Cellular metabolic alterations after focal cerebral ischemia with or without bone marrow transplantation determined by cytochrome oxidase histochemistry
To determine the degree and extent of changes in cellular metabolic demand after stroke and bone marrow cell transplantation, a histochemistry assay of cytochrome oxidase (COx) which correlates with neuronal activity was employed,. Adult Wistar rats (n=9) were subjected to transient (2 h) middle cerebral artery occlusion (MCAo). At 1 d after ischemia, bone marrow stromal cells (MSCs, 4x105 in 10 :l) were transplanted intracerebrally into the ischemic boundary zone in the striatum and the cortex. The ischemia rats with (n=4) or without (n=5) MSC transplantation were sacrificed at 14 d after MCAo. Bone marrow cells were harvested from normal donor adult rats and cultured in Iscove s Modified Dulbecco s medium supplemented with 10% fetal bovine serum. MSCs were isolated by their adherence to the plastic dishes from the whole bone marrow cells at 72 h of incubation. Subsequently, the MSCs were cultured for 2 weeks and bromodeoxyuridine (BrdU, as a tracer to identify cells derived from bone marrow) was added into the medium at 3 d before transplantation. Cellular metabolic activity in the ischemic damaged brain was measured on a standard section per animal using quantitative histochemistry of COx with the Global Lab Image Analysis system. Immunohistochemistry staining was employed to identify BrdU-MSCs. COx was absent in the ischemic core in all experimental animals. However, COx activity was intense and prominent in the areas of BrdU-MSCs, as well as the subventricular zone (SVZ) of the adult forebrain. COx (72.8% of the normal hemisphere) was significantly increased (p=0.004) in the ipsilateral hemisphere after MCAo with MSC transplantation, compared with MCAo alone (30.4%). Since MSCs contain a population of marrow stem-like cells and the SVZ of the adult forebrain contains a population of neural stem-like cells, our data indicate that COx provides cellular metabolic evidence that stem-like cells (MSCs and SVZ cells) are activated in response to ischemic brain damage.