Reperfusion following focal cerebral ischemia alters distribution of neuronal cells with DNA fragmentation in mice

doi:10.1016/S0006-8993(97)00029-2

Brain Research

Volume 751, Issue 1, 14 March 1997, Pages 160-164

https://doi.org/10.1016/S0006-8993(97)00029-2 Get rights and content

Abstract

To clarify the role of reperfusion in DNA damage following focal cerebral ischemia, we determined the distribution of cells with DNA fragmentation following permanent or transient focal ischemia. Less DNA-damaged cells were observed in the permanent than in the transient group in the ischemic core, but there was no difference in the boundary zone. We conclude that reperfusion following transient ischemia may exacerbate neuronal death following DNA damage.

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Acknowledgements

The present study was supported by National Institutes of Health Grants NS 14543, NS 25372 and NOI-NS-52334. The authors thank S. Chen, L. Reola and B. Calagui for their technical assistance and C. Christensen for her editorial assistance.

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The aim of this study was to investigate the role of apoptosis or necrosis in the development of delayed infarct, and the relationship between the level of XIAP gene, caspase-3 activation and ischemic cell death following transient focal cerebral ischemia. Adult male Sprague–Dawley rats underwent right middle cerebral artery occlusion (MCAo) for 50 min and reperfusion for 0.5, 4, 8, 24 h, 3, 7, 14 days. On TTC-stained coronal sections, delayed infarct was observed to develop in the whole MCA territory, especially in frontoparietal cortex after ischemia. Near total infarct was shown in striatum 24 h after MCAo, while delayed infarct was evident in the cortex. By day 3, the infarct had progressively expanded to the nearly whole area of the frontoparietal cortex. Flow cytometric analysis of Annexin-V (marks apoptosis) and PI (propidium iodide, marks necrosis) labeling cells showed that MCAo dominantly induced necrosis in ischemic core, striatum. Apoptosis contributed to delayed infarct and cell death in the border zone, dorsolateral cortex and hippocampus. The time-course of caspase-3 activation was consistent with the changes of apoptosis and infarct following MCAo. Further RT-PCR experiments indicated that there was a biphasic regulation of XIAP in time- and region-dependent manner after ischemia. In the infarct core (striatum), following a transient and slight increase during 0.5 h to 4 h post-MCAo, expression of XIAP mRNA markedly decreased. On the other hand, a longer and larger upregulation of XIAP was observed at early time points in border zone (0.5 to 8 h, in dorsolateral cortex; 0.5 to 24 h in hippocampus), then the level of XIAP reduced. A negative correlation was observed between apoptosis and regulation of XIAP gene in these regions. Our findings suggest a possible association between expression of XIAP gene, apoptosis and delayed infarct following ischemia.
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Citation Excerpt :
One of the important properties which the experimental models of cerebral ischemia should be characterized by is the resemblance of their underlying pathological changes to those taking place in the cerebrovascular diseases in which delayed neuronal cell death, possibly apoptosis associated with DNA fragmentation [9,22,24], is the hall mark change taking place.
The spatial memory impairment and expression of apoptotic cells in hippocampal CA1 cells were investigated in rats using single and repeated ischemia models. The neuroprotective and memory-improving effect of YM-90K, an α-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) receptor antagonist, was compared to MK-801, an N-methyl-d-aspartate (NMDA) receptor antagonist. Twice-repeated ischemia, but not single ischemia, impaired the spatial memory and increased expression of apoptotic cells. YM-90K, given before and 6 h after the second reperfusion, significantly improved the memory and reduced the apoptotic cells 7 days after the second reperfusion in repeated ischemia. MK-801 neither improved the spatial memory nor reduced apoptotic cells. The present study showed that delayed expression of apoptotic cells is mediated by mechanisms involving AMPA receptors, but not by NMDA receptor, during the late phase after reperfusion. YM-90K could provide neuroprotective activity and improve the spatial memory impaired by repeated ischemia.
Lidocaine attenuates apoptosis in the ischemic penumbra and reduces infarct size after transient focal cerebral ischemia in rats
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Lidocaine is a local anesthetic and antiarrhythmic agent. Although clinical and experimental studies have shown that an antiarrhythmic dose of lidocaine can protect the brain from ischemic damage, the underlying mechanisms are unknown. In the present study, we examined whether lidocaine inhibits neuronal apoptosis in the penumbra in a rat model of transient focal cerebral ischemia. Male Wistar rats underwent a 90-min temporary occlusion of middle cerebral artery. Lidocaine was given as an i.v. bolus (1.5 mg/kg) followed by an i.v. infusion (2 mg/kg/h) for 180 min, starting 30 min before ischemia. Rats were killed and brain samples were collected at 4 and 24 h after ischemia. Apoptotic changes were evaluated by immunohistochemistry for cytochrome c release and caspase-3 activation and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) for DNA fragmentation. Cytochrome c release and caspase-3 activation were detected at 4 and 24 h after ischemia and DNA fragmentation was detected at 24 h. Double-labeling with NeuN, a neuronal marker, demonstrated that cytochrome c, caspase-3, and TUNEL were confined to neurons. Lidocaine reduced cytochrome c release and caspase-3 activation in the penumbra at 4 h and diminished DNA fragmentation in the penumbra at 24 h. Lidocaine treatment improved early electrophysiological recovery and reduced the size of the cortical infarct at 24 h, but had no significant effect on cerebral blood flow in either the penumbra or core during ischemia. These findings suggest that lidocaine attenuates apoptosis in the penumbra after transient focal cerebral ischemia. The infarct-reducing effects of lidocaine may be due, in part, to the inhibition of apoptotic cell death in the penumbra.
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We previously reported differential alterations of the μ, δ, and κ opioid receptors following permanent middle cerebral artery occlusion. The present work studied the evolution of opioid receptor types following transient focal cerebral ischemia (tMCAO), as well as the putative predictive potential of early neurochemical alterations on the delayed ischemic damage. δ Receptors were significantly decreased as early as 6 h post tMCAO (−22% ∼ −57% vs. sham group), followed by a decrease in the μ binding site density at 24 h post tMCAO (−18% ∼ −65%), in infarcted and penumbral cortices. Finally, early decreases in cortical opioid μ and δ receptor densities were found to significantly correlate (P < 0.001, r² = 0.48 and 0.75, respectively) with the occurrence of delayed histological damage. The high correlation between decreases in μ and δ receptor densities at 6 h post tMCAO and the histological damage that occurred at 24 h post tMCAO suggests that these early neurochemical alterations could be used as predictive markers of delayed ischemic damage.
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Angiotensin II (Ang II) type 2 receptors (AT2Rs) have been associated with apoptosis. We hypothesized that AT2Rs are increased in stroke and may contribute effects of stroke to the brain. To test this, we have examined the expression of Ang II type 1 receptor (AT1R), AT2R and Ang II levels in the brain 24 h after transient middle cerebral artery occlusion (MCAO). The densities of AT1R and AT2R were measured by quantitative autoradiography (n=6). The levels of Ang II were measured by radioimmunoassay (RIA) (n=6) and by immunohistochemistry (n=3). AT1R levels on autoradiography showed a significant decrease (0.87±0.06 to 1.39±0.07 fmol/mg, p<0.01) in the ventral cortex of the stroke side compared to the cortices of non-stroke (NS) rats (n=4). There was no significant difference on ATIR in the contralateral verbal cortex of the stroke rats compared to NS control. In contrast, levels of AT2R in the ventral cortex of both the stroke and the contralateral sides were significantly increased (0.77±0.06, p<0.05 and 0.91±0.05, p<0.01 compared to 0.60±0.03 fmol/mg tissue, respectively). RIA showed that Ang II in the ventral cortex of both the stroke and the contralateral sides were significantly increased (241.63±47.72, p<0.01 and 165.51±42.59, p<0.05 compared to 76.80±4.10 pg/g tissue, respectively). Also, Ang II in the hypothalamus was significantly increased (179.50±17.49 to 118.50±6.65 pg/g tissue, p<0.05). Immunohistochemistry confirmed the increase of Ang II. These results demonstrate that brain Ang II and AT2Rs are increased whereas AT1Rs are decreased after transient MCAO in rats. We conclude that in stroke, Ang II and AT2R are activated and may contribute neural effects to brain ischemia.

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Short communicationReperfusion following focal cerebral ischemia alters distribution of neuronal cells with DNA fragmentation in mice

Abstract

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Short communication
Reperfusion following focal cerebral ischemia alters distribution of neuronal cells with DNA fragmentation in mice