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Spectroscopic NMR Studies in a Cat Stroke Model

Joel H. Greenberg / Department of Neurology, University of Pennsylvana

29 May 2011

Until the mid 1980’s, much of what was known about brain energy metabolism under pathological conditions was obtained by classical biochemical techniques that were extremely invasive (Siesjo, 1978). Britton Chance was part of a program project grant from NIH headed by my colleague, Martin Reivich, and so we were well aware of his contributions and expertise in phosphorus-31 nuclear magnetic resonance. We therefore met with Brit in late 1985 to plan some studies examining cerebral energy metabolism in the cat following focal cerebral ischemia induced by middle cerebral artery occlusion. He was very enthusiastic to collaborate with us on this project and together with Shoko Nioka and Satoru Komatsumoto, a visiting scientist working in my laboratory from Keio University Medical School in Japan, we embarked on a series of studies to examine the relationship between stroke severity and cerebral energy metabolism (Komatsumoto et al, 1987).

For these studies we instrumented cats with a device that we fabricated from 6-0 silk suture, a small sponge, and a 20 cm piece of PE10 polyethylene tube along with a larger, longer tube. The middle cerebral artery (MCA) was isolated by a transorbital approach and this device was used to remotely occlude the MCA while the animal was in the bore of the magnet. There permitted measurements to be made prior to MCA occlusion, during two hours of occlusion, and for four hours of recovery. NMR spectra were obtained with a two-turn 17 mm diameter surface coil placed on the surface of the brain over the MCA territory using a 10.5-inch bore 2.1 T superconducting magnet and a Phospho-Energetics NMR Spectrometer. This system yielded seven peaks including beta, alpha, gamma phosphorous in ATP, creatine phosphate (PCr), phosphodiester (PDE), inorganic phosphate (Pi), and phosphomonoester (PME). Focal ischemia was produced by pulling on the PE10 tube and reperfusion by reducing the tension on this tube.Stroke severity was assessed from the EEG obtained from brass electrodes implanted in the skull over the MCA territory. When the ratio of the EEG amplitude decreased to below 20% of the pre-ischemic level, the stroke was considered severe, and when the amplitude was above 70% of control, the stroke was graded as mild.

The EEG ratio in the severe animals fell to below 20% of control and stayed depressed through the ischemia and reperfusion period rising to about 35% at the end of the four hour recovery, whereas the EEG ratio in the mild stroke animals dropped to approximately 45% of control almost immediately after MCA occlusion, but rose to about 90% at one hour and remained at that level. In the severely stroke animals the PCr/Pi ratio exhibited a precipitous decrease in concert with the drop in the EEG ratio both during MCA occlusion and the recovery period, while the mild stroke animals showed only a small decrease in PCr/Pi. The Pi peak in the severe stroke animals exhibited a splitting and an appreciable shift immediately after MCA occlusion, with the Pi peak increasing rapidly and remaining elevated for both the ischemic and recovery periods. Although the Pi peak did increase in the mild stroke animals, the increase was small. The chemical shift of the Pi peak indicated that pH decreased in the animals with severe strokes from about 7.1 to 6.2-6.3, while no shift was observed in the animals with mild ischemia. A similar pattern was seen with ATP – animals with severe ischemia exhibited a decrease starting at about 60 minutes after MCA occlusion and persisting throughout ischemia and reperfusion, while animals with mild ischemia did not show any ATP changes.

This was one of the first studies to show that 31P-NMR can be used to study dynamic processes in brain bioenergetics during stroke and revealed a good correlation between severity of ischemia and metabolic changes in the tissue. Brit’s advice during the planning and execution of these studies was invaluable. Due to the time-consuming surgical preparation for these studies and the long period of measurements, these studies would extend into the late evening. Every few hours Brit would poke his head into the room where the studies were being done, look at our data and make insightful comments as to the interpretation of our results; his suggestions and advice were almost always ‘right on’. Meanwhile he was in other room developing optical techniques for exploring tissue metabolism in vivo. His enthusiasm for discovery was infectious and it was an honor, and certainly an education, to have the opportunity to work with him.

– Joel Greenberg

 

Komatsumoto S, Nioka S, Greenberg JH, Yoshizaki K, Subramanian VH, Chance B, Reivich M (1987) Cerebral energy metabolism measured in vivo by 31P-NMR in middle cerebral artery occlusion in the cat – relation to severity of stroke. J Cereb Blood Flow Metab 7(5):557-562.

Siesjo BK (1978) Brain Energy Metabolism. New York, John Wiley & Sons