Neuronal subclass-selective loss of pyruvate dehydrogenase immunoreactivity following canine cardiac arrest and resuscitation.

Chronic impairment of aerobic energy metabolism accompanies global cerebral ischemia and reperfusion and likely contributes to delayed neuronal cell death. Reperfusion-dependent inhibition of pyruvate dehydrogenase complex (PDHC) enzyme activity has been described and proposed to be at least partially responsible for this metabolic abnormality. This study tested the hypothesis that global cerebral ischemia and reperfusion results in the loss of pyruvate dehydrogenase immunoreactivity and that such loss is associated with selective neuronal vulnerability to transient ischemia. Following 10 min canine cardiac arrest, resuscitation, and 2 or 24 h of restoration of spontaneous circulation, brains were either perfusion fixed for immunohistochemical analyses or biopsy samples were removed for Western immunoblot analyses of PDHC immunoreactivity. A significant decrease in immunoreactivity was observed in frontal cortex homogenates from both 2 and 24 h reperfused animals compared to samples from nonischemic control animals. These results were supported by confocal microscopic immunohistochemical determinations of pyruvate dehydrogenase immunoreactivity in the neuronal cell bodies located within different layers of the frontal cortex. Loss of immunoreactivity was greatest for pyramidal neurons located in layer V compared to neurons in layers IIIc/IV, which correlates with a greater vulnerability of layer V neurons to delayed death caused by transient global cerebral ischemia.

Distribution of neuronal populations containing neurofilament protein and calcium-binding proteins in the canine neocortex: regional analysis and cell typology.

Neurophysiological experiments in carnivores have revealed the existence of a large number of cortical regions and an organization of sensory systems quite similar to that found in primates. However, the cyto- and chemoarchitecture of the cerebral cortex is relatively poorly known in carnivores. We analyzed the distribution and typology of classes of neurons containing neurofilament protein or the calcium-binding proteins parvalbumin, calbindin, and calretinin in six neocortical regions of the dog. In all these areas, neurofilament protein was present in a subpopulation of medium-to-large size pyramidal neurons predominantly distributed in layers III and V. Parvalbumin was present in a large population of morphologically diverse interneurons. Small ovoid and multipolar neurons were observed throughout the cortical layers, but predominated in layers II and IV. Layers III and V-VI were characterized by the presence of larger and intensely immunoreactive neurons with bitufted or multipolar morphology, and layers V-VI also contained large multipolar neurons. Calbindin was observed in small round and multipolar interneurons in layer II, and typical double bouquet cells in layer III. Layers IV-VI contained isolated double bouquet cells and large multipolar neurons. A few calbindin-immunoreactive pyramidal neurons were also observed in layer V. Calretinin was localized in bipolar and double bouquet cells in layers II and upper III. The lower part of layer III and layers IV-VI contained rare calretinin-immunoreactive neurons. In some areas, layer III displayed a few large isolated multipolar neurons and pyramidal neurons containing calretinin. In addition, the results show that there is a substantial degree of variability in the distribution of these proteins among cortical regions, and that although they are found in morphologically comparable neuronal types in dog, monkeys, and humans, many differences exist in their regional distribution patterns between carnivores and primates.

Postischemic inhibition of cerebral cortex pyruvate dehydrogenase.

Postischemic, mitochondrial respiratory impairment can contribute to prolonged intracellular lactic acidosis, secondary tissue deenergization, and neuronal cell death. Specifically, reperfusion-dependent inhibition of pyruvate dehydrogenase (PDH) may determine the degree to which glucose is metabolized aerobically vs. anaerobically. In this study, the maximal activities of pyruvate and lactate dehydrogenase (LDH) from homogenates of canine frontal cortex were measured following 10 min of cardiac arrest and systemic reperfusion from 30 min to 24 h. Although no change in PDH activity occurred following ischemia alone, a 72% reduction in activity was observed following only 30 min of reperfusion and a 65% inhibition persisted following 24 h of reperfusion. In contrast, no significant alteration in LDH activity was observed in any experimental group relative to nonarrested control animals. A trend toward reversal of PDH inhibition was observed in tissue from animals treated following ischemia with acetyl-L-carnitine, a drug previously reported to inhibit brain protein oxidation, and lower postischemic cortical lactate levels and improve neurological outcome. In vitro experiments indicate that PDH is more sensitive than LDH to enzyme inactivation by oxygen dependent free radical-mediated protein oxidation. This form of inhibition is potentiated by either elevated Ca2+ concentrations or substrate/cofactor depletion. These results suggest that site-specific protein oxidation may be involved in reperfusion-dependent inhibition of brain PDH activity.

Prevention of postischemic canine neurological injury through potentiation of brain energy metabolism by acetyl-L-carnitine.

BACKGROUND AND PURPOSE: Mechanisms of ischemia/reperfusion brain injury include altered patterns of energy metabolism that may be amenable to pharmacological manipulation. The purpose of this study was to test the effectiveness of postischemic acetyl-L-carnitine administration on potentiation of metabolic recovery and prevention of neurological morbidity in a clinically relevant model of complete, global cerebral ischemia and reperfusion. METHODS: Neurological deficit scoring as well as spectrophotometric and fluorescent assays of frontal cortex lactate and pyruvate levels were used in a canine model employing 10 minutes of cardiac arrest followed by restoration of spontaneous circulation for 2 or 24 hours. RESULTS: Dogs treated with acetyl-L-carnitine exhibited significantly lower neurological deficit scores (p = 0.0037) and more normal cerebral cortex lactate/pyruvate ratios than did vehicle-treated control animals. CONCLUSIONS: Postischemic administration of acetyl-L-carnitine potentiates normalization of brain energy metabolites and substantially improves neurological outcome in a clinically relevant model of global cerebral ischemia and reperfusion.