M. Smith-Barbour

Characterization of glutamate, aspartate, and GABA release from ischemic rat cerebral cortex

The purpose of this study was to evaluate potential mechanisms of ischemia-evoked amino acid transmitter release. Changes in extracellular levels of transmitter amino acids and lactic acid dehydrogenase (LDH) in rat cerebral cortex during and following four-vessel occlusion elicited global cerebral ischemia were examined using a cortical cup technique. Ischemia-evoked release of glutamate, aspartate and gamma-amino-butyric acid (GABA) was compared in control vs. drug-treated animals. Tetrodotoxin and antagonists of glutamate receptors (DNQX, MK-801, and AP-3) depressed the initial rate of increase in extracellular glutamate and aspartate without altering the total amount of these amino acids collected in the cortical superfusates. Cobalt, a calcium channel antagonist, failed to alter efflux. Acidic amino acid transport inhibitors (dihydrokainate, L-trans-PDC) depressed the rate of onset of glutamate and aspartate release and dihydrokainate depressed total release by 44%. PD 81723, an allosteric enhancer at the A1 adenosine receptor, depressed glutamate efflux, as did L-NAME, an inhibitor of nitric oxide synthase. Extracellular increases in GABA levels were depressed by tetrodotoxin and L-trans-PDC. The GABA transport inhibitor, nipecotic acid, increased the initial rate of onset of GABA release. Increases in LDH levels in the extracellular fluid became apparent during the period of ischemia and continued to increase during the subsequent 90 min of reperfusion. These results suggest that ischemia evokes a release of neurotransmitter amino acids that is only partially dependent upon Ca2+ influx activation or the reversal of amino acid transporters. Nonselective mechanisms, resulting from the disruption of plasma membrane integrity, may contribute significantly to the total ischemia-evoked release of excitatory amino acids.

Reduction of postsurgical adhesion formation in the rabbit uterine horn model with use of hyaluronate/ carboxymethylcellulose gel

OBJECTIVE To assess the efficacy of a bioabsorbable gel for reducing primary postoperative adhesions. DESIGN A randomized, prospective, blinded study. SETTING Academic research environment. ANIMALS Forty-one New Zealand Rabbits. INTERVENTION(S) A chemically modified hyaluronate and carboxymethylcellulose (HA/CMC) gel formulation was applied to a bilateral uterine horn injury. Postoperative adhesions were assessed at a second-look laparoscopy. MAIN OUTCOME MEASURE(S) The uterine horn model was shown to be adhesiogenic, with 29 (70%) of 42 untreated uterine horns found to have adhesions. After gel treatment, 22 (55%) of 40 uterine horns were free of adhesions compared with 12 (30%) of 42 controls. RESULT(S) Animals treated with HA/CMC gel had significantly reduced postsurgical adhesion scores when compared with controls. CONCLUSION(S) Treatment of injured uterine horn with HA/CMC gel resulted in a significant reduction in postoperative surgical adhesions.

A possible role for phospholipases in the release of neurotransmitter amino acids from ischemic rat cerebral cortex.

The involvement of phospholipases in ischemia-evoked release of aspartate, glutamate, glycine, and GABA from the cerebral cortex was studied in a four vessel occlusion rat model of cerebral ischemia/reperfusion. In comparison with the control group, the phospholipase A2 inhibitor mepacrine significantly decreased the ischemia-evoked efflux of transmitter amino acids into cortical superfusates. Direct application of phospholipases A2 or C to the cerebral cortex of non-ischemic animals resulted in a significant increase in amino acid levels. These results suggest that neurotransmitter release following cerebral ischemia may involve phospholipase induced plasma membrane disruption.

CHANGES IN EXTRACELLULAR AMINO ACID NEUROTRANSMITTERS AND PURINES DURING AND FOLLOWING ISCHEMIAS OF DIFFERENT DURATIONS IN THE RAT CEREBRAL CORTEX

The time courses of changes in extracellular levels of the transmitter amino acids, glutamate, aspartate and gamma-aminobutyric acid (GABA), and of the purines, adenosine and inosine, during 20 or 40 min periods of four vessel occlusion rat cerebral ischemia, followed by reperfusion, were investigated using a cerebral cortical cup technique. During a 20 min period of ischemia, superfusate amino acids increased by 10-30-fold and adenosine levels increased five-fold. Reperfusion was followed by the return of amino acids and purines towards pre-ischemic levels. Significantly greater increases in glutamate and aspartate levels were observed during a 40 min ischemia and, in contrast to the 20 min ischemia, the efflux of all compounds remained elevated throughout the 40 min reperfusion period. These results suggest that longer periods of ischemia are associated with increasing degrees of plasma membrane disruption allowing for a greater leakage of intracellular contents. The failure of extracellular levels of amino acids and purines to return towards pre-ischemic levels indicates that cells may be unable to effectively reconstitute their membranes after longer periods of ischemia.

Amino Acid and Purine Release in Rat Brain Following Temporary Middle Cerebral Artery Occlusion

Excitatory amino acid release and neurotoxicity in the ischemic brain may be reduced by endogenously released adenosine which can modulate both glutamate or aspartate release and depress neuronal excitability. The present study reports on the patterns of release of glutamate and aspartate; the inhibitory amino acids GABA and glycine; and of the purine catabolites adenosine and inosine from the rat parietal cerebral cortex during 20 and 60 min periods of middle cerebral artery (MCA) occlusion followed by reperfusion. Aspartate and glutamate efflux into cortical superfusates rose steadily during the period of ischemia and tended to increase even further during the subsequent 40 min of reperfusion. GABA release rose during ischemia and declined during reperfusion, whereas glycine efflux was relatively unchanged during both ischemia and reperfusion. Adenosine levels in cortical superfusates rose rapidly at the onset of ischemia and then declined even though MCA occlusion was continued. Recovery to pre-occulusion levels was rapid following reperfusion. Inosine efflux also increased rapidly, but its decline during reperfusion was slower than that of adenosine.

Oxypurinol‐Enhanced Postischemic Recovery of the Rat Brain Involves Preservation of Adenine Nucleotides

Abstract: The present study investigated the effect of the administration of oxypurinol (40 mg/kg), an inhibitor of xanthine oxidase, on adenosine and adenine nucleotide levels in the rat brain during ischemia and reperfusion. The brains of the animals were microwaved before, at the end of a 20‐min period of cerebral ischemia, and after 5, 10, 45, and 90 min of reperfusion. Cerebral ischemia was elicited by four‐vessel occlusion with arterial hypotension to 45–50 mm Hg. Adenosine and adenine nucleotide levels in the oxypurinol‐pretreated (administered intravenously 20 min before ischemia) rats were compared with those in nontreated animals exposed to the same periods of ischemia and reperfusion. Oxypurinol administration resulted in significantly elevated ATP levels at the end of ischemia and 5 min after ischemia, but not at 10 min after ischemia. ADP levels were also elevated, in comparison with those in the control rats, at the end of the ischemic period. Conversely, AMP levels were significantly reduced at the end of ischemia and during the initial (5 min) period of reperfusion. Adenosine levels were lower in oxypurinol‐treated rats, during ischemia, and in the initial reperfusion phase. Oxypurinol administration resulted in a significant increase in the energy charge both during ischemia and after 5 min of reperfusion. Physiological indices, namely, time to recovery of mean arterial blood pressure and time to onset of respiration, were also shortened in the oxypurinol‐treated animals. These beneficial effects of oxypurinol may have been a result of its purine‐sparing (salvage) effects and of its ability to inhibit free radical formation by the enzyme xanthine oxidase. Preservation of high‐energy phosphates during ischemia likely contributes to the cerebroprotective potency of oxypurinol.

The adenosine kinase inhibitor, 5-iodotubercidin, is not protective against cerebral ischemic injury in the gerbil

5-Iodotubercidin is a potent inhibitor of the enzyme adenosine kinase. It has a recognized ability to enhance interstitial fluid levels of the cerebroprotective purine, adenosine, in the hypoxic brain, and an anticonvulsant action, which is thought to be a consequence of its ability to increase extracellular adenosine levels. 5-Iodotubercidin (1 mg/kg, i.p.) was therefore tested for its ability to reduce cerebral ischemic injury in a gerbil model. Unanesthetized gerbils were subjected to a 5 min period of bilateral carotid artery occlusion and then maintained in an environmental chamber at 30 degrees C for 5 hr to counteract the hypothermia-inducing action of 5-iodotubercidin. As estimated from the extent of the increases in locomotor activity and the magnitude of hippocampal CAI layer pyramidal cell loss, 5-iodotubercidin (1 mg/kg) failed to have a cerebroprotective effect against ischemic injury.

Indomethacin modulates ischemia-evoked release of glutamate and adenosine from the rat cerebral cortex

The effects of indomethacin (10 mg/kg) on the release of the transmitter amino acids, glutamate, aspartate, GABA, and of the purines, adenosine and inosine, from the cerebral cortex was studied in a four-vessel occlusion rat model of cerebral ischemia/reperfusion. In comparison with the control group, indomethacin significantly attenuated the ischemia-evoked release of glutamate and aspartate, but not of GABA. Adenosine levels in the cortical superfusates were significantly elevated following indomethacin administration. As indomethacin is a potent inhibitor of adenosine uptake, these results suggest that, by blocking adenosine uptake, indomethacin could elevate extracellular adenosine levels and depress glutamate and aspartate efflux as a consequence of the activation of adenosine A1 receptors.

Transmitter amino acid release from rat neocortex: Complete versus incomplete ischemia models

Release of the excitotoxic amino acids, glutamate and aspartate, from the ischemic rat cerebral cortex was compared in two models; the seven vessel occlusion model (7VO) of complete cerebral ischemia and the four vessel occlusion model (4VO) of incomplete cerebral ischemia. Amino acid efflux into cortical superfusates was measured using cortical cups placed on both hemispheres. Whereas a 20 min period of ischemia causes a pronounced release of glutamate and aspartate from the 4VO model, efflux was significantly reduced in the 7VO model. Release of the inhibitory transmitter GABA, was similar in the two models. This result suggests that excitotoxic amino acid efflux into the extracellular spaces of the cerebral cortex may be enhanced by the residual blood flow in an incomplete ischemia.

Acetylcholine output from the ischemic rat cerebral cortex: effects of adenosine agonists.

The efflux of acetylcholine (ACh) from the ischemic rat cerebral cortex was examined using the cortical cup technique and an HPLC with electrochemical detection assay. Four vessel occlusion of the cerebral circulation caused a rapid increase in ACh efflux into the cortical superfusates, which was then sustained during the 20 min period of occlusion. Reperfusion was associated with a rapid return of ACh efflux to basal levels. The A1 and A2 selective adenosine receptor agonists, N6-cyclopentyladenosine (10(-8) and 10(-10) M) and CGS 21680 (10(-8)), failed to significantly alter ischemia-evoked release of ACh. Because ACh is known to enhance NMDA receptor mediated neuronal depolarization and intracellular Ca2+ levels, and to potentiate L-glutamate-induced neural degeneration, the present findings suggest that ACh could contribute to ischemic brain injury.