Subcellular distribution of calcium and ultrastructural changes after cerebral hypoxia-ischemia in immature rats

Recent data imply that mitochondrial regulation of calcium is critical in t he process leading to hypoxic-ischemic brain injury. The aim was to study t he subcellular distribution of calcium in correlation with ultrastructural changes after hypoxia-ischemia in neonatal rats. Seven-day-old rats were su bjected to permanent unilateral carotid artery ligation and exposure to hyp oxia (7.7% oxygen in nitrogen) for 90 min. Animals were perfusion-fixed aft er 30 min, 3 h or 24 h of reperfusion. Sections were sampled for light micr oscopy and electron microscopy combined with the oxalate-pyroantimonate tec hnique. At 30 min and 3 h of reflow, 3 progressive accumulation of calcium was detected in the endoplasmic reticulum, cytoplasm, nucleus and, most mar kedly, in the mitochondrial matrix of neurons in the gray matter in the cor e area of injury. Some mitochondria developed a considerable degree of swel ling reaching a diameter of several mum at 3 h of reflow whereas the majori ty of mitochondria appeared moderately affected. Chromatin condensation was observed in nuclei of many cells with severely swollen mitochondria with c alcium deposits. A whole spectrum of morphological features ranging from ne crosis to apoptosis was seen in degenerating cells. After 24 h, there was e xtensive injury in the cerebral cortex as judged by breaks of mitochondrial and plasma membranes, and a general decrease of cellular electron density. In the white matter of the core area of injury, the axonal elements exhibi ted varicosity-like swellings filled with calcium-pyroantimonate deposits. Furthermore, the thin myelin sheaths were loaded with calcium. Numerous oli godendroglia-like cells displayed apoptotic morphology 'with shrunken cytop lasm and chromatin condensation, whereas astroglial necrosis was not seen. In conclusion, markedly swollen 'giant' mitochondria with large amounts of calcium were found at 3 h of reperfusion often in neuronal cells with conde nsation of the nuclear chromatin. The results are discussed in relation to mitochondrial permeability transition and activation of apoptotic processes . (C) 2000 Elsevier Science B.V. All rights reserved.