Fluorescent histochemical localization of lipid peroxidation during brain reperfusion following cardiac arrest

Abstract

Rats were subjected to cardiac arrest and resuscitation, 90 min of reperfusion, and in situ perfusion fixation. Thiobarbituric acid (TBA) was included in the aldehyde-free perfusion fixative, the TBA reaction was driven in situ by heating, and fluorescence microscopy was utilized to characterize the location of products of the TBA reaction. Absorbance-difference spectra were performed on butanol-extracted brain homogenates to confirm in situ formation of TBA adducts with aldehydic products of lipid peroxidation. Nissl-stained sections revealed good cellular fixation without shrinkage artifacts. Fluorescence was not seen microscopically when TBA was omitted from the perfusion fixative, and little fluorescence was present in normal brains or brains after ischemia only. However, after 90-min reperfusion, intense granular fluorescence was seen in the neuronal perikarya (especially at the base of the apical dendrite) of numerous pyramidal neurons in cortical layers 5 and 6 and in the pyramidal layer of Ammon's horn in the hippocampus. The nuclei of these cells exhibited no fluorescence. Fluorescence was also present in some striatal neurons, but was absent in the adjacent radial bundles. Neither glia nor white matter exhibited similar fluorescence. These observations indicate that neurons in the selectively vulnerable zones of the cortex and hippocampus are early and specific targets of lipid peroxidation during post-ischemic reperfusion.

Mechanoporation Induced by Diffuse Traumatic Brain Injury: An Irreversible or Reversible Response to Injury?

“…Significance is also attached to the LM observation that these neurons revealed morphological features consistent with irreversible damage, including cellular distortion, vacuolization, and pyknotic nuclei. The parallel EM findings of increased electron density, together with the dilation of the Golgi and SER and increased numbers of dilated vesicles, were also consistent with a necrotic cascade triggered by the translocation of lysosomal cathepsins and other hydrolytic enzymes (White et al, 1993;Chan, 1996;Yamashima et al, 1998;Yamashima, 2004). However, we cannot rule out that this organelle and vesicular dilation are linked to an albeit abortive attempt at membrane repair mediated by the lysosomes and the synaptotagmin found on their surfaces (Andrews, 2005).…”

Sustained calpain activation associated with lysosomal rupture executes necrosis of the postischemic CA1 neurons in primates

“…De Duve and Wattiaux (1966) first reported that the lethal cell injury occurs by the release of hydrolytic enzymes from damaged lysosomes. Spreading of hydrolytic enzymes into the cytoplasm by the lysosomal membrane injury or rupture, was confirmed in both heart (Brachfeld, 1969;Ichihara et al, 1987;Molchanova et al, 1991) and brain (White et al, 1992;Chan, 1996) ischemic injuries. The translocation of cathepsin B from lysosomes to cytosol and nucleus was confirmed also in the bile salt-induced and tumor necrosis factor (TNF)-triggered hepatic apoptosis Guicciardi et al, 2001).…”

Neuronal injury in rat model of permanent focal cerebral ischemia is associated with activation of autophagic and lysosomal pathways

“…40 The lysosomal enzymes are involved in autophagic activity. Spreading of hydrolytic enzymes from lysosomes into the cytoplasm due to the lysosomal membrane injury or rupture had been suggested in both heart 49-51 and brain 52,53 ischemic injuries. The lysosomal system could contribute to cell death in the following ways: (1) rupture of the lysosome, (2) excess of autophagy, (3) accumulation of secondary lysosomes or residual bodies, or (4) lysosome dysfunction.…”