Caspase activation as an apoptotic evidence in the gerbil hippocampal CA1 pyramidal cells following transient forebrain ischemia

doi:10.1016/S0304-3940(01)01559-2

Neuroscience Letters

Volume 300, Issue 2, 9 March 2001, Pages 103-106

https://doi.org/10.1016/S0304-3940(01)01559-2 Get rights and content

Abstract

To determine whether apoptotic process is involved in the delayed neuronal death in hippocampal CA1 region following forebrain ischemia in gerbils, time dependent activation of caspase and DNA fragmentation were evaluated by immuno-staining and terminal dUTP nick-end-labeling staining, respectively. After transient forebrain ischemia in gerbils, activation of apoptosis related caspase, including caspase-3, was apparent, and it preceded DNA fragmentation in CA1 region. These observations suggest that apoptotic process is involved in hippocampal delayed neuronal death.

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Acknowledgements

Supported in part by Grant-in-Aid for Scientific Research from Gifu Life Science Research Promotion Council and by Grant-in-Aid for Scientific Research (No. 12470015) from the Ministry of Education, Science, Sports and Culture, Japan. The authors wish to thank Ms Kyoko Takahashi and Chikako Usui for their technical assistance.

References (20)

A Hara et al.
Immunohistochemical detection of Bax and Bcl-2 proteins in gerbil hippocampus following transient forebrain ischemia
Brain Res.
(1996)
A Hara et al.
Neuronal apoptosis studied by a sequential TUNEL technique: a method for tract-tracing
Brain Res. Protocol.
(1999)
T Iwai et al.
Temporal profile of nuclear DNA fragmentation in situ in gerbil hippocampus following transient forebrain ischemia
Brain Res.
(1995)
T Kirino
Delayed neuronal death in the gerbil hippocampus following ischemia
Brain Res.
(1982)
J.P MacManus et al.
Global ischemia can cause DNA fragmentation indicative of apoptosis in rat brain
Neurosci. Lett.
(1993)
H Nakatsuka et al.
Release of cytochrome c from mitochondria to cytosol in gerbil hippocampal CA1 neurons after transient forebrain ischemia
Brain Res.
(1999)
M Niwa et al.
Relationship between magnitude of hypothermia during ischemia and preventive effect against post-ischemic DNA fragmentation in the gerbil hippocampus
Brain Res.
(1998)
F Colbourne et al.
Continuing postischemic neuronal death in CA1: influence of ischemia duration and cytoprotective doses of NBQX and SNX-111 in rats
Stroke
(1999)
J.A Collins et al.
Major DNA fragmentation is a late event in apoptosis
J. Histochem. Cytochem.
(1997)
M Enari et al.
A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD
Nature
(1998)

There are more references available in the full text version of this article.

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Cytochrome c is a small heme protein, found loosely associated with inner membrane of the mitochondrion and is involved in initiation of apoptosis. The cytoplasmic concentration of cytochrome c has been also found to increase after ischemia/reperfusion injury (Inoue et al., 2007; Niwa et al., 2001). The release of cytochrome c has been found to activate caspase 9, which further activates caspase 3 in the cytochrome c apoptosis pathway after cerebral ischemia.
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Generation of hydrogen peroxide mediates hanging death-induced neuronal cell apoptosis in the dentate gyrus of the rat brain
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Although few neuronal cells underwent apoptosis in CD group during 1–2 h post CD, most of the neuronal cells underwent necrosis as early as 3 h post CD and continued till 24 h post CD. The global brain ischemia produced by other methods have been reported to induce DNA fragmentation (Heron et al., 1993; MacManus et al., 1993; Sharma and Gupta, 2007; Niwa et al., 2001; Han et al., 2011). In conclusion, data of the present study suggest that the permanent global brain ischemia generated due to HD increased production of H2O2 and thereby neuronal cell apoptosis through caspases-mediated pathway during early period (0–9 h) followed by necrosis during later period (12–24 h).
Present study was aimed to find out whether hanging death (HD) induces generation of reactive oxygen species (ROS) and neuronal cell apoptosis in the dentate gyrus (DG) region of rat brain. Permanent global brain ischemia was generated by HD in experimental rats and the brain was isolated after 0, 1, 2, 3, 4, 5, 6, 9, 12 and 24 h post- HD and cervical dislocation (CD). The histology, hydrogen peroxide (H₂O₂) concentration, catalase, caspase-9 and caspase-3 activities and DNA fragmentation were analyzed in neuronal cells of DG region of the brain. Permanent global brain ischemia generated due to HD induced generation of H₂O₂ as well as catalase activity. The increased level of H₂O₂ was associated with the increased caspase-9 and caspase-3 activities. The increased caspase-3 activity induced neuronal cell apoptosis during early period (0–9 h) of HD as compare to CD group. The neuronal cells necrosis was observed only 12 h post-HD, while CD induced necrosis as early as 3 h post-CD and the histoarchitecture of DG region was gradually disrupted after 6 h of CD. In conclusion, data of the present study suggest that the permanent global brain ischemia induces neuronal cell apoptosis during early period of HD through ROS-mediated pathway, while CD induces neuronal cell necrosis and disruption of the histoarchitecture of the DG region. Thus, neuronal cell apoptosis may be used to develop a cellular marker to find out the exact timing of HD.
Long-term changes in neuronal degeneration and microglial activation in the hippocampal CA1 region after experimental transient cerebral ischemic damage
2010, Brain Research
Delayed neuronal death following transient cerebral ischemia is mixed with apoptosis and necrosis, and the activation of microglia are activated after the ischemic insult. In the present study, we examined the long-term changes in neuronal degeneration and microglial activation in the gerbil hippocampal CA1 region after 5 min of transient cerebral ischemia using specific markers for neuronal damage and microliosis. Transient ischemia-induced neuronal death was shown in CA1 pyramidal cells 4 days after ischemia/reperfusion (I/R). However, neuronal degeneration of the pyramidal cells were observed up to 45 days in the CA1 region after I/R. Microglial activation was also observed in the CA1 region after I/R. Isolectin B4- (IB4) immunoreactive (⁺) microglia appeared in the CA1 region 4 days after I/R. On the other hand, ionized calcium-binding adapter molecule 1 (Iba-1)⁺ microglia was markedly increased after I/R, and peaked at 15 days after I/R. Thereafter, Iba-1 immunoreactivity was decreased with time-dependant manner in the ischemic CA1 region. These results indicate that neuronal degeneration of CA1 pyramidal cells may last about 45 days in the CA1 region after ischemic damage, and microglial activation may be diverse according to their function, such as phagocytosis, after I/R.
Effects of citicoline used alone and in combination with mild hypothermia on apoptosis induced by focal cerebral ischemia in rats
2010, Journal of Clinical Neuroscience
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In our study, we aimed to prevent ischemia/reperfusion injury and inhibit caspase-related apoptosis by the combined effect of citicoline and hypothermia. Up-regulation and activation of caspase-3 precedes the death of neurons, especially in the hippocampus and caudate–putamen.20,21 Cao et al. reported that transient global ischemia caused caspase-3-mediated cleavage of inhibitor of caspase-3-activated DNase (ICAD), resulting in the apoptotic degradation of DNA by caspase-3-activated DNase (CAD).22
The effects of citicoline used either alone or in combination with hypothermia on the suppression of apoptotic processes after transient focal cerebral ischemia were investigated. Middle cerebral artery occlusion (MCAo) was performed for 2 hours on Sprague-Dawley (SD) rats using intraluminal thread insertion. The treatment groups were as follows: Group 1, sham-operated; Group 2, saline; Group 3, citicoline (400 mg/kg intraperitoneal.); Group 4, hypothermia (34 ± 1 °C); Group 5, citicoline + hypothermia. All rats were reperfused for 24 hours, and after sacrifice and transcardiac perfusion, immunohistochemical studies were performed for markers of apoptosis. In Group 2, the Bcl-2 immunostaining score (mean ± standard deviation, 0.71 ± 0.75) was lower compared to Groups 3, 4 and 5 (2.33 ± 0.81; 3.00 ± 0.00; 2.20 ± 0.83; p < 0.05). There was higher expression of caspase-3 proteins in Group 2 (2.28 ± 0.95) compared to Group 5 (1.50 ± 0.83; p < 0.05). Bax proteins were also increased in Group 2 (1.85 ± 1.06) compared to Group 5 (0.40 ± 0.54) and in Group 4 (2.00 ± 0.00) compared to Group 5 (0.40 ± 0.54; p < 0.05). Significant differences in caspase-9 immunostaining scores were found in Group 2 (2.29 ± 0.96) compared to Group 5 (0.20 ± 0.44) (p < 0.05); Group 3 (1.00 ± 0.70) compared to Group 5 (0.20 ± 0.44; p < 0.05); and Group 4 (3.00 ± 0.00; p < 0.05) compared to Group 5 (0.40 ± 0.54; p < 0.05). Thus by suppressing apoptotic processes citicoline with hypothermia is more effective than either used alone in ameliorating cerebral damage after transient focal ischemia.
Curcuma oil modulates the nitric oxide system response to cerebral ischemia/reperfusion injury
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The antioxidant activity of C.oil in cerebral stroke has been reported earlier. We have attempted here to clarify the mechanisms underlying the neuroprotection against experimental cerebral ischemia by Curcuma oil (C.oil), isolated from the rhizomes of Curcuma longa. C.oil (250 mg/kg i.p.) was given 30 min before focal ischemia in rats caused by occlusion of the middle cerebral artery (1 h of occlusion, 24 h of reflow). Ischemia, leads to elevation in [Ca²⁺] this sets into motion a cascades of ischemic injury which was attenuated by C.oil. C.oil reduced post-ischemic brain neutrophil infiltration in the ischemic area, controlled tissue NOx levels and the neuronal levels of nitric oxide, peroxynitrite and reactive oxygen species when measured after 24 h of reflow. Double immunofluorescence staining analysis and Western immunoblot analysis with C.oil treatment showed that the expression of nitric oxide synthase (NOS) isoforms were decreased significantly compared to the untreated ischemia group. Ischemia is associated with increased in TUNEL (TdT-mediated dUTP nick-end labeling) positive cells in brain sections indicating DNA fragmentation. The C.oil treated group showed a significant decrease in numbers of apoptotic cells compared to the untreated ischemia group, as seen in the flowcytometric analysis of the neurons. Results of immunohistochemistry and Western immunoblot indicate that C.oil suppressed the elevated protein level of Bax, and aided mitochondrial translocation and activation of Bcl-2 by altered mitochondrial membrane potential. It also inhibits the cytosolic release of apoptogenic molecules like cytochrome c, inhibits the activation of caspase-3 and the expression of p53 ultimately inhibiting apoptosis. Our observations suggest that high levels of NO generated by NOS isoforms are partially responsible for exacerbating the neuronal damage induced by MCAo by intraluminal filament.

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Caspase activation as an apoptotic evidence in the gerbil hippocampal CA1 pyramidal cells following transient forebrain ischemia

Abstract

Section snippets

Acknowledgements

Brain Res.

Brain Res. Protocol.

Brain Res.

Brain Res.

Neurosci. Lett.

Brain Res.

Brain Res.

Continuing postischemic neuronal death in CA1: influence of ischemia duration and cytoprotective doses of NBQX and SNX-111 in rats

Stroke

Major DNA fragmentation is a late event in apoptosis

J. Histochem. Cytochem.

A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD

Nature