Protein Kinase C Expression and Activity After Global Incomplete Cerebral Ischemia in Dogs

Sieber, Frederick E. MD
Traystman, Richard J. PhD
Brown, Phillip R. DVM
Martin, Lee J. PhD

Received December 29, 1997; final revision received February 23, 1998; accepted March 27, 1998.
From the Departments of Anesthesiology and Critical Care Medicine (F.E.S., R.J.T.) and the Department of Comparative Medicine (P.R.B.), the Johns Hopkins Medical Institutions, and the Department of Pathology and Neuroscience, Johns Hopkins University School of Medicine (L.J.M.), Baltimore, Md.
Reprint requests to Frederick E. Sieber, MD, Departments of Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine, 600 N Wolfe St, Meyer 8-134, Baltimore, MD 21287-7834. E-mail [email protected].

Stroke 29(7):p 1445-1452, July 1998.

Background and Purpose

Studies suggest that protein kinase C (PKC) activation during ischemia plays an important role in glutamate neurotoxicity and that PKC inhibition may be neuroprotective. We tested the hypothesis that elevations in the biochemical activity and protein expression of Ca^2+-dependent PKC isoforms occur in hippocampus and cerebellum during the period of delayed neurodegeneration after mild brain ischemia.

Methods

We used a dog model of 20 minutes of global incomplete ischemia followed by either 6 hours, 1 day, or 7 days of recovery. Changes in PKC expression (Western blotting and immunocytochemistry) and biochemical activity were compared with neuropathology (percent ischemically damaged neurons) by means of hematoxylin and eosin staining.

Results

The percentage of ischemically damaged neurons increased from 13 +/- 4% to 52 +/- 10% in CA1 and 24 +/- 11% to 69 +/- 6% in cerebellar Purkinje cells from 1 to 7 days, respectively. The occurrence of neuronal injury was accompanied by sustained increases in PKC activity (240% and 211% of control in hippocampus and cerebellum, respectively) and increased protein phosphorylation as detected by proteins containing phosphoserine residues. By Western blotting, the membrane-enriched fraction showed postischemic changes in protein expression with increases of 146 +/- 64% of control in hippocampal PKC alpha and increases of 138 +/- 38% of control in cerebellar PKC alpha, but no changes in PKC beta and PKC gamma were observed. By immunocytochemistry, the neuropil of CA1 and CA4 in hippocampus and the radial glia in the molecular layer of cerebellum showed increased PKC alpha expression after ischemia.

Conclusions

This study shows that during the period of progressive ischemic neurodegeneration there are regionally specific increases in PKC activity, isoform-specific increases in membrane-associated PKC, and elevated protein phosphorylation at serine sites. (Stroke. 1998;29:1445-1453.)