| ObjectiveThe local cerebral cold - injury model, one kind of cortical contusion, can induce dramatic blood -brain barrier (BBB) breakdown and evident brain edema formation. Fukuda et al. have observed some morphological changes of perivascular macrophages in rat cerebral cortex of this model by transmission e-lectron microscopy.Perivascular macrophages are situated in the perivascular space of the central nervous system ( CNS). The cells express the phenotypes common to the macrophage lineage, including ED2 antigen , the scavenger receptor and MHC - class II antigens. The cells can take up horseradish peroxidase and ferritin administered via the femoral vein or cerebral ventricles. Moreover, the cells are activated in models of CNS inflammation and autoimmune disease. The scavenger and immunoregulatory function are important roles of perivascular macrophages in the CNS.The most important morphological character of perivascular macrophages is the presence of large - sized lysosomal inclusion bodies (IBs) in their cytoplasm. The IBs contain lysosomal enzymes, such as acid phosphatase and pro-teinases. Perivascular macrophages may remove the edema fluid by endocytosis, and may break down protein molecules in the IBs. Fukuda et al. reported that the activity of acid phosphatase in the IBs was decreased after cold injury. But, the quantitative changes of proteinases in the IBs have not been reported.Cathepsins are acidic proteinases residing within endosomal/lysosomal compartments , and participate in cellular protein turnover and in the degradation of proteins that enter the cell by endocytosis. Cathepsin D is the most important ly-sosomal aspartic proteinase, having been implicated in antigen processing. Therefore, the amount of cathepsin D may be relevant to the function of perivas-cular macrophages, both protein degradation and immunoregulation.The aim of this research is to reexamine the morphological changes of perivascular macrophages in cold - induced brain edema. Especially, the a-mount of cathepsin D was investigated by quantitative immunogold electron microscopic method. The functions of perivascular macrophages are also discussed.Material and Methods1. Local cerebral cold injury modelA total of 40 Wistar male rats, aged 4 months, were used in this study. Cerebral edema was induced by dry ice through a drilled hole at the parietal bone of rat for 20 minutes. As controls, sham operated animals were used. At 5 hours, 10 hours, 1 days, 2 days, 3 days, 1 week and 2 weeks after the operation, animals were perfused from the heart with a fixative containing 4% paraformaldehyde in 0.1 M phosphate buffer. Coronal sections including the injury area were used for immunofluorescence microscopy. Small pieces of cerebral cortex near the injury (2-4 mm from the injury regions) were excised, and were prepared for post - embedding immunogold labeling as well as conventional transmission electron microscopy.2. Horseradish peroxidase injectionIn order to examine the breakdown of blood - brain barrier after cold injury , three rats were received intravenous injection of 10 mg of horseradish peroxidase. Two animals were injected HRP just before cold injury, and one was fixed at 1 hour after the injury, the other was fixed at 2 hours. Another one rat was injected at 1 day after the injury, and at 2 hours after the injection, the animal was fixed as the same way as above. After rinsing with phosphate buffer, coronal sections were cut at 40 microns with a vibratome. These sections were incubated in the substrate solution. After counterstaining with 0. 1% Kernechtrot nuclearfast red, sections were observed by light microscopy.3. Transmission electron microscopySmall pieces of cerebral cortex excised from cerebral cortex near the injured area were immersed in a fixative containing 2% paraformaldehyde, 2.5% glut-araldehyde in 0. 1M phosphate buffer for 8 hours at 4 . .After rinsing briefly with cold PBS, the specimens were postfixed with 1 % osmic acid buffered with 0. 05 M phosphat... |
PDF Full Text Request