated in the present study, but our data indicates that this should be determined in co-morbid animal stroke models before moving to large-scale clinical studies. Future preclinical stroke studies on co-morbid animals should also include groups of healthy animals in order to determine whether negative results can be attributed to the comorbid condition
Allogeneic Non-Adherent Bone Marrow Cells Facilitate Hematopoietic Recovery but Do Not Lead to Allogeneic Engraftment
Background Non adherent bone marrow derived cells (NA-BMCs) have recently been described to give rise to multiple mesenchymal phenotypes and have an impact in tissue regeneration. Therefore, the effects of murine bone marrow derived NA-BMCs were investigated with regard to engraftment capacities in allogeneic and syngeneic stem cell transplantation using transgenic, human CD4+, murine CD4?/?, HLA-DR3+ mice. Methodology/Principal Findings Bone marrow cells were harvested from C57Bl/6 and Balb/c wild-type mice, expanded to NA-BMCs for 4 days and characterized by flow cytometry before transplantation in lethally irradiated recipient mice. Chimerism was detected using flow cytometry for MHC-I (H-2D[b], H-2K[d]), mu/huCD4, and huHLA-DR3). Culturing of bone marrow cells in a dexamethasone containing DMEM medium induced expansion of non adherent cells expressing CD11b, CD45, and CD90. Analysis of the CD45+ showed depletion of CD4+, CD8+, CD19+, and CD117+ cells. Expanded syngeneic and allogeneic NA-BMCs were transplanted into triple transgenic mice. Syngeneic NA-BMCs protected 83% of mice from death (n = 8, CD4+ donor chimerism of 5.8±2.4% [day 40], P<.001). Allogeneic NA-BMCs preserved 62.5% (n = 8) of mice from death without detectable hematopoietic donor chimerism. Transplantation of syngeneic bone marrow cells preserved 100%, transplantation of allogeneic bone marrow cells 33% of mice from death. Conclusions/Significance NA-BMCs triggered endogenous hematopoiesis and induced faster recovery compared to bone marrow controls. These findings may be of relevance in the refinement of strategies in the treatment of hematological malignancies
Effizienz einer Kombinationstherapie aus G-CSF und mononukleÀren Knochenmarkzellen in einem prÀklinischen Schlaganfallmodell
Eine Vielzahl prĂ€klinischer Schlaganfallstudien zeigte die neuroprotektive und neuroregenerative Wirkung des hĂ€matopoetischen Wachstumsfaktors G-CSF (Granulozyten-Kolonie stimulierender Faktor). Ein Wirkungsmechanismus des G-CSF ist die Mobilisation von protektiven Knochen-markzellen in die ischĂ€mische LĂ€sion, wobei diese zeitverzögert nach G-CSF-Gabe stattfindet. Eine zusĂ€tzliche frĂŒhzeitige Transplantation mononukleĂ€rer Knochenmarkzellen (BM MNC) könnte diese therapeutische LĂŒcke fĂŒllen. Ziel der vorliegenden Studie war es, die Wirksamkeit dieser Kombinations-therapie in einem Schlaganfallmodell der spontan hypertensiven Ratte (SHR) zu testen. Syngene BM MNC wurden aus dem Knochenmark von SHRs durch immunmagnetische Depletion der Granulozyten isoliert. Nach Verschluss der Arteria cerebri media wurde den Tieren ĂŒber insgesamt 5 Tage G-CSF verabreicht und zusĂ€tzlich zu einem frĂŒhen (6h nach Schlaganfall) oder spĂ€teren (48h nach Schlaganfall) Zeitpunkt BM MNC intravenös appliziert. Unbehandelte Schlaganfalltiere sowie Tiere mit alleiniger G-CSF-Therapie dienten als Kontrolle. Das Infarktvolumen wurde weder durch die alleinige G-CSF-Gabe noch durch die zusĂ€tzliche Zelltherapie verĂ€ndert. Dennoch wiesen Tiere mit G-CSF-Einzeltherapie eine anhaltende funktionelle Verbesserung des sensomotorischen Defizites auf. WĂ€hrend die zusĂ€tzliche frĂŒhzeitige Zelltransplantation (6h) keinen weiteren Therapieeffekt zeigte, fĂŒhrte die Zelltransplantation nach 48h zu einer Aufhebung des protektiven G-CSF Effektes. Die G-CSF-Therapie bewirkte erwartungsgemÀà einen deutlichen Anstieg der zirkulierenden Leukozyten. Interessanterweise wurde der Granulozytengehalt im Blut und in der Milz durch die einmalige Zelltherapie nach 48h signifikant erhöht. Ein GroĂteil der transplantierten BM MNC (48h) konnte in der Milz nachgewiesen werden und fĂŒhrte dort vermutlich zu einer kompetitiven Hemmung des Granulozytenabbaus. Dies hatte sowohl den Anstieg der zirkulierenden Granulozyten als auch deren vermehrte Infiltration in das ischĂ€mische Hirngewebe zur Folge und könnte schlieĂlich den negativen Einfluss auf die funktionelle Verbesserung erklĂ€ren. Die beobachteten Interaktionsmechanismen werfen ein interessantes Licht auf die mögliche Wirkungsweise von Zelltherapien und unterstreichen die entscheidende Rolle des Immunsystems in der Pathophysiologie des Schlaganfalls
Effizienz einer Kombinationstherapie aus G-CSF und mononukleÀren Knochenmarkzellen in einem prÀklinischen Schlaganfallmodell
Eine Vielzahl prĂ€klinischer Schlaganfallstudien zeigte die neuroprotektive und neuroregenerative Wirkung des hĂ€matopoetischen Wachstumsfaktors G-CSF (Granulozyten-Kolonie stimulierender Faktor). Ein Wirkungsmechanismus des G-CSF ist die Mobilisation von protektiven Knochen-markzellen in die ischĂ€mische LĂ€sion, wobei diese zeitverzögert nach G-CSF-Gabe stattfindet. Eine zusĂ€tzliche frĂŒhzeitige Transplantation mononukleĂ€rer Knochenmarkzellen (BM MNC) könnte diese therapeutische LĂŒcke fĂŒllen. Ziel der vorliegenden Studie war es, die Wirksamkeit dieser Kombinations-therapie in einem Schlaganfallmodell der spontan hypertensiven Ratte (SHR) zu testen. Syngene BM MNC wurden aus dem Knochenmark von SHRs durch immunmagnetische Depletion der Granulozyten isoliert. Nach Verschluss der Arteria cerebri media wurde den Tieren ĂŒber insgesamt 5 Tage G-CSF verabreicht und zusĂ€tzlich zu einem frĂŒhen (6h nach Schlaganfall) oder spĂ€teren (48h nach Schlaganfall) Zeitpunkt BM MNC intravenös appliziert. Unbehandelte Schlaganfalltiere sowie Tiere mit alleiniger G-CSF-Therapie dienten als Kontrolle. Das Infarktvolumen wurde weder durch die alleinige G-CSF-Gabe noch durch die zusĂ€tzliche Zelltherapie verĂ€ndert. Dennoch wiesen Tiere mit G-CSF-Einzeltherapie eine anhaltende funktionelle Verbesserung des sensomotorischen Defizites auf. WĂ€hrend die zusĂ€tzliche frĂŒhzeitige Zelltransplantation (6h) keinen weiteren Therapieeffekt zeigte, fĂŒhrte die Zelltransplantation nach 48h zu einer Aufhebung des protektiven G-CSF Effektes. Die G-CSF-Therapie bewirkte erwartungsgemÀà einen deutlichen Anstieg der zirkulierenden Leukozyten. Interessanterweise wurde der Granulozytengehalt im Blut und in der Milz durch die einmalige Zelltherapie nach 48h signifikant erhöht. Ein GroĂteil der transplantierten BM MNC (48h) konnte in der Milz nachgewiesen werden und fĂŒhrte dort vermutlich zu einer kompetitiven Hemmung des Granulozytenabbaus. Dies hatte sowohl den Anstieg der zirkulierenden Granulozyten als auch deren vermehrte Infiltration in das ischĂ€mische Hirngewebe zur Folge und könnte schlieĂlich den negativen Einfluss auf die funktionelle Verbesserung erklĂ€ren. Die beobachteten Interaktionsmechanismen werfen ein interessantes Licht auf die mögliche Wirkungsweise von Zelltherapien und unterstreichen die entscheidende Rolle des Immunsystems in der Pathophysiologie des Schlaganfalls
Isolation and flow cytometric analysis of immune cells from the ischemic mouse brain
Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed
Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain
Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed
Sterile inflammation after permanent distal MCA occlusion in hypertensive rats
The pathophysiology of stroke is governed by immune reactions within and remote from the injured brain. Hypertension, a major cause and comorbidity of stroke, entails systemic vascular inflammation and may influence poststroke immune responses. This aspect is, however, underestimated in previous studies. Here we aimed to delineate the sequence of cellular inflammation after stroke in spontaneously hypertensive (SH) rats. Spontaneously hypertensive rats were subjected to permanent middle cerebral artery occlusion and killed after 1 or 4 days. Immune cells of the peripheral blood and those which have infiltrated the injured brain were identified and quantified by flow cytometry. The spatial distribution of myeloid cells and T lymphocytes, and the infarct volume were assessed by histology. We observed a concerted infiltration of immune cells into the ischemic brain of SH rats. At day 1, primarily neutrophils, monocytes, macrophages, and myeloid dendritic cells entered the brain, whereas the situation at day 4 was dominated by microglia, macrophages, lymphatic dendritic cells, and T cells. Postischemic inflammation did not cause secondary tissue damage during the subacute stage of experimental stroke in SH rats. Considering the intrinsic vascular pathology of SH rats, our study validates this strain for further translational research in poststroke inflammation
Flow cytometric characterization of brain dendritic cell subsets after murine stroke
Background
Sterile inflammation is a substantial element of post-stroke pathophysiology with the determination of autoimmunity versus tolerance being one of its most important aspects. It is believed that this determination is initiated relatively early after stroke onset by clearing macrophages and migratory dendritic cells (DC). However, the phenotypic differentiation of macrophages and DC is intricate particularly in the disease context. Here, we utilized a set of surface markers used in mucosal immunity research to investigate the involvement of macrophages and DC subpopulations in post-stroke inflammation in mice.
Findings
Photothrombotic stroke induced a significant increase of lineage (CD3, B220, Ly6G and CD49b) negative CD11b+âcells in the brain primarily consisting of F4/80+ macrophages and, to a lesser extent, F4/80-/CD11c-/CD11b+âmonocytes and F4/80-/CD11c+âDC. The latter could be differentiated into the classical migratory DC subpopulations (CD11b+âand CD103+), but no CD4 or CD8+ DC were found. Finally, stroke caused a significant increase of CD11b/CD103 double-positive DC in the affected brain hemisphere.
Conclusions
The surface marker combination used in this study allowed a phenotypic differentiation of macrophages and DC subpopulations after stroke, thus providing an important prerequisite to study post-stroke immunity and tolerance