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 the process leading to hypoxic-ischemic brain injury. The aim was to study the subcellular distribution of calcium in correlation with ultrastructural changes after hypoxia-ischemia in neonatal rats. Seven-day-old rats were subjected to permanent unilateral carotid artery ligation and exposure to hypoxia (7.7% oxygen in nitrogen) for 90 min. Animals were perfusion-fixed after 30 min, 3 h or 24 h of reperfusion. Sections were sampled for light microscopy and electron microscopy combined with the oxalate-pyroantimonate technique. At 30 min and 3 h of reflow, 3 progressive accumulation of calcium was detected in the endoplasmic reticulum, cytoplasm, nucleus and, most markedly, in the mitochondrial matrix of neurons in the gray matter in the core area of injury. Some mitochondria developed a considerable degree of swelling reaching a diameter of several mum at 3 h of reflow whereas the majority of mitochondria appeared moderately affected. Chromatin condensation was observed in nuclei of many cells with severely swollen mitochondria with calcium deposits. A whole spectrum of morphological features ranging from necrosis to apoptosis was seen in degenerating cells. After 24 h, there was extensive 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 exhibited varicosity-like swellings filled with calcium-pyroantimonate deposits. Furthermore, the thin myelin sheaths were loaded with calcium. Numerous oligodendroglia-like cells displayed apoptotic morphology 'with shrunken cytoplasm 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 condensation 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.