B. J. Mršulja

Experimental cerebral ischemia in Mongolian gerbils

SummaryBehaviour of biogenic amines was studied in the brains of Mongolian gerbils subjected to unilateral occlusion of the common carotid artery. Assays on the hemispheres ipsilateral to occlusion revealed in symptom-positive animals a progressive decrease in norepinephrine and dopamine, and an increase in serotonin throughout the duration of an ischemic insult. In post-ischemic periods following the release of the clip, changes in biogenic amine levels generally conformed to the principles of a previously described “maturation” phenomenon, with delayed reactions occurring after the shorter ischemic insults.

Isolation of brain capillaries: a simplified technique.

Few different procedures have been described for the isolation of brain capillaries recently. The reported methods were simple but cumbersome, requiring the repeated use of nylon cloth sieve or glass beads column for the elimination of nonvascular cerebral tissue or debris in order to obtain a pure or relatively pure fraction of capillaries1, 9x°,1~. In this communication we will outline an easy system (based on the techniques of Goldstein et al. and Joo and Karnushina, see refs. 9 and 10) for the separation of a pure cerebral microvascular fraction which is morphologically intact and enzymatically active, as well as suitable for transport studies. Rabbit cerebral tissue (forebrain freed from pia arachnoid membrane) was homogenized in 20 volumes of cold Ringers solution containing 1 ~ bovine serum albumin (SBA, Sigma Chemical Co., St. Louis, Mo.) and 10 m M Hepes (Calbiochem, San Diego, Calif.) at pH 7.4 in a glass homogenizer using a slow speed motor driven pestle. Six to seven upwards and downwards strokes were applied during the homogenization. The homogenate was centrifuged at 1500 x g for 15 min, the supernatant was discarded and the resuspended second pellet in 1 ~ SBA-Ringers solution was again centrifuged at 1500 x g for 10 rain. The third pellet was suspended in 10 ml of cold 0.25 M sucrose at pH 7.0 and layered over (90 min in advanced prepared) 1.0-1.5 M sucrose gradient (each 12 ml) and centrifuged at 58,000 x g using a Spinco SW-27 rotor in Beckman L2-65B ultracentrifuge for 30 min. This procedure yielded three morphologically distinct fractions. Only the pellet containing microvessels was used for further histological and histochemical investigations, as well as for transport studies. The amount of 1 ~ SBA-Ringer solution used for the final suspension of this fraction varied from 1-2 ml depending on the experimental needs.

ALTERATIONS OF CYCLIC NUCLEOTIDE-RELATED ENZYMES AND ATPase DURING UNILATERAL ISCHEMIA AND RECIRCULATION IN GERBIL CEREBRAL CORTEX

Abstract— Several enzyme activities were determined in gerbil cerebral cortex during unilateral ischemia or in the post‐ischemic period following 1 h of ischemia. Adenylate cyclase and Na + ‐K + ‐activated ATPase showed essentially the same pattern. Neither enzyme changed during ischemia but the activities decreased on recirculation to 40–60% of right side control by 5 h. The ATPase had returned to control level by 20h; the adenylate cyclase by 7 days of recirculation. Particulate cyclic AMP‐dependent protein kinase in the ischemic left hemisphere decreased throughout the 6h of ischemia. It remained depressed in the first 5 h of the post‐ischemic period but returned to control by 20 h. The soluble protein kinase activity, the soluble cyclic AMP and cyclic GMP phosphodiesterase and the Mg2+ dependent ATPase did not change significantly during the ischemic or post‐ischemic periods. The results suggest that ischemia and/or recirculation may affect cellular membranes and membrane‐bound enzymes, in particular. Furthermore, the results imply that despite apparent metabolite recovery during the post‐ischemic period, enzymatic changes are occurring that may be important for both the quality of recovery and the response to further ischemic insult.

POST-ISCHEMIC CHANGES IN CERTAIN METABOLITES FOLLOWING PROLONGED ISCHEMIA IN THE GERBIL CEREBRAL CORTEX

‐Eight metabolites were measured in the post‐ischemic period following either 1 or 3 h of unilateral ischemia in the gerbil cerebral cortex. The levels of ATP, P‐creatine, glucose, glycogen and GABA were essentially restored by 1 h after ischemia. In the 3 h ischemic animals. glycogen continued to increase to greater than control values aftcr 5 and 20 h of recirculation. The Icvels of glutamate were unchanged during the ischemic episode, but decreased to 60% of control at Smin and 1 h after either period of ischemia. The concentrations of cyclic AMP, which were 4‐to 5‐fold elevated during ischemia. increased an additional 6‐fold 5 min after recirculation in both groups. Arter 1 h of recovery. the levels were not different from control values. After the 1 h ischemic period, lactate levels recovered between 5 and 20 h of recirculation. In the 3 h ischemic animals. lactate concentrations were still elevated even after 20 h of recirculation. These data suggest that with the exception of lactate. recovery of metabolites is not sevcrely compromiscd by either 1 or 3 h of ischemia. Furthermore, the changes in glycogen. glutamate and cyclic AMP after recirculation suggest that the recovery process is not just a rcversal of the changes observed during ischemia.

Ultracytochemical localization of ouabain-sensitive K+-dependent, p-nitrophenyl phosphatase in myelin.

Ouabain-sensitive, K+-dependent p-nitrophenyl phosphatase (K-NPPase) activity was demonstrated ultracytochemically in the myelin of nerve fibers in peripheral and central white matter. Enzyme activity was more prominent in paranodal than compact myelin, and it was absent from nodal and interparanodal axolemma. Since K-NPPase is part of the Na-KATPase complex, we consider myelin as an important site of the sodium pump and believe that myelin participates in cationic regulation of the nervous tissue.

Brain glycogen following experimental cerebral ischemia in gerbils (Meriones unguiculatus)

Cortical glycogen levels decreased after both 1 and 3 h of unilateral ischemia. After 1 h of recirculation, the levels of glycogen were restored to control values in both groups. Subsequently, glycogen increased above normal levels after 1 week of recirculation in the 1 h ischemic groups, and after 5 h in the 3 h ischemic group. Thus, the onset of the excess glycogen accumulation appears to be dependent on the intensity of the ischemic insult.

Monoamines in Cerebral Ischemia in Relation to Brain Edema

Cerebral ischemia was produced by unilateral ligation of the common carotid artery in Mongolian gerbils. Levels of monoamines and their metabolites, with and without pretreatment with modulators of monoamine turnover, were measured. The results suggest that cerebral ischemia is accompanied by: (1) decreased rate of monoamine synthesis, (2) increased synaptosomal release of monoamines, and (3) accumulation of monoamine metabolites due to changes in the active transport of these substances out of the brain.

Histochemical investigation of the Mongolian gerbil's brain during unilateral ischemia.

SummaryThe ischemic effect on cerebral enzymes and glycogen content was histochemically evaluated in mongolian, gerbils subjected to unilateral common carotid artery occlusion for various periods of time from 1/2 to 9h. In early stages (up to 2h), the only enzyme affected was the phosphorylase which revealed a decreased activity. Thereafter, the observed changes inclusive of glycogen and other enzymes such as the dehydrogenase, nonspecific acid and alkaline phosphatases, leucine aminopeptidase and thiamine pyrophosphatase progressed proportionally to the duration of ischemia. There was an overall inverse appearance of histochemically demonstrated enzymatic disturbances between the severely damaged ischemic regions and its marginal zones; the former revealing a conspicuous decrease and/or loss of enzymatic activities while the latter showing an increase of the same enzymes. Correlating the various ischemic responses of the intracellular organelles it appears that the changes in the lysosomes and Golgi apparatus occurred slower than those of mitochondria.

Recovery of decreased synaptosomal 2-deoxy-D-[3H]glucose uptake after cerebral ischemia in mongolian gerbils

Deprivation of oxygen reduces the brain uptake of glucose or glucose analogues from blood to brain in dog and rabbit, respectively 1,3. The altered glucose brain uptake could have occurred in one or more sites in which glucose transport may have taken place, such as capillary endothelium, neurons and glia. Synaptosomes (pinched off nerve endings) which are enclosed by membrane derived from neurons and dendrites exhibit some of the biochemical and histological characteristics of in vivo nerve endings and may reflect the same transport properties as the one present in neurons, axons and dendrites, and thus, can serve as excellent in vitro model to study these structures 5-9,2°. Since the synaptosomal high affinity glucose transport may be a general property of neuronal membranes and may be responsible for the greater oxidative metabolism observed in neurons than glia 6, it was thought to be of great importance to investigate the functional t ransport studies of synaptosomes in cerebral ischemic and postischemic period. In this communication, we will demonstrate a complete recovery of the decreased 2-deoxy-D-glucose uptake in the synaptosomes l h following ischemia of 1 and 3 h duration. Cerebral ischemia ipsilateral to the left common carotid artery occlusion was produced in adult Mongolian gerbils for 30-180 min. The recovery from the ischemic insult was studied in animals with reestablished cerebral circulation for 5 and 60 min after 1 h and 3 h of left carotid artery clipping. Mongolian gerbils with clinical signs of cerebral infarction 11 were decapitated at the end of each experimental period. Left (ischemic) and right (control) hemispheres were removed quickly and immediately homogenized in 0.32 M sucrose pH 7.0. Synaptosomes were prepared according to the described method of Whittaker and Barker 21. The synaptosomal fractions obtained from the 0.8 to 1.2 M density sucrose gradient were diluted with equal volume of dis-

Cytochemistry of Hippocampus Following Cerebral Ischemia

The feature of selective vulnerability to ischemia displayed by certain topistic units and most clearly observed in the hippocampus, the brain region particularly sensitive to ischemia (12, 14), was the important finding brought out by the light microscopic observation on ger-bils. The most striking lesion was the “reactive change”, confined to the H3 sector of the hippocampus (26). This change occurred only in the animals subjected to relatively slight ischemic insult and fully developed only upon recirculation. The neurons of the H3 sector were characterized by a peripheral shift of the nucleus whereas the voluminous cytoplasm showed a central chro-matolysis. The reversibility of these changes was evident from the almost normal appearance of the H3 sector in the animals sacrificed 1 week after the reestablishment of blood supply to the brain. It is thus evident that “reactive change” represents a cellular reaction to ischemia in which the neurons are capable of full recovery from an ischemic injury.