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Cerebral ischemia and umbilical stem cell transplantation in chronically prepared fetal sheep



CEREBRAL ISCHEMIA AND UMBILICAL STEM CELL TRANSPLANTATION IN CHRONICALLY PREPARED FETAL SHEEP Y. GARNIER1, H.-M. VAIHINGER1, J. MIDDELANIS1, O. BRÜSTLE2, A. JENSEN1 1Department of Obstetrics & Gynecology, University of Bochum 2Institute of Reconstructive Neurobiology, University of Bonn Objective: Hypoxic-ischemic cerebral damage is an important contributor to perinatal mortality and morbidity including long-term neurological sequalae in term and preterm fetuses. Hypoxic-ischemic insults are usually causing parasagittal brain lesions and affect the parietal and occipital regions in particular. This form of damage has been reproduced in the chronically prepared fetal sheep model, which has been in use for years in Bochum. Experimental evidence has shown that neuroprotective strategies using pharmacologic agents may alleviate perinatal brain damage. However, so far no convincing strategies are available for regeneration of damaged nervous structures in the perinatal period. Therefore, one of the most urgent tasks for scientists and clinicians will be to explore the enormous potential of cell replacement therapies using stem cells in general and umbilical cord blood stem cells in particular to provide a future therapeutic approach for perinatal neuronal repair. Material and Methods: Fetal sheep will be chronically instrumented at 0.7 of gestation. The ewe will be anesthetized by subarachnoid injection of 0.75% bupivacaine at the lower spine, and will be operated under sterile conditions. The fetal hindlimbs will be exposed through a small incision of the uterus. Using local anesthesia polyvinyl catheters will be inserted into the inferior vena cava of the fetus. Furthermore, a catheter will be placed in a cotyledonary vein and its tip will be advanced to the umbilical vein. The uterine incision will be closed and a second uterine incision will be performed over the fetal snout in order to exteriorize the head and neck of the fetus. Catheters will be inserted into the fetal ascending aorta via the right brachial artery. Furthermore, both fetal common carotic arteries will be prepared. After measurements of blood gases and acid base balance global cerebral ischemia will be induced by bilateral occlusion of the carotid arteries for 30 min. All catheters will be filled with heparin, plugged, and passed subcutaneously to the ewe’s flank, where they will be exteriorized and protected by a pouch sewn to the skin. Sampling of fetal blood and flushing of the catheters will be repeated daily. At +24 h, approx. 1-2 x 107 mononuclear cells prepared from human umbilical cord blood will be intravenously transfused to the fetus at a rate of 0.5 ml/min (10 ml total volume). The delay between the ischemic insult and the transplantation of cells will be extended up to two weeks in further experiments. At the end of the experiment (at +120 h or later in further experiments) the ewe will be anaesthetized for perfusion fixation. The brain will be dissected and immediately placed in ice-cold fixative without removal of the meninges. Furthermore, organ samples will be taken from spleen, liver, lung, bone marrow and cotyledons. Results: In a promising pilot experiment, cerebral ischemia was induced by occlusion of both carotid arteries in utero in chronically prepared fetal sheep. One day after the insult, mononuclear cells from human umbilical cord blood were applied to the fetus via the umbilical vein. Three days after transplantation, the fetal brain was examined for both damage and evidence of incorporated human cells. Preliminary evaluation indicates Page 2 severe parasaggital neuronal degeneration in the host cortex. DNA in situ hybridization detecting a human alu-repeat element revealed numerous labeled cells, some of which also expressed the neural progenitor specific marker glial fibrillary acidic protein (GFAP). Conclusion and Outlook: Our preliminary results point to the migration of transplanted cells and, furthermore, indicate that differentiation commences in vivo. Thus, the planned series of studies is essential to solve important questions regarding therapeutical approaches using umbilical cord blood derived stem cells for the regeneration of damage of the nervous system. (PDF) Garnier et al., 2002 Abstract Symposium 2002, p. 7 and 8. Available from: [accessed Sep 24 2020].
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