The Neuroprotective Effects Of Combination Therapy With HBO And Memantine Against Focal Cerebral Ischemic Injury In Rats

Ischemic cerebrovascular diseases often lead to neurologic deficits which seriously influence the quality of life and even threaten the survival of patients. Recently, with the progress of study on the molecular mechanisms and histopathology of cerebral ischemia/reperfusion injury, a number of measures and drugs have been proved to protect neurons from ischemia/reperfusion injury. However, the clinical efficacy is still in doubt.Hyperbaric oxygen (HBO) therapy is a promising treatment strategy to improve tissue oxygenation in ischemic stroke.Multiple studies have demonstrated the neuroprotective effect of HBO applied early in focal cerebral ischemia. However, accumulating evidence also indicated that HBO treatment beyond 6 hours after onset of ischemia is ineffective. In fact, the narrow therapeutic time window has limited the use of HBO treatment in stroke patients. Therefore, it would be beneficial to find a way to prolong the narrow therapeutic window for HBO treatment. Memantine is an uncompetitive NMDA receptor antagonist and have been approved for clinical use in the U.S. Memantine has also proved to be a promising drug in the treatment of cerebral ischemia. However, adverse effect of high-dose memantine also limited the use of memantine treatment in stroke patients. This study was designed to explore the neuroprotective effect of combination therapy with HBO and memantine against focal cerebral ischemic injury, and to investigate whether the combination therapy can extend the therapeutic time window of HBO and lower the dosage requirement of memantine for neuroprotection, and explore its possible mechanism.Part 1 The neuroprotective effects of combination therapy with HBO and memantine against focal cerebral ischemic injury in ratsExperiment I The therapeuic time window of HBO against focal cerebral ischemia in ratsAim To evaluate the therapeuic time window of HBO treatment aginst focal cerebral ischemic injury in ratsMethods 50 male Spraque-Dawley rats , weighing 280～320 g, were radomly divided into five groups as follows(n=10 in each): control, T1 h, T3 h, T6 h and T12 h group. Control group underwent focal cerebral ischmia induced by middle cerebral artery occlusion(MCAO)for 2h. T1 h, T3 h, T6 h and T12 h groups were administered the HBO teatment following MCAO and 1 h, 3 h, 6 h and 12 h (1h,2.5ATA,100%O2) after reperfusion respectively. After 24 h period of reperfusion, the animals were examined for neurological scores by the method of Garcia and then killed to measured infarct volumes.Results The neurological scores were higher in T1 h, T3 h, T6 h group than those in T12h and control groups; no statistical difference was found between T12 h group and control group.The infarct volumes in T1 h, T3 h, T6 h group were significantly smaller than those in T12 h and control groups, but no statistical difference was found between T12 h group and control group.Experiment II Neuroprotection of MEM in MCAO ratsAim To evaluate the neuroprotection of MEM in MCAO rats.Methods 40 male Spraque-Dawley rats,weighing 280～320g, were radomly divided into four groups (n=10 in each): control group (received normal saline); memantine therapeutic groups (received 5 mg/kg,10 mg/kg and 20 mg/kg memantine respectively). Each groups were subjected to 120min of middle cerebral artery occlusion (MCAO) with 3-0 nylon monofilament. 0.9% saline, memantine 5 mg/kg,10 mg/kg and 20mg/kg were injected intraperitoneally 15 min after MCAO. After 24h period of reperfusion, the animals were examined for neurological scores by the method of Garcia and and then killed to measured infarct volumes.Results The neurological scores were higher in memantine 10 mg/kg and 20 mg/kg groups than those in memantine 5 mg/kg and control groups,and the neurological scores was higher in 20 mg/kg group than in memantine 10mg/kg group,no statistical difference was found between memantine 5mg/kg group and control group.The infarct volumes in memantine 10 mg/kg and 20 mg/kg groups were significantly smaller than that in control groups, and the infarct volumes in memantine 20 mg/kg group was smaller than memantine 10 mg/kg group,but no statistical difference was found between memantine 5 mg/kg and control group.Experiment III The synergic neuroprotective effects of HBO and MEM therapy in MCAO ratsAim To evaluate the synergic neuroprotective effects of HBO and MEM therapy in MCAO ratsMethods 30 male Spraque-Dawley rats were randomly divided into three groups as follows(n=10 in each): control(underwent focal cerebral ischmia induced by MCAO), combined treatment A group (20 mg/kg of MEM were injected intraperitoneally 15 min after MCAO, and then administered the HBO teatment after reperfusion 1 h); combined treatment B group (5 mg/kg of MEM were injected intraperitoneally 15 min after ischemia, and then administered the HBO teatment after reperfusion 1 h). After 24 h period of reperfusion, the animals were examined for neurological scores by the method of Garcia and then killed to measured infarct volumes.Results The neurological scores were higher in combined treatment A group, and combined treatment B group than that in control group. The infarct volumes in combined treatment A group and combined treatment B group were also signaficantly smaller than that in control group.However, compared to combined treatment A group, there is no no statistical differences in combined treatment B group between the neurological score and the infarct volume. The effect of combined treatment of HBO and MEM on Blood-Brain Barrier in tegrity in MCAO ratsExperiment IV The effect of combined treatment of HBO and MEM on Blood-Brain Barrier integrity in MCAO ratsAim To evaluate the effect of combined treatment of HBO and MEM on Blood-Brain Barrier integrity in MCAO ratsMethods 48 male Spraque-Dawley rats were randomly divided into eight groups as follows(n=6 in each): sham, control group underging focal cerebral ischmia induced by middle cerebral artery occlusion(MCAO), HBO treatment T1 h and T12 h groups administered the HBO (1 h,2.5ATA,100%O2) teatment following MCAO and 1h and 12h after reperfusion, memantine 5 mg/kg and 20 mg/kg groups injected memantine intraperitoneally 15 min after MCAO, combined treatment A group injected 20 mg/kg of MEM intraperitoneally 15 min after MCAO, and then administered the HBO teatment after reperfusion 1h, combined treatment B group injected 5 mg/kg of MEM intraperitoneally 15 min after MCAO, and then administered the HBO teatment after reperfusion 12 h. Just at the beginning of reperfusion, EB (2% in saline, 4 ml/kg) was injected from tail vein in all of groups. BBB permeability was measured at 24 h after repefusion by measuring the extravasation of Evans blue.Results EB content of the ischemic ipsilateral hemisphere were significantly increased in control group compared with that in sham group (p<0.01). There are no statistical differences between HBO T12 h group, MEM 5 mg/kg group and control group. EB content of the ischemic ipsilateral hemisphere were Part 2 significantly decreased in HBO treatment T1 h group, MEM treatment 20 mg/kg group and combined treatment A and B groups compared with that in control group. (p<0.01), however, there are no statistical differences among HBO T1 h group, MEM 20 mg/kg group and combined treatment A and B groups. EB content of the ischemic contralateral hemisphere were not significantly different among each group(p>0.05).Experiment V The effect of combined treatment of HBO and MEM on cytokines in MCAO ratsAim To evaluate the effect of combined treatment of HBO and MEM on cytokines in MCAO ratsMethods 48 male Spraque-Dawley rats were divided into eight groups randomly as follows (n=6 in each): sham, control group underging focal cerebral ischmia induced by middle cerebral artery occlusion(MCAO), HBO treatment T1 h and T12 h groups administered the HBO (1h,2.5ATA,100%O2) teatment following MCAO and 1 h and 12 h after reperfusion, memantine 5 mg/kg and 20 mg/kg groups injected memantine intraperitoneally 15 min after MCAO, combined treatment A group injected 20mg/kg of MEM intraperitoneally 15 min after MCAO, and then administered the HBO teatment after reperfusion 1h, combined treatment B group injected 5 mg/kg of MEM intraperitoneally 15 min after MCAO, and then administered the HBO teatment after reperfusion 12 h. After 24 h reperfusion, the brain tissues were harvested and using ABC-ELISA method to measure the content of IL-1β,TNFα,IL-6,IL-8,IL-10.Results The contents of IL-1β,TNFα,IL-6 and IL-8 were significantly increased in control group compared with those in sham group (p<0.05). The contents of IL-1β,TNFα,IL-6 and IL-8 were signaficantly decreased in HBO treatment T1 h group, MEM treatment 20 mg/kg group and combined treatment A and B groups compared with those in control group, the content of IL-10 were signaficantly increased in HBO treatment T1 h group, MEM treatment 20 mg/kg group and Combined treatment A and B groups compared with those in control group (p<0.05),however, there are no statistical differences among HBO T1 h group, MEM 20 mg/kg group and Combined treatment A and B groups(p>0.05).Conclusions1. HBO treatment has neuroprotective effect and the therapeutic time window is within 6 h after reperfusion following transient focal cerebral ischemia in rats.2. Administration of MEM 15 min after MCA occlusion decreases infarct size and improves neurological deficit in a dose-dependent manner in rats.3. Combination of MEM and HBO therapy prolongs the therapeutic window of HBO from 6 to 12 h after reperfusion and lowers the dosage requirement of memantine for neuroprotection from 10 mg/kg to 5 mg/kg.4. Combination of MEM and HBO therapy reduces the permeability of blood-brain barrier and the mechanism is at least partially similar between MEM and HBO.5. Combination of MEM and HBO therapy has significant effect of reducing the levels of IL-1β, TNFα, IL-6, IL-8, increacing the levels of IL-10, thereby reducing the permeability of blood-brain barrier.