Bone Marrow Irradiation and Replacement
Bone marrow is isolated from donor mice (either genetically manipulated, e.g. CCR2 -/- or ROCK-1 -/- mice, or mice carrying a marker construct such as lacZ) by flushing the tibia and femur with cold Hanks’ Balanced Salt Solution including two percent fetal bovine serum and 10 mM HEPES. Cells are pressed through an 18 gauge needle to create a single cell suspension before they are filtered through a 70 µm cell strainer. Cells are counted and diluted to the appropriate volume in flushing buffer.
Recipient mice (C57BL/6, age 6-8 weeks) are irradiated (10 Gy in 2 sessions at least 2 h apart) according to standard procedures. 1x106 white blood cells (in a volume of 300 µl) from donor mice are immediately injected retro orbital into recipient mice. The chimeric mice are allowed to recover, and are closely monitored on a daily basis during the first 14 days, then once a week. 6-8 weeks after bone marrow transplantation, the animals appear to have been fully recovered.
This protocol was utilized in our previous experiments with Dr. Goodell who is a long-term collaborator [Goodell et al, 2001; Jackson et al, 2001] and our recent studies of non-adaptive fibrosis [Haudek et al, 2006] will consult in these studies.
Determination of Engraftment
Peripheral blood from chimeric mice is isolated via retro orbital bleeding, and white blood cells are separated. Cells are stained with antibodies against the marker construct (CCR2, ROCK-1 or fluorescein di(β-D-galactopyranoside) in case of lacZ staining) and analyzed by flow cytometry. Approximately 8-10 weeks following bone marrow transplantation, mice with ³ 60 percent engraftment are randomly assigned to treatment groups and are subjected to heart surgery according to above described protocols.
Cells, nucleotide vectors, autocoids and pharmaceutical agents are frequently administered or infused in the various cardiovascular models described above. The laboratory has experience with providing this technology for a variety of different purposes as follows.
Animals are instrumented on a chronic basis with a catheter in the left jugular vein that allows for injection or infusion. This technique has been most frequently used for the delivery of cells into the circulation which is the cardiac precursor cells utilized in our collaboration with Dr. Michael Schneider [Oh et al, 2003; Oh et al, 2004].
Aortic Root Injection
Acute injection into the aortic root to favor coronary distribution is done acutely in the open-chested mouse to allow a very brief aortic occlusion at the time of injection. This technique has been utilized to inject plasmids and viral vectors and has been successful in achieving cardiac expression of intended genes. This method has not been published and the work is proprietary with our corporate collaborators.
Direct Cardiac Injection
Cells and plasmids have been injected directly into the prewall of the left ventricle at the level of the mid-left anterior descending coronary artery. This technique has been performed in an area of myocardial infarction at the LAD obstruction and in normal myocardium. While these studies have been successful, the degree of attendant myocardial injury has not been sufficiently predictable; we are currently improving this technique.
Alzet Pump for Chronic Infusion
This technique is available in the laboratory and is currently under active use in our studies in angiotensin injury to the heart.