B. B. 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.

Ischemic Modification of Cerebrocortical Membranes: 5-Hydroxytryptamine Receptors, Fluidity, and Inducible In Vitro Lipid Peroxidation

Abstract: The effect of ischemia on the properties of 5‐hydroxytryptamine1A+B (5‐HT1A+B) and 5‐hydroxytrypt‐amine1B5‐HT1B) binding sites, physical‐state “fluidity” of the membrane, and its susceptibility to peroxidation in vitro was investigated in the cerebral cortex of gerbils. Ischemia was induced by bilateral carotid artery occlusion for 15 min alone or with release for 1 h. Ischemia both with and without reflow decreased the number of 5‐HT1A+B and 5‐HT1B binding sites, whereas ischemia and reflow altered the affinity for 5‐HT1B binding sites. Resistance to the temperature‐dependent increase in “fluidity” of the membrane was detected (by fluorescence anisotropy using l,6‐diphenyl‐l,3,5‐hexatriene as a probe) after ischemia and reflow but not in ischemia alone. Susceptibility of the membranes to Fe2+‐ and ascorbic acid‐stimulated lipid peroxidation in vitro was decreased following ischemia and recirculation only. These findings strongly suggest that the composition and the function of the membrane are markedly disturbed during recirculation after ischemia.

Monoamines and related enzymes in cerebral cortex and basal ganglia following transient ischemia in gerbils

SummaryThe post-ischemic effects on cerebral cortex and basal ganglia monoamine levels and monoamine oxidase (MAO A and B) and catechol-O-methyl transferase (COMT) activities were evaluated in Mongolian gerbils (Meriones unguiculatus) subjected to bilateral common carotid arteries occlusion for 15 min and reflow for 7 days. Disorders of monoamine metabolism was found in ischemic brain which persisted during the long-term post-ischemia. A rebound increase of norepinephrine and serotonin appeared in early stages (up to 1 h) of post-ischemia both in cerebral cortex and basal ganglia; a rebound increase of dopamine was found only in cerebral cortex. Thereafter, the serotonin level was enhanced over the control level during the whole post-ischemic period whereas the levels of catecholamines were reduced particularly in basal ganglia. With respect to monoamine content and activities of monoamine degraded enzymes an oscillatory behavior was observed in post-ischemia. Disorder of the monoamine metabolism found during post-ischemic period possibly contributes to neurological dysfunction after an ischemic insult.

Liposome-entrapped superoxide dismutase reduces ischemia/reperfusion ‘oxidative stress’ in gerbil brain

Bilateral common carotid artery occlusion (15 min.) followed by two hours of recirculation reduced mitochondrial superoxide dismutase (SOD) and glutathione reductase (GR) activities, and increased susceptibility of mitochondrial membranes to in vitro lipid peroxidation in brain regions (i.e., cortex, striatum and hippocampus) of Mongolian gerbil. Intraperitoneal bolus injection (2 mg/kg b.w.) of liposome-entrapped CuZn superoxide dismutase (l-SOD) increased the endogenous SOD activity in normal brain tissue and, when given at the end of ischemia, counteracted both the ischemic reduction of endogenous SOD and the increased peroxidation of mitochondrial membranes. 1-SOD treatment was ineffective in reducing brain swelling, suggesting that superoxide radicals are not a main participant in the process of (post)ischemic brain edema formation.

Brain serotonin after experimental vascular occlusion

Unilateral ligation of a common carotid artery in gerbils causes a decreased rate of serotonin synthesis and degradation but an increased release of this monoamine. In the brain, reduction of cerebral serotonin content during ischemia is followed by accumulation of its main metabolite, 5-hydroxyindolacetic acid. These data support the contention that serotonin plays an important role in the progression of cerebral infarction.

Dopamine metabolism and free-radical related mitochondrial injury during transient brain ischemia in gerbils

Regional extracellular release of dopamine (DA) and its metabolites, 3,4-dihydroxy-phenylacetic acid (DOPAC), homovanillic acid (HVA) and 3-methoxytyramine (3-MT) was measured in gerbils (with or without pargyline pretreatment) subjected to bilateral carotid artery occlusion (15 min) and various periods of recirculation (up to 6 hr), utilizing intracerebral microdialysis and high-performance liquid chromatography (HPLC) with electrochemical detection. Mitochondrial monoamine oxidase (MAO) and superoxide dismutase (SOD) activities andin vitro stimulated lipid peroxidation (TBARM) were determined in separate experimental groups of animals. The ischemically induced DA release, decrease of MAO-derived DA metabolites DOPAC and HVA, and accumulation of 3-MT were potentiated and prolonged by pargyline pretreatment. Mitochondrial MAO and SOD activities were significantly reduced during ischemia alone and up to 1 hr of reperfusion, whereas TBARM was enhanced during reflow only. The data suggest that reduced activity of mitochondrial antioxidative enzyme(s) but not DA metabolism by MAO may contribute to free radical-mediated injury of (mitochondrial) membranes.

Cerebral ischemia: Changes in monoamines are independent of energy metabolism

The relationship of neurotransmitters and neuroeffectors to the energy state of the brain was examined in the gerbil model of ischemia after 5 and 15 min of bilateral common carotid artery occlusion only or with 1 hr of reperfusion. The gerbil brains were fixed by microwave irradiation and a total of 15 metabolites were measured from a single piece of tissue from either the hippocampus or the striatum. The rapid alterations in energy-related compounds and cyclic nucleotides appeared to be directly related both to the loss of oxygen and glucose during ischemia and the resupply of these nutrients during reflow. Significant reduction in the level of monoamines occurred prinicipally during reflow, at a time when the energy-related metabolites were restored. It is proposed that the changes in monoamines were triggered by other ischemic-induced events unrelated to energy depletion.