Quanzhong Chang, Shuling Zhang, Jinbao Yin

Department of Physiology, Zhuhai Campus of Zunyi Medical College, Zhuhai  519041, Guangdong Province, China

Quanzhong Chang☆, Doctor, Professor, Master’s supervisor, Department of Physiology, Zhuhai Campus of Zunyi Medical College, Zhuhai  519041, Guangdong Province, China

Corresponding author: Quanzhong Chang, Department of Physiology, Zhuhai Campus of Zunyi Medical College, Zhuhai  519041, Guangdong Province, China
cqzchang@tom.com

Supported by: the Science and Technology Department of Guizhou Province, No. (2007)2127*; the Key Development Program of Science and Technology Department of Guizhou Province, No. [2009]3075*; A Grant from the Science and Technology Department of Zhuhai, No. PC20081010*

www.crter.cn
www.nrronline.org

doi:10.3969/j.issn.1673-5374.2010.09.009

Abstract
BACKGROUND: Chloride channels participate in non-neuronal apoptosis. However, it remains unclear whether chloride channels are involved in ischemic neuronal apoptosis.
OBJECTIVE: To explore the effects of 4-acetamido-4'-isothiocyanatostilbene-2, 2'-disulfonic acid (SITS) and 4, 4'-diisothiocyanostilbene-2, 2'-disulfonic acid (DIDS), two chloride channel blockers, on the hippocampal neuronal apoptosis induced by 3-morpholinosydnonimine (SIN-1) based on the nitric oxide toxicity theory of neuronal apoptosis following ischemic brain injury.
DESIGN, TIME AND SETTING: Comparative observation and in vitro experiments were performed at the laboratory of Zhuhai Campus of Zunyi Medical College from January to May 2009.
MATERIALS: SIN-1, SITS, and DIDS were purchased from Sigma, USA.
METHODS: Hippocampal neurons from Sprague-Dawley rats, aged 1 day, were cultured in vitro for 12 days and randomly assigned to control, SIN-1, or chloride channel blocker groups. SIN-1 group neurons were induced by SIN-1 for 18 hours to establish a model of ischemic neuronal apoptosis. Neurons in chloride channel blocker groups were treated with SITS or DIDS plus SIN-1 for 18 hours. The controls were cultured in DMEM/Ham’s F12 complete medium alone.
MAIN OUTCOME MEASURES: The apoptotic neurons and nuclear appearance were detected by Hoechst 33258 fluorescence staining; neuronal viability was quantitatively determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide analysis. Caspase-3 activity was analyzed by Western blot.
RESULTS: SIN-1 (1 mmol/L) dramatically induced apoptosis (50%-60%). SITS and DIDS inhibited nitric oxide-induced neuronal injury in a dose-dependent manner, suppressed caspase-3 activation, reduced neuronal apoptosis, and improved neuronal survival.
CONCLUSION: Chloride channel blockers can protect against neuronal injury induced by NO. Chloride channels might be involved in neuronal apoptosis following cerebral ischemia.
Key Words: chloride channel; nitric oxide; hippocampal neuron; rats; cerebral ischemia; neural regeneration

INTRODUCTION
  
Ion studies of ischemic cerebral neuronal death mainly focus on the roles of Ca2+ and K+ in neuronal apoptosis, and Cl- channels are rarely explored. Cl- may play an important role in cell apoptosis[1-4]. Outwardly rectifying Cl- channels participate in T lymphocytic apoptosis, and an opener of the outwardly rectifying Cl- channel can induce human B lymphocyte and HeLa cell swelling[5]. 4, 4'-diisothiocyanostilbene-2, 2'-disulfonic acid (DIDS), a chloride channel blocker, can inhibit staurosporine or tumor necrosis factor/cycloheximide-induced non-neuronal apoptosis[6-8]. DIDS also protects against staurosporine-induced mouse cortical injury or N-methyl-D-aspartate-induced rat hippo-campal neuronal injury[9-11]. Chloride but not potassium or calcium channels mediate this staurosporine-induced rat cortical neuronal injury[12].
This study induced rat hippocampal neu-ronal apoptosis in vitro by 3-morpholinosydnonimine (SIN-1), a nitric oxide (NO) donor that induces ischemic brain injury, to investigate the role of chloride channel activities in neuronal apoptosis as well as the relationship between ischemic/hypoxic neuronal apoptosis and chloride channel activities.

MATERIALS AND METHODS

Design
Comparative observation and in vitro experiments.
Time and setting
This study was performed at the Laboratory of Zhuhai Campus of Zunyi Medical College from January to May 2009.
Materials
A total of 60 Sprague-Dawley rats, aged 1 day, weighing (12.31 ± 2.15) g, were pro-vided by the Animal Experimental Center of Sun Yat-sen University (2005A055). The 
animal procedures were performed in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of the People’s Republic of China[13].
Reagents and instruments are listed as follows:

Methods
Hippocampal neuronal cultures
The rats were sacrificed and the brain was harvested and placed in cold D-Hank’s solution. Bilateral hip-pocampi were isolated, cut into blocks (1 mm3) after excising the vessels and meninges, and trypsinized (0.25%) at 37 °C for 15 minutes. The reaction was terminated by adding DMEM/Ham’s F12 basal medium. Cells were mechanically pipetted, filtered through a 500-mesh screen to prepare single cell suspension, centrifuged at 1 000 r/min at room temperature for 10 minutes, placed in DMEM/Ham’s F12 complete me-dium after discarding the supernatant, and pipetted to prepare a single cell suspension. The suspension at a density of 4×105/mL was seeded in 24- or 96-well culture plate coated with polylysine and incubated in saturated humidity, 5% CO2, at 37 °C. The culture medium contained 90% DMEM/Ham’s F12, 10% fetal bovine serum, 2 mmol/L glutamine, and 100 IU/mL penicillin-streptomycin solution and was exchanged after 48 hours of culture. Cytarabine (1×10-5 mol/L) was added for 48 hours to inhibit glial cell proliferation. Culture medium was exchanged twice a week[14-15].
Grouping and intervention
Hippocampal neurons cultured for 12 days were ran-domly assigned to control, SIN-1, and chloride channel blockers groups with 6 wells in control and SIN-1 groups and 60 wells in chloride channel blockers. Control cells were cultured in DMEM/Ham’s F12 complete medium alone. The SIN-1 group was treated with the NO donor SIN-1, 1.0 mmol/L, for 18 hours to induce hippocampal neuronal apoptosis in vitro to establish models of ischemic neuronal apoptosis. Blockers were treated with SIN-1 under similar condi-tions. The SITS group was treated with 0.05, 0.3, 0.5, 1, and 2 mmol/L SITS, with six wells in each group; the DIDS group was treated with 10, 50, 100, 200, and 300 μmol/L DIDS, with six wells in each group.
Determination of cell survival rate
Hippocampal neurons were cultured in 96 wells for 12 days and mixed with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide to a terminal concen-tration of 0.5 mg/mL and incubated at 37 °C for 4 hours. The culture solution was discarded, and dimethyl sulfoxide was mixed with the neurons and shaken for 10 minutes. Culture wells with no cell culture solution served as blank control. Absorbance (A) at 570 nm was determined by a microplate reader[16].
Cell survival rate (%)=(Atreated group-Ablank control group)/  (Acon-trol group-Ablank control group) × 100%.
The procedures were performed in triplicate to calcu-late the mean value.
Morphological observation
Hippocampal neurons were washed with 0.01 mmol/L phosphate buffered saline (PBS, pH 7.4) once, fixed with 4% paraformaldehyde for 15 minutes, dried, stained with 5 μg/mL Hoechst 33258 for 15 minutes, washed with 0.01 mmol/L PBS (pH 7.4) twice, ob-served by inverted fluorescence microscopy (200-fold), photographed, and quantified to calculate apoptosis percentage[17-18].
Caspase-3 protein detected by Western blot
Hippocampal neurons from each group were washed with cold 0.1 mmol/L PBS twice, mixed with loading buffer at 85 °C to collect cells, electrophoresed on sodium dodecyl sulfate polyacrylamide gels, trans-ferred to a polyvinylidene fluoride membrane, blocked with 3% fetal bovine serum overnight at 4 °C, incu-bated with rabbit anti-caspase-3 polyclonal antibody at room temperature for 2 hours, washed with Tris-buffered saline Tween, 3 × 10 minutes, incubated with horseradish peroxidase-labeled goat anti-rabbit polyclonal antibody at room temperature for   2 hours, washed with Tris-butfered saline Tween, 3×10 minutes, followed by enhanced chemiluminescence reagent developing and exposure[19].
Main outcome measures
Hippocampal neuronal survival, apoptosis and cas-pase-3 activity.
Design, enforcement and evaluation
The experiment was designed by Quanzhong Chang, performed by Shuling Zhang, and evaluated by Jinbao Yin.
Statistical analysis
Origin 7.0 software was used for plotting. The data were expressed as Mean ± SD and analyzed by SPSS 11.0 software (SPSS, Chicage, IL, USA). Intergroup differences were compared using one-way analysis of variance. P < 0.05 was considered statistically sig-nificant.

RESULTS

Dose effect of SITS and DIDS on inhibiting NO-induced apoptosis
Neuronal apoptosis was occasionally observed in the control group. SIN-1 (1.0 mmol/L) induced 50%-60% neuronal apoptosis. SITS (1 mmol/L) and DIDS (10-200 μmol/L) significantly inhibited apoptosis (Figures 1, 2).

Influence of SITS and DIDS on neuronal appearance
The nucleus of normal neurons was ovate and fluo-rescent; apoptotic neurons displayed shrinking or round cell bodies, chromatin aggregation or breakage or apoptotic bodies, with stronger blue fluorescence (Figure 3). SIN-1 induced more apoptotic neurons (P < 0.05), but SITS and DIDS treatment blocked this apoptosis (P < 0.05; Figures 3, 4).
Influence of chloride channel blockers on the apoptotic signaling molecule, caspase-3
Activated caspase-3 levels are low in normal neurons, with a 32 kD band on Western blot. SIN-1 treatment caused two activated caspase-3 segments of 17 kD and 11 kD. SITS and DIDS inhibited caspase-3 activity to various extents (Figure 5). Pro-caspase-3 domi-nated in the control group; SIN-1 could activate pro-caspase-3 into two segments of 17 kD and 11 kD; SITS (1.0 mmol/L) and DIDS (0.1mmol/L) blocked this activation.

DISCUSSION

NO is involved in neuronal apoptosis following ischemic brain injury[20]. We simulated ischemic brain injury to induce rat hippocampal neuronal apoptosis in vitro by SIN-1. Hoechst 33258 fluorescence staining showed that NO induces apoptosis, consistent with previous results[21]. Cl- activities are involved in STS-induced rat cortical neuronal apoptosis[9, 22-23]. To validate whether chloride channel activities participate in ischemic neuronal apoptosis, we tested whether SITS or DIDS blocked SIN-1-treated neuronal apop-tosis. SITS and DIDS dose-dependently protected neurons, reduced apoptosis, and improved survival rates. SITS at 1.0 mmol/L and DIDS at 0.2 mmol/L exhibited the best effects. The anti-apoptotic effects of chloride channel blockers are consistent with pre-vious studies[24-26].

Western blot suggests that caspase-3 is inactive under normal conditions and is activated following apopto-sis. Activated caspase-3 activates two nuclear factors, DNA repairase and cytoskeletal protein, and induces morphological changes, such as cell shrinking, DNA splits, chromosome condensation, and apoptotic body formation. Moreover, caspase-3 activation is the final common pathway following injury-induced apoptosis[27]. Results from the present study show that SITS and DIDS inhibited caspase-3 activation, indi-cating the anti-apoptotic effect of chloride channel blockers.
Cl- inflow is dependent on extracellular fluid Cl- and Na+, but independent of extracellular fluid Ca2+[28]. The relationship between chloride channel activities and other ion channels, as well as the role of other ions in NO-induced neuronal apoptosis, requires further investigation. In addition, as there are no specific chloride channel blockers, it remains unclear whether chloride channel activities participate in NO-induced neuronal apoptosis.

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 (Edited by Li DF, Gao DM/Su LL/Song LP)