Delayed neuronal death after global incomplete ischemia in dogs is accompanied by changes in phospholipase C protein
F E Sieber1, R J Traystman, L J Martin
1Department of Anesthesiology, Johns Hopkins Medical Institutions, Baltimore, Maryland, USA.
Abstract:
Activation of phospholipase C (PLC) increases intracellular Ca2+ and may play a role in delayed neuronal death after ischemia. Because changes in intracellular Ca2+ are believed to participate in ischemic neuronal injury, we tested the hypothesis that PLC beta protein levels are temporally altered in brain regions that undergo neurodegeneration after global incomplete ischemia. Dogs (n = 12) were subjected to 20 minutes of global incomplete ischemia followed by recovery of either 1 (n = 5) or 7 days (n = 7). Six sham-operated animals were used as nonischemic controls. In hematoxylin and eosin-stained brain sections, neuronal density at 1 day after ischemia was unchanged relative to nonischemic controls in hippocampus CA1, caudate, and cerebellar cortex (anterior lobule). However, at 7 days after ischemia, neuronal densities were decreased to 56 +/- 15% (mean +/- SD) and 75 +/- 17% of control in CA1 and caudate, respectively. At 1 and 7 days after ischemia, the percentage of neurons showing ischemic injury increased from 13 +/- 10 to 40 +/- 35% in CA1, 24 +/- 25 to 59 +/- 16% in cerebellum, and 4 +/- 2 to 18 +/- 12% in caudate. Densitometric analysis of immunocytochemically stained brain sections from controls (n = 3). 1 day after ischemia (n = 3), and 7 days after ischemia (n = 5) revealed that PLC beta immunoreactivity was increased in cerebellum at 1 day (0.274 +/- 0.013 v 0.295 +/- 0.005 optical density units [OD] in control and 1 day, respectively) and 7 days (0.108 +/- 0.009 v 0.116 +/- 0.005 O.D. in control and 7 days, respectively). PLC beta immunoreactivity was unchanged after ischemia in caudate and hippocampus. Western blot analysis of PLC beta immunoreactivity in the cerebellar cortex and hippocampus in the control (n = 3), 1 day (n = 2), and 7 days after ischemia (n = 2) groups showed that PLC beta levels were increased after ischemia in cerebellum 266% and 227% above control at 1 and 7 days, respectively. However, in hippocampus, PLC expression after ischemia was unchanged at 97% and 84% of control at 1 and 7 days, respectively. These results show that delayed neuronal degeneration after global incomplete ischemia is accompanied by regional abnormalities in PLC levels. Elevated PLC levels early may represent an aberrant signal transduction mechanism resulting in delayed cell damage, whereas decreased PLC levels later after ischemia may reflect ongoing neurodegeneration.
