Key words: angiogenesis --cultured endothelial cells --[3H]thymidine autoradiography -endothelial cell growth factor --rat brain development Brain capillary proliferation in postnatal rats was measured in vivo by [3H]thymidine autoradiography. Maximal capillary proliferation occurred between 5 and 9 postnatal days, and was 40 times greater than in the adult. To test the hypothesis that soluble angiogenesis factors play a role in this developmental vascularization of brain, we prepared extracts from the brains of 6-day-old rats at the peak of proliferative activity, and from adults when it was lowest. We assayed them using an in vitro growth system measuring [3H]thymidine incorporation into cultured brain capillary endothelial cells. Extracts prepared from either 6-day or adult rats and containing 150/~g/ml protein caused more than a 4-fold stimulation of the endothelial cells, increasing to 8-fold at a concentration of 150011g/ml. The presence of growth-promoting activity in brain extracts from both adult and immature rats suggests that soluble angiogenesis factors may be present in the brain throughout life, but are unavailable for stimulation of in vivo capillary growth unless released or activated by an appropriate stimulus.
In order to study cell proliferation after ischemic infarction, a model of bilateral common carotid artery occlusion in the gerbil was developed. A comparison of survival rates after 15, 30, 45 and 60 min of occlusion revealed that 45 min was the maximum duration of ischemia after which most (72%) of the gerbils were alive at 1 week. The administration of pentobarbital (single dose, 30 mg/kg) postoperatively to badly seizing animals increased survival to 100%. Large, well-demarcated infarcts were present in posterior thalamus or midbrain in 62% of gerbils subjected to 45 min bilateral occlusion. In 60% of these animals the infarcts were unilateral; in 40% they were bilateral. To quantitate cell proliferation in the infarcts from 12 h to 25 days after ischemia, gerbils were injected with [3H]thymidine 4 h prior to sacrifice, and autoradiographs were prepared from sectioned brains. Proliferation took place from 2 to 7 days after occlusion, with a maximum of 24% labeled cells at 6 days.
Tritiated thymidine autoradiography was used to measure cellular proliferation after ischemic injury in gerbil brain. Gerbils were subjected to bilateral occlusion of the common carotid arteries which resulted in areas of necrosis, or infarcts, in the posterior thalamus or midbrain. From 12 h to 10 days following the ischemia, gerbils were injected with 3H thymidine, sacrificed 4 h later, and the brains sectioned. In order to identify astrocytes and monocytes/macrophages, immunocytochemistry was performed prior to autoradiography, using antisera against glial fibrillary acidic protein and endothelial-monocyte reticuloendothelial antigen, respectively. Immunocytochemistry was also used to visualize microvessel laminin, myelin, and leakage of serum albumin. Lastly, a histochemical procedure for acid phosphatase activity was employed to verify cellular phagocytic activity in the wound. A reproducible sequence of reactions took place during the first 10 days after ischemia. Early changes included leakage of albumin and myelin breakdown, followed by arrival of monocytes at 2 days and their differentiation into macrophages by 5 days. These cells exhibited intense proliferation from 2 to 6 days post-ischemia. Microvessel endothelial cells were maximally labeled at 4 days post-ischemia. Hypertrophied astrocytes were apparent at 2 days and proliferated from 3 to 7 days post-ischemia, and by 10 days the wound was replaced by a "glial scar".
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