SUMMARY Mongolian gerbils were treated with alpha-methyl-para-tyrosine methyl ester (AMPT, a tyrosine hydroxylase inhibitor), in order to decrease brain levels of catecholamines. Six hours later, unilateral ischemic stroke was induced by ligation of the left common carotid artery. The delayed degeneration of nerve terminals was studied sixteen hours later by measuring the high-affinity uptake of radiolabeled transmitters by isolated synaptosomes. Dopamine, serotonin and glutamate terminals were studied. AMPT-treated gerbils were compared to untreated (no AMPT) animals; 220 gerbils were studied. AMPT pretreatment (100, 250 and 400 mg/kg) produced a dose-dependent protection of all three types of nerve terminals. In the absence of AMPT pretreatment, the uptake of radiolabeled transmitters by the ischemic hemisphere, expressed as a percentage of that seen in the contralateral (unaffected) side of the brain, was as follows (mean ± SEM): 27.3 ± 5.2% for dopamine terminals, 49.5 ± 6.2% for serotonin terminals, and 42.7 ± 5.3% for glutamate terminals. Protection was essentially complete at a dose of 400 mg AMPT per kg. The number of animals with significant damage to nerve terminals was reduced from 38.5% in untreated animals to 11.1% in animals treated with AMPT 400 mg/kg. Although the nerve terminals were protected, gerbils still showed the behavioral signs of unilateral stroke due to the permanent occlusion of the left carotid. These results indicate that endogenous dopamine may play a significant role in ischemic damage to nerve terminals in the cerebrum. Stroke Vol 16, No 5, 1985 THE MEASUREMENT of high affinity uptake of radiolabeled neurotransmitters by isolated synaptosomes can be used to evaluate damage to nerve terminals in experimental animals with cerebral ischemia. The uptake and accumulation of neurotransmitter against a concentration gradient is a complex neuronal function that requires energy and the structural integrity of the axonal membrane. The neuronal cell type is specified by the high-affinity binding of neurotransmitter to the axonal transport system. Prior studies have determined that the catecholamine nerve terminals that take up dopamine (DA) and norepinephrine (NE) are damaged to a significantly greater degree by cerebral ischemia than terminals incorporating serotonin (5-HT), GABA or glutamate. 1 -2 All nerve terminals were able to function normally in vitro when synaptosomes were isolated for up to eight hours after carotid ligation.1 However, by sixteen hours, severe reduction in uptake had occurred. Since the potassium-stimulated release of radiolabeled transmitter proceeded normally, the reduction in uptake appeared to be due to loss of terminals, rather than to reduced uptake in existing terminals. This reduction in uptake probably represents irreversible damage to the nerve terminals because function cannot be restored by incubation in vitro with adequate supplies of substrate and oxygen. Measurement of the levels of transmitters in the ischemic hemisphere of gerbils with irreversi...

SUMMARYThe energy-dependent uptake of ( 3 H)-dopamine (DA), ( 3 H)-norepinephrine (NE) and ( 3 H)-serotonin (5-HT) was measured in synaptosomes isolated from either the whole cerebral hemispheres or the striata of gerbils after cerebral ischemia. Ischemic stroke was induced in the Mongolian gerbil by left common carotid ligation. Uptake values in the affected hemisphere (expressed as a percent of the corre sponding control hemisphere) were 32.6% for DA, 35.1% for NE, and 52.0% for 5-HT, 16 hours after stroke. The differential reduction in uptake of the catecholamines relative to 5-HT was significant (p < 0.005). This differential persisted when measures were made on isolated striata from the ischemic and control hemispheres. In the latter measurements, uptake of DA was 20.7% of control and uptake of 5-HT was 44.7% of control. Uptake of both DA and NE were significantly reduced in animals exhibiting milder circling behavior, while uptake of 5-HT was not. There was no significant reduction of uptake in animals subjected to left common carotid ligation not exhibiting signs of stroke. These studies indicate a selective sensitivity of catecholamine nerve terminals to damage in ischemic stroke. Stroke Vol 14, No 6, 1983PRIOR STUDIES IN THIS LABORATORY exam ined the active uptake of dopamine, GABA and glutamate into synaptosomes that were prepared from ger bils at 16 hours after unilateral carotid ligation. 1The high-affinity uptake of neurotransmitters by synapto somes is an energy-dependent process that requires the integrity of neuronal membrane function. No changes in uptake were noted for up to 8 hours after carotid ligation, even though animals exhibited signs of stroke. However, by 16 hours, the uptake of dopamine (DA) into synaptosomes prepared from the ischemic hemisphere was reduced to 15.2% of the unaffected hemisphere. GABA uptake was reduced to 28.0% and glutamate uptake to 47.5%.' The greater effect of is chemia on the uptake of dopamine relative to gluta mate was significant (p < 0.001). Potassium-stimulat ed release of radiolabelled transmitter from the synaptosmal preparation, following labeling of the tis sue via uptake, was not affected. the residual uptake of transmitter was into normally functioning synaptosomes and that the reduction in uptake was most probably due to loss of nerve termi nals. Marked reductions in the levels of the catechola mines DA and norepinephrine (NE) have been ob served in the ischemic hemispheres of gerbils exhibit ing stroke after unilateral carotid ligation.2-5 Lavyne et al 4 and Meyer et al 6 have proposed that increased re lease of NE 4 -6 and 5-HT 6 occurs during ischemia, and accounts for the decreased levels in the brain. Howev er, this would not explain why 5-HT levels are not reduced to the same extent as dopamine and NE in cerebral ischemia.2 Therefore, we studied the highaffinity active uptake of DA, NE and 5-HT into synap tosomes prepared from gerbils 16 hours after unilateral carotid ligation, in order to determine if there was a differential susceptibility of...

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The time course of changes in monoamine metabolism in ischemic striatum was assessed by measurement of levels of dopamine (DA), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), serotonin (5-HT) and 5-hydroxy-indole-acetic acid (5-HIAA) 2, 4, 7 and 16 hours after irreversible unilateral carotid ligation in Mongolian gerbils with stroke. DA was reduced to 30% of the level in the contralateral non-ischemic striata by 2 hours after stroke, but DOPAC was significantly elevated (p less than 0.01) to 227%, while HVA remained equal to control. At 4 hours after stroke, DOPAC was 86% of the contralateral non-ischemic striata but HVA had risen to 130%. At 7 hours after stroke, DOPAC in the ischemic striata was 148% of control, while HVA remained at 133%. By 16 hours after stroke, DA, DOPAC and HVA were depleted from the ischemic striata, corresponding to the time course for irreversible damage to the neurotransmitter uptake function of nerve terminals. 5-HT levels in the ischemic striata were 30% of control at 2 hours, 46% at 4 hours, 30% at 7 hours and 21% at 16 hours, while 5-HIAA remained equal to control throughout the time course. These studies indicate that monoamine metabolism continues in ischemic striatum for up to 8 hours after the onset of stroke following irreversible unilateral carotid ligation in the Mongolian gerbil, but metabolism of DA is disrupted by 16 hours after stroke while metabolism of 5-HT continues.

Evolution of infarction following cerebral ischemia is a delayed process, with spongiform degeneration of the neuropil occurring 6 to 8 hours after onset of ischemia. The brains of gerbils with stroke following unilateral carotid artery ligation were examined for catecholamine-derived fluorescence (CADF) by the Falck-Hillarp technique to study the relationship of catecholamine (CA) metabolism with damage to the neuropil. CADF could still be identified in the striatum for up to 16 hours after stroke and there appeared to be spongiform degeneration of the neuropil in relation to accumulations of CADF at 7 and 16 hours after stroke. Pretreatment of gerbils with a-methyl-p-tyrosine 400 mg/kg 6 hours prior to carotid ligation depleted the striatum of CADF until 16 hours after stroke and appeared to reduce the spongiform degeneration of the neuropil, though it did not affect ischemic degeneration of neuronal cell bodies. The continued presence of CADF in the striatum for up to 16 hours after stroke supports the previously reported findings that CA nerve terminals are still functional for 8 hours after stroke and that CA metabolism continues even though levels of CA are reduced immediately after onset of ischemia due to carotid artery ligation.