Amiloride but not memantine reduces neurodegeneration, seizures and myoclonic jerks in rats with cardiac arrest-induced global cerebral hypoxia and reperfusion

Abstract

It has been reported that both activation of N-methyl-D-aspartate receptors and acid-sensing ion channels during cerebral ischemic insult contributed to brain injury. But which of these two molecular targets plays a more pivotal role in hypoxia-induced brain injury during ischemia is not known. In this study, the neuroprotective effects of an acid-sensing cation channel blocker and an N-methyl-D-aspartate receptor blocker were evaluated in a rat model of cardiac arrest-induced cerebral hypoxia. We found that intracisternal injection of amiloride, an acid-sensing ion channel blocker, dose-dependently reduced cerebral hypoxia-induced neurodegeneration, seizures, and audiogenic myoclonic jerks. In contrast, intracisternal injection of memantine, a selective uncompetitive N-methyl-D-aspartate receptor blocker, had no significant effect on cerebral hypoxia-induced neurodegeneration, seizure and audiogenic myoclonic jerks. Intracisternal injection of zoniporide, a specific sodium-hydrogen exchanger inhibitor, before cardiac arrest-induced cerebral hypoxia, also did not reduce cerebral hypoxia-induced neurodegeneration, seizures and myoclonic jerks. These results suggest that acid-sensing ion channels play a more pivotal role than N-methyl-D-aspartate receptors in mediating cerebral hypoxia-induced brain injury during ischemic insult.

Figure 1. The time course of cardiac arrest-induced cerebral hypoxic neurodegeneration and seizures.

(A) The number of FJ-positive degenerating neurons in the hippocampal CA1, the cerebellum, and the TRN of the saline-injected rats subjected to cardiac arrest-induced cerebral hypoxia. (B) The number of animals that developed seizures after cardiac arrest-induced cerebral hypoxia. * (p<0.05) indicates significantly different from that of day 1 after cardiac arrest-induced cerebral hypoxia. Values are mean ± S.D., n = 6.

Figure 2. Amiloride but not memantine or zoniporide dose-dependently reduced cardiac arrest-induced cerebral hypoxic neurodegeneration.

(A) The number of FJ-positive degenerating neurons in the hippocampal CA1, the cerebellum and the TRN of the rats that received 0.5, 1.5 and 5 nmole of amiloride, memantine or zoniporide before cardiac arrest-induced cerebral hypoxia. * (p<0.05) indicates significantly different from that of the saline-, memantine- or zoniporide-injected groups. Values are mean ± S.D., n = 6.

Figure 3. Intracisternal injection of amiloride but not memantine or zoniporide reduces cardiac arrest-induced cerebral hypoxic neurodegeneration.

FJ staining of representative coronal brain sections showing the hippocampal CA1, the cerebellum, and the TRN of the rats that received intracisternal administration of saline or 5 nmole of amiloride, memantine or zoniporide before cardiac arrest-induced cerebral hypoxia. The arrows indicate some of the FJ-positive degenerating neurons in the rat brain sections. Pictures were taken at 20X for the hippocampal CA1, the TRN and at 10X for the cerebellum.

Figure 4. Amiloride but not memantine or zoniporide reduces the number of posthypoxic rats developed seizures and the severity of audiogenic-myoclonic jerks.

(A) The number of the rats in the saline-injected, amiloride-, memantine- or zoniporide-treated group that developed seizures after cardiac arrest-induced cerebral hypoxia. (B) The myoclonus scores of the rats before and after cardiac arrest-induced cerebral hypoxia. Cardiac arrest-induced cerebral hypoxia was introduced on day 6. Rats were received intracisternal administration of 5 nmole of amiloride, memantine, zoniporide or 5 µl saline before cardiac arrest. * (p<0.05) indicate significantly different from that of the memantine-, zoniporide- or saline-injected control group at the corresponding time point. Values are mean ± S.D., n = 6.

Figure 5. Memantine does not reduce cardiac arrest-induced cerebral hypoxic neurodegeneration, seizures and audiogenic myoclonic jerks when sodium pentobarbital was used as the anesthetic agent in this animal model.

Rats were anesthetized with a single intraperitoneal injection of sodium pentobarbital (Nembutal) supplemented with a single subcutaneous injection of buprenorphine. (A) The number of FJ-positive degenerating neurons in the hippocampal CA1, the cerebellum and the TRN. (B) The number of posthypoxic rats that developed seizures. (C) The myoclonus scores recorded in the rats before and after cardiac arrest. The rats were received intracisternal injection of saline or 5 nmole of memantine, amiloride or zoniporide before cardiac arrest. * (p<0.05) indicates significant difference between the saline-injected group and the amiloride-pretreated group. Values are mean ± S.D., n = 6.