| | Possible mechanisms of Cyclosporin A ameliorated the ischemic microenvironment and inhibited mitochondria stress in tree shrews’ hippocampusAbstract Objective: The ischemic brain damage is always accompanied by the significant accumulation of glutamate and calcium ions (Ca2+). Our objectives were to observe the effects of glutamate and Ca2+ overloading in tree shrew's hippocampal microenvironment on mitochondrial stress resulting in cytochrome C release and caspase apoptotic gene activation, and to explore the possible mechanism of Cyclosporin A (CsA) inhibiting mitochondrial stress. Methods: The thrombotic focal cerebral ischemia was induced by photochemical reaction in tree shrews. The extracellular contents of amino acidic neurotransmitters and Ca2+ were determined, respectively, with high performance liquid chromatography (HPLC) and atomic absorption spectrophotometry at 4, 24 and 72 h after cerebral ischemia. The glutamate–calcium chloride solutions were microperfused into hippocampus by a kind of single-pumped push–pull perfusion (SPPP) system under three-dimensional orientation instrument in tree shrews. At 24h, the expression of cytochrome C was observed in perfused lateral hippocampus by immunochemistry. Also, the hippocampus was removed, then mitochondria and cytoplasmic fragment were divided by low temperature centrifugation and the distribution of cytochrome C was assessed through Western blot. Real time fluorescence polymerase chain reaction was used to evaluate the relative amounts of caspase-3 and caspase-9 mRNA. In the treated group, CsA (40mg/kg) was intravenously injected at 6h after the microperfuse or cerebral ischemia. The glutamate–calcium solutions were perfused into the hippocampus and inspected the above-mentioned items at 24h. Data were compared between the two groups (ischemia group vs. sham group, or ischemia group vs. CsA group). Results: Thrombotic cerebral ischemia led to significant increase in extracellular glutamate and Ca2+ level of hippocampus (P<0.01). The cerebral ischemia group and the microperfusion group, which cytochrome C immunoreactivity increased and Western blot analysis demonstrated that the cytochrome C content in the mitochondria of hippocampal cells decreased (P<0.01), but the cytochrome C in the cytosol increased (P<0.01). When CsA was intravenously injected at 6h after the microperfusion or cerebral ischemia, the cytochrome C expression weakened and its release was diminished to a lesser extent. By real time PCR, in relation to the control group, the caspase-3 and caspase-9 mRNA was higher in the glutamate–calcium chloride solution perfused group. CsA treatment cut down the contents of caspase-3 mRNA and caspase-9 mRNA (P<0.01). Conclusions: It is a primary factor that glutamate and Ca2+ accumulate in hippocampal microenvironment, which results in proapoptotic protein cytochrome C release from mitochondria into cytoplasm and caspase cascade activation, and finally mitochondria stress and neuronal secondary injury appear. The neuroprotection of CsA is in relation to inhibiting glutamate receptor overactivation and reducing the Ca2+ influx, which can decrease cytochrome C release and caspase mRNA transition. When isolated mitochondria are exposed to high concentrations of calcium ions, a mitochondrial megachannel (MMC) opens in the inner mitochondrial membrane. This event, also named the mitochondrial permeability transition pore (mPTP), is considered to be an early event in apoptosis in some cells [1] and possibly a trigger of cell death in ischemia–reperfusion damage [2]. The current research suggests that mPTP and mitochondrial swelling is inhibited by Cyclosporin A (CsA, a mPTP inhibitor), which may also inhibit apoptosis in some cells. Heretofore, the microenvironment changes leading to mitochondrial stress and the neurons secondary injury after cerebral ischemia is still unclear. However, most of the experimental results were from stroke models on mechanical occlusion of brain arteries and therefore do not simulate the clinical condition of this disease and this implies that little has been known about the exact correlation between mPTP opening and caspase activation in abnormal microenvironment after cerebral ischemia or the microperfusion. Furthermore, caspase inhibitors have shown promising effect in attenuating brain injury after ischemia [2]. Moreover, information concerning the dysfunction of and glutamate accumulate in hippocampus induced by thrombotic cerebral ischemia is poorly understood. This study is focused on hippocampal CA1 area, where pyramidal neurons of hippocampus CA1 are susceptible to ischemic damage, and glutamate and Ca2+ accumulation often occurs in ischemic area where the regional cerebral blood flow decreases [3]. Hausenloy [2] found that preventing mPTP opening during the first few minutes of myocardial reperfusion using CsA improved post-ischemic contractile function in human atrial trabeculae harvested from patients undergoing cardiac surgery. CsA possesses cardioprotection effects by improving mitochondrial metabolism [4]. Further investigations should clarify whether treatment with CsA after photochemical lesioning can inhibit the release of cytochrome C and improve extracellular microecosystem of hippocampus. 1. Materials and methods  1.5. Measurements of MPT pore opening Animals were decapitated, and the brain tissue was transferred to ice-cold isolation buffer (0.25mol/L sucrose, 0.0005mol/L EDTA-K2, 0.01mol/L Tris–HCl, pH 7.40). The neuronal mitochondria were isolated according to the Matsumoto's method [9]. The homogenates were centrifuged at 2000×g in 4°C for 3min, and the suspension was obtained and centrifuged at 12,500×g in 4°C for 8min. The sediment was in turn added to a gradient buffer A (0.12mol/L mannitol, 0.03mol/L sucrose, 0.0025mmol/L EDTA-K2, pH 7.4) and buffer B (0.24mol/L mannitol, 0.06mol/L sucrose, 50μmol/L EDTA-K2 pH 7.4) and centrifuged according to above authors’ method. Mitochondria were activated by a 0.0025mol/L glutamate in a buffer (0.25mmol/L sucrose, 0.02mol/L Mops, 0.01mol/L Tris, 0.15mol/L KCl, 26°C, pH 7.0) for 3min. MPT pore opening was induced by addition of CaCl2 0.0001mol/L M. Then CsA 5μmol/L was added, a variance of fluorescent spectrophotometer was performed to measure the scattering of light of the mitochondrial suspension, which can reflect the swelling degree of mitochondria. 1.7. Western blot analysis of Cyt C The decapitated execution was performed when it was 24h after the animal microperfusion. Perfused hippocampus tissues was weighed and cut and homogenized with 1:7 pre-cooling buffer (0.02mol/L Tris–HCl, pH 7.5, 0.25mol/L sucrose, 0.01mol/L KCl, 0.0015mol/L MgCl2, 0.001mol/L EDTA, PMSF, protein kinase and phosphokinase inhibitor 5μL/mL). Homogenized tissues were centrifuged at 750×g 4°C 3min, supernatant was centrifuged again at 8000×g 4°C 20min, finally, the precipitation was mitochondria. This supernatant was centrifuged again at 100,000×g 4°C 30min and the precipitation was cytoplasm part. Mitochondria and cytoplasm part were added 1× SDS (2:1), respectively, and boiled for 5min. Protein was quantified with Bradford and separated by SDS-PAGE and then transferred to a PVDF membrane. The membrane was blocked for 30min at 37°C. mAB-Cyt was diluted in 1:2000 and incubated with the membrane for 1h at 25°C and then overnight at 4°C. After incubation with the secondary antibody (1:5000) for 1.5h at 25°C, Cyt protein was visualized by the ECL chemiluminescent kit and analyzed by Scion Image software on each specific picture of the value of the number of grey zone [13]. 1.8. RT-PCR analysis of caspase-3 and caspase-9 mRNA Total RNA was isolated from hippocampus tissues on the right side in each group using the TRIZOLasy Mini Kit according to the manufacturer's instructions. cDNA was generated using the real time PCR as primers. Primer Design (based on gene sequences known from NCBI (U.S. National Center for Biotechnology Information) with primer premier 5.0 design primers, by the Biological Engineering Company Limited-Dalian synthesis. Primers were β-actin (human), caspase-3 (mouse), cNCBI (U.S. National Center for Biotechnology Information aspase-9 (mouse) mRNA sequences). Primer Premier 5.0 software used to design PCR primers, β-actin: 5′-CCTGTACGCCAACACAGTGC-3′ (upstream), 5′-ATACTCCTGCTTGCTGATCC-3′ (downstream) caspase-3: 5′-GAGCTGGACTGTGGCATTGAG-3′ (upstream), 5′-CAAAGGGACTGGATGAACCA-3′ (downstream); caspase-9: 5′-TCTCAGACCAGAAACACCCAG-3′ (upstream), 5′-GGGCAGAAGTTCACATTGTTG-3′ (downstream); primer is synthesis by the Shanghai Kang biological company. PCR was performed at 94°C, 5min, for 40 cycle (94°C, 20s; 58°C, 20s; 72°C, 20s; 85°C, 10s). PCR product for the establishment of the melting curve, after the end of each amplification reactions are to continue to slow from 72°C heated to 95°C (every 5s increased 1°C). Finally, the results of the sample copies were calculated by the Ct value from the standard curve. Real time PCR was used to analyse the samples in the target gene and the housekeeper gene (β-actin), using a standard curve of the samples in the target gene and housekeeper of a quantitative difference. Housekeeper gene through the correction, large intestine was detected in sample gene expression of the relative. 2. Results 2.2. Effects of CsA on glutamate-induced mPTP opening Fig. 1 shows the addition of calcium chloride initiated swelling of mitochondria from neurons. In the presence of CsA, there was a distinct but incomplete inhibition of calcium chloride induced mitochondrial swelling as compared to sham control. 3. Discussion 3.1. Changes of hippocampal microenvironment during cerebral ischemia The hippocampus is perhaps the most studied structure in the brain. The hippocampus is formed by two interlocking sheets of cortex and in cross-section has a very defined laminar structure with layers visible where rows of pyramidal cells are arranged. The connections within the hippocampus generally follow this laminar format and, as a rule, are uni-directional. They form well-characterised closed loops that originate mainly in the adjacent entorhinal cortex. Thus there are defined routes for information flow making the hippocampus a very popular target for the study of synaptic function. The mPTP, a megachannel in the inner mitochondrial membrane, opens and causes isolated mitochondria to swell. In previous studies, we have shown that thrombotic cerebral ischemia with photochemical reaction induced a marked decrease in blood flow [10], a disturbance of energy metabolism, and a pronounced reduction in mitochondria respiration [5] and monoaminergic neurotransmitter in the ischemic area [6]. When release of glutamate and depolarization lead to activation of ion conductance, calcium equilibrates across membranes and intracellular calcium  concentration rise [11]. Because the cell membrane is depolarized, an equilibration of the ionic gradients across the membrane occurs. The extracellular calcium ion concentration in the ischemic group decreases from 2.15±0.08μmol/L to 1.88±0.10μmol/L at 4h after cerebral iashemia. Hereafter, deregulation of the intracellular Ca2+ homeostasis by mPTP activation leads to neuronal cell death. 3.2. Inhibitory action of CsA on mPTP Mitochondria may play an active role in physiological cellular signal transduction and in pathophysiological events [12], [13]. It is known that excessive Ca2+ sequestration, as well as other stimuli, can induce the opening of mPTP in the mitochondrial inner membrane. Based on the behavior of isolated mitochondria suspended in isotonic buffers, sustained mPTP opening may result in mitochondrial swelling (which is the result of a high concentration of relatively fixed charges associated with the proteins and phospholipids within and surrounding the matrix) induced by extrinsic glutamate. Induction of the mPTP by Ca2+ is a critical event in mediating cell death. The presented results indicate that mPTP can be detected using fluorescent spectrophotometer, which would allow the study of the physiological role of mPTP in cell death. Our data showed that the mPTP can be inhibited by the CsA, and their action may be relate to the reperfusion injury salvage kinase (RISK) pathway. But the molecular mechanism of this change remains unclear. 3.3. Effects of CsA on in hippocampus Cyt C level Under the influence of many kinds of stress factors such as excitability amino acid accumulating and Ca2+ overloading, mitochondrial ultrastructure and its function were easily damaged, then the active substance originally in mitochondria related to apoptosis including cytochrome C, was released into cytoplasm [13]. Cytochrome C is an approximate 13-kd electron transport protein normally localized to the mitochondrial intermembrane space, where it electrostatically associates with the outer leaflet of the inner mitochondrial membrane. Furthermore, MPT pore opened, mitochondrial matrix was swollen and ruptured due to hyperosmotic effect, cytochrome C was released in great amount, electron transferring respiratory chain was blocked, ATP content was radically reduced, effective supply of energy could not be realized and cell necrosed [14], [15]. Under other circumstances, a separate mitochondrion-independent pathway also operates. In the present study, the accumulate of glutamate in the hippocampus was accompanied by a significant mitochondria stress. Therefore, mPTP seems to play a role in mitochondria-mediated accumulate of glutamate and Ca2+ over loading of the hippocampus. This second increase of cytosolic cytochrome C was prevented by CsA, the neuroprotective effect of mPTP inhibitor may be related to the close of megachannel in inner mitochondrial membrane and the decrease of cytochrome C release. In this study, we used glutamate and Ca2+ to investigate the effects of glutamate on cytosolic and mitochondrial cytochrome C levels in hippocampus and found this second increase of cytosolic cytochrome C may be prevented by CsA. 3.4. Effects of CsA on caspase-3 and caspase-9 mRNA level in the hippocampus Apoptosis, mediated by caspases family, has attracted great attention [1]. Caspase-3 and caspase-9 have been identified as the key executor of apoptotic cell death. Recent progress in studies on apoptosis has revealed that cytochrome C is a pro-apoptotic factor. It is released at early stage of apoptosis and, by combining with some cytosolic proteins, could activate conversion of the latent apoptosis-promoting protease to its active form. In broad view, apoptosis can operate via two pathways, one mitochondria-mediated and the other receptor-mediated but mitochondria-independent. Cytosolic cytochrome C can then bind apoptotic protease-activating factor 1 (APAF-1), which then binds to the inactive form of caspase-9. Mitochondria-mediated activation of caspase-9 can also occur via external (extracellular) receptor-mediated signals to target various ligands on the mitochondrial membrane, thereby causing cytochrome C release and binding of APAF-1. Once cytochrome C was released, the latter and Apaf-1 and caspase-9 were combined, and then Cytc–Apaf-1–caspase-9 compound was formed, which would lead to the activation of pro-caspase-9, and subsequently, downstream caspase-3 was activated, and apoptic cascade reaction was promoted. At the same time, part of the unreleased cytochrome C was steadily combined with cytochrome b-c1 and C oxidase, and adequate ATP was produced to provide energy for apoptosis [14], [16]. Consequently, the change of mitochondrial structure and function could determine the cells to go into apoptosis or necrosis [14], [17]. Hostettler [15] investigated the cultured astrocytes and oligodendrocytes which were treated with 0.02–2mmol PAF for 72h and showed the significant levels of cell death in both cell types, moreover this effect was blocked by the PAF receptor antagonists. Immunocytochemistry demonstrated that PAF at all concentrations caused activation of caspase-3 at 24, 48, and 72h after treatment in both cell types. The present results showed that when CsA was administered by intravenous injection, the caspase-3 and caspase-9 mRNA levels decreased significantly. It may be related to the close of mPTP and decrease of cytochrome C release. CsA can inhibit effectively the increase of cytochrome C and caspases, suggesting that the caspases are fundamental factors of the mechanism of hippocampal neurons injury and cytochrome C is a prototypical component of the apoptotic mechanism. 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Department of Pathophysiology, Kunming Medical College, Kunming 650031, China  Corresponding author. Tel.: +86 871 013987111684.
PII: S0928-4680(09)00023-6 doi:10.1016/j.pathophys.2009.02.014 Crown Copyright © 2009. Published by Elsevier Inc. All rights reserved. | |
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