Antioxidant action of a Chrysanthemum morifolium extract protects rat brain against ischemia and reperfusion injury.

The present study evaluated the potential neuroprotective effect and underlying mechanism of the total flavones extracted from Chrysanthemum morifolium (TFCM) against ischemia/reperfusion (I/R) injury. An animal model of cerebral ischemia was established by occluding the right middle cerebral artery for 90 minutes followed by reperfusion for 22 hours. The neurobehavioral scores, infarct area, and hemispheric edema were evaluated. The superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, and reactive oxygen species (ROS) level in brain were also measured. The results showed that pretreatment with TFCM significantly decreased the neurological deficit scores, percentage of infarction, and brain edema and attenuated the decrease in SOD activity, the elevation of MDA content, and the generation of ROS. In isolated brain mitochondria, Ca(2+)-induced swelling was attenuated by pretreatment with TFCM, and this effect was antagonized by atractyloside. These results showed that pretreatment with TFCM provides significant protection against cerebral I/R injury in rats by, at least in part, its antioxidant action and consequent inhibition of mitochondrial swelling.

Bicyclol attenuates oxidative stress and neuronal damage following transient forebrain ischemia in mouse cortex and hippocampus.

To assess its potential neuroprotective effect against ischemia/reperfusion (IR) injury in mice, bicyclol was administered intragastrically once a day for 3 days. After 6h of bicyclol pretreatment on the third day, forebrain ischemia was induced for 1h by bilateral occlusion of the carotid arteries. After different times of reperfusion, the histopathological changes and the levels of mitochondria-generated reactive oxygen species (ROS), malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) in the cortex and hippocampus were measured. We found that extensive neuronal death occurred in the cortex and the CA1 area of the hippocampus at day 7 after IR and that bicyclol significantly attenuated IR-induced neuronal death in a dose-dependent manner. We also found that pretreatment with bicyclol dose dependently decreased the generation of ROS and the MDA content and reduced the compensatory increase in SOD activity in the cortex and hippocampus at 4h of reperfusion. These results suggest that bicyclol protects the mouse brain against cerebral IR injury by attenuating oxidative stress and lipid peroxidation.

Genistein attenuates oxidative stress and neuronal damage following transient global cerebral ischemia in rat hippocampus.

Oxidative stress is believed to contribute to neuronal damage induced by cerebral ischemia/reperfusion (I/R) injury. The present study was undertaken to evaluate the possible antioxidant neuroprotective effect of genistein against neuronal death in hippocampal CA1 neurons following transient global cerebral ischemia in the rat. Transient global cerebral ischemia was induced in male Sprague-Dawley rats by four-vessel-occlusion for 10min. At various times of reperfusion, the histopathological changes and the levels of mitochondria-generated reactive oxygen species (ROS), malondialdehyde (MDA), cytosolic cytochrome c and caspase-3 activity in hippocampus were measured. We found extensive neuronal death in the CA1 region at day 5 after I/R. The ischemic changes were preceded by increases in ROS generation and MDA concentration and followed by increased cytosolic cytochrome c, and subsequently caspase-3 activation and apoptosis. Treatment with genistein (15mg/kg, i.p.) significantly attenuated ischemia-induced neuronal death. Genistein administration also decreased ROS generation, MDA concentration and the apoptotic indices. These results suggest that genistein protects neurons from transient global cerebral I/R injury in rat hippocampus by attenuating oxidative stress, lipid peroxidation and the signaling cascade leading to apoptotic cell death.

Ischemic postconditioning protects against global cerebral ischemia/reperfusion-induced injury in rats.

BACKGROUND AND PURPOSE: Ischemic postconditioning has been found to decrease brain infarct area and spinal cord ischemic injury. In this study, we tested the hypothesis that ischemic postconditioning reduces global cerebral ischemia/reperfusion-induced structural and functional injury in rats. METHODS: Ten-minute global ischemia was induced by 4-vessel occlusion in male Sprague-Dawley rats. The animals underwent postconditioning consisting of 3 cycles of 15-second/15-second (Post-15/15), 30-second/30-second (Post-30/30), or 60-second/15-second (Post-60/15) reperfusion/reocclusion or 15-second/15-second reperfusion/reocclusion applied after a 45-second reperfusion (Post-45-15/15). RESULTS: Ten minutes of ischemia and 7 days of reperfusion destroyed 85.8% of CA1 hippocampal neurons and 64.1% of parietal cortical neurons. Three cycles of Post-15/15, Post-30/30, and Post-45-15/15 reperfusion/reocclusion markedly reduced neuronal loss after 7 days or 3 weeks of reperfusion and diminished the deficiency in spatial learning and memory. After reperfusion, a period of hyperperfusion followed by hypoperfusion was observed, both of which were blocked by postconditioning. The cytosolic level of cytochrome c increased significantly after 48 hours of reperfusion, and this was inhibited by Post-15/15, Post-30/30, and Post-45-15/15. However, 3 cycles of 60-second/15-second reperfusion/reocclusion failed to protect against neuronal damage, behavioral deficit, or cytochrome c translocation. CONCLUSIONS: Our data provide the first evidence that an appropriate ischemic postconditioning strategy has neuroprotective effects against global cerebral ischemia/reperfusion injury and a consequent behavioral deficit and that these protective effects are associated with its ability to improve disturbed cerebral blood flow and prevent cytochrome c translocation.

[Effect of activation of mitochondrial ATP sensitive potassium channel and calcium activated potassium channel on the permeability transition of mitochondria from both normal and ischemic rat brain].

AIM: To clarify whether the activation of mitochondrial ATP sensitive potassium channel and calcium activated potassium channel can influence the permeability transition of normal and ischemic brain mitochondria. METHODS: spectrophotometry was used to determine the effect of the two mitochondrial potassium channel agonists on the swelling of normal and ischemic brain mitochondria respectively. RESULTS: In normal mitochondria, diazoxide and NS1619 could inhibit the decrease of calcium induced mitochondrial absorbance at 520 nm (A520), which were blocked by atractyloside. When compared with the normal mitochondria, mitochondrial A520 decrease in ischemic brain was even more rapid. Diazoxide and NS1619 could still inhibit the calcium induced mitochondrial A520 decrease, which were blocked by atractyloside. CONCLUSION: Activation of mitochondrial ATP sensitive potassium channel and calcium activated potassium channel can protect brain mitochondria in vitro probably via influencing the mitochondrial permeability transition.

Reactive oxygen species mediate the neuroprotection conferred by a mitochondrial ATP-sensitive potassium channel opener during ischemia in the rat hippocampal slice.

Reactive oxygen species (ROS) are known to mediate the protection conferred by the opening of mitochondrial ATP-sensitive potassium channels (mitoK(ATP)) during ischemia in heart, but this has not been demonstrated in brain. The present study examined whether ROS mediate the neuroprotection conferred by a mitoK(ATP) opener during ischemia in rat hippocampal slices. Ischemia was simulated by oxygen and glucose deprivation. The direct current potential and population spike were recorded in the stratum pyramidale of the CA1 region, and lactate dehydrogenase (LDH) efflux into the medium was assayed. ROS generation was measured spectrophotofluorometrically. Pretreatment of slices with diazoxide (DIA, 300 microM), a mitoK(ATP) opener, (i) prolonged the latency to ischemic depolarization and decreased its amplitude, (ii) delayed the onset of population spike disappearance and enhanced its recovery after reperfusion, (iii) decreased LDH efflux and (iv) increased ROS levels. The effects induced by DIA were attenuated by 5-hydroxydecanoic acid (200 microM), a mitoK(ATP) blocker. Pretreatment with N-2-mercaptopropionyl glycine (MPG, 500 microM), a ROS scavenger, also abrogated the effects induced by DIA, while treatment with MPG alone had no effect during normoxia and ischemia. These results indicate that ROS participate in the neuroprotection conferred by a mitoK(ATP) opener during ischemia.

Mitochondrial permeability transition dynamics: an indicator of mitochondrial potassium channel opener.

Mitochondrial permeability transition (MPT) is an intracellular event that is closely related to apoptosis and necrosis. However, whether this process underlies the recently reported neuroprotective potency of mitochondrial potassium channel openers applied in vivo remains uncertain. This study aims to clarify this issue by determining the effects of potassium channel openers on MPT dynamics in vitro along with their in vivo effects. Male Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO) for 90 min, followed by reperfusion. 30µl of diazoxide, an opener of the mitochondrial adenosine triphosphate-sensitive K+channel (mitoKATP), or NS1619, an opener of the mitochondrial Ca2+-activated potassium channel (mitoKCa) (2 mM and 0.1 mM respectively), was infused into the right lateral cerebral ventricle 15 min before the induction of ischemia. Neurological scores were assessed 24 h after MCAO and then infarct area was determined by standard 2,3,5-triphenyltetrazolium chloride staining techniques. To further clarify the capacity of diazoxide and NS1619 to protect mitochondria from Ca2+-induced MPT, we isolated brain-derived non-synaptosomal mitochondria and evaluated the effects of diazoxide and NS1619 on Ca2+-induced MPT dynamics through measurement of spectrophotometric alterations in light scattering at 520 nm. Neurological scores and infarct size were improved in animals pretreated with diazoxide and NS1619. In isolated mitochondria, MPT was readily induced by 200 µM Ca2+and was effectively inhibited by diazoxide and NS1619. The specific MPT pore opener atractyloside abolished the inhibitory effects. According to time-constant analysis, MPT dynamics was in accordance with the neuroprotective effects of channel openers in vivo.

[Changes in field excitatory postsynaptic potential and population spike in CA1 region of rat hippocampal slices following low-frequency stimulation].

The technique of extracellular recording was used and the changes in the slope of field excitatory postsynaptic potential (S-EPSP) and the amplitude of population spike (A-PS) were observed when homosynaptic long-term depression (LTD) was induced by low-frequency stimulation (LFS) in the CA1 region of rat hippocampal slices. After LFS of 900 pulses at 1 Hz was delivered, S-EPSP and A-PS were reduced by 35.4 +/- 5.3% and 68.0 +/- 7.2%, respectively. When LFS of 450 pulses at 1 Hz was delivered, S-EPSP and A-PS were reduced by 14.3 +/- 2.3% and 36.8 +/- 6.7%, respectively. In both groups, the change in A-PS was significantly greater than that in S-EPSP (P<0.01). The changes in both indexes in the group of 900 pulses were greater than those in the group of 450 pulses (P<0.05). High Mg(2+) (4 mmol/L) could attenuate the synaptic transmission, but did not affect the induction of LTD. In the high Mg(2+) medium, the change in A-PS induced by LFS was also markedly greater than that in S-EPSP (P<0.01). These results indicate that the level of homosynaptic LTD induced by LFS is dependent not only on the numbers of pulses of LFS delivered, but also on the selection of evaluating index.

[Studies on properties of depotentiation of long-term potentiation induced by low frequency stimulation in CA1 neurons of rat hippocampal slices].

AIM AND METHODS: The parameters of low frequency stimulation (LFS) were altered systematically (frequencies of 1, 3 or 5 Hz; number of pulses of pulses of 300 or 900; and time lag after high frequency stimulation (HFS) of 20 or 100 min) and examined their effects on depotentiation (DP) of long-term potentiation (LTP) of synaptic transmission in CA1 neurons in hippocampal slices of rat. RESULTS: LTP could be induced by HFS (two trains of 100 Hz, 100 pulses, separated by 30 s) and be reversed to produce DP by a train of LFS of 900 pulses at 3 Hz given 20 min after HFS. DP induced by LFS could be blocked by NMDA receptor antagonist AP5 (50 micromol/L). And significantly reduced effect was observed for LFS at 1 Hz or 5 Hz, with smaller numbers of pulses or a longer time lag from LFS to HFS. CONCLUSION: The above results indicate that DP induced in CA1 neurons of rat hippocampal slices is strongly dependent on the parameters of LFS, and the process may be mediated through the NMDA receptor.