Experimental animals
Sprague-Dawley rats weighting 200-to 250g were obtained from the Animal Center of China Medical University (Shenyang, China). All rats were preacclimatized 7 days before surgery. They were housed in standard cages under a 12-h light/dark cycle with the temperature at 23-24°C, humidity at 40-50%. The experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (United States National Institutes of Health publication number 85-23, National Academy Press, Washington DC, revised 1996).
Rat IR model establishment and experimental groups
The rat IR model was preformed by occluding the aortic arch for 14 minutes [4, 29]. Briefly, after being anesthetized, the rats were catheterized at the left carotid artery and the tail artery to measure proximal and distal blood pressure (BP), respectively. Following exposing the aortic arch, the clamp was placed between the left common carotid artery and the left subclavian artery for 14 min to induce ischemia. The ischemia was confirmed as a 90% decrease in distal BP. Then the clamp was removed to induce the reperfusion for 12h. The sham-operated rats were preformed the same procedures without inducing ischemia.
MiR microarray analysis
As we previously reported, the rat miRNA microarray analysis was performed with the miRNeasy mini kit (Qiagen, West Sussex, United Kingdom) [29,31]. The L4–6 segments of the spinal cord were collected at 4h after reperfusion. According to the manufacturers’ instructions, 2.5μg total RNA samples were firstly labeled with the miRCURY™ Hy3™/Hy5™ Power labeling kit (Exiqon, Vedbaek, Denmark) and hybridized on a miRCURY™ LNA Array (version 18.0, Exiqon, Vedbaek, Denmark).
After removing the nonspecific bindings, the fluorescent images of microarray slides were scanned by an Axon GenePix 4000B microarray scanner (Axon Instruments, CA, USA). The fluorescent intensity of the scanned images were loaded into the GenePix Pro 6.0 program (Axon Instruments) for feature extraction. The average of the replicated miRs with intensities of 50 or more were used to calculate a normalization factor. After normalized by the median normalization method, the significantly different miRs were identified by Volcano Plot filtering. Finally, the hierarchical clustering was performed to determine the differences of the miR expression by MEV software (version 4.6, TIGR ).
Intrathecal injection and drug delivery
All treatments in vivo including the synthetic miRs (Dharmacon,Chicago, IL, USA) and recombinant rat calpain-2 (rr-CALP2,B71107, 150U/L, Calbiochem, China ) were diluted into 20 µl in total volume and intrathecally injected, as we previously described [5,6]. Briefly, the needle of a 25μ microsyringe was inserted into the L5-6 segment of spinal cords by the sign of a tail flick. Then, the concentration of 100 μmol/L of miR-137-3p mimic, 125 μmol/L miR-137-3p inhibitor or 100 μmol/L negative control (NC) was co-administered with Lipofectamine 3000 (Invitrogen, USA) at a 24h-interval for five consecutive days before surgery. Likewise, the rr-CALP was dissolved into a final concentration of 75 U/L immediately before injection. The number of days and the dosage used in this stud were evaluated by the overall effects in vivo by PCR and Western blotting in preliminary experiments. Only the rats displayed normal motor function were included for further study.
Motor function assessment
All being fully preacclimatized to the testing environment, the hind-limb motor functions were scored with Tarlov scores by two observers by the double-blind method [5].
Luciferase reporter assay
The target interaction between miR-137-3p and CAPN-2 was verified by the luciferase reporter assay[5]. Briefly, 293T cells were seeded in a 96-well plate at 4×104 cells/well. Using Lipofectamine 3000, the cells were co-transfected with 100 nM miR-137-3p mimic or 100 nM NC and 180 ng luciferase reporter vector containing the wild-type (WT) 3′UTR (5′-ACATCGTCTCTCATAGCAATAT-3′) or mutant (MT) 3′UTR (5′-ACATCGTCTCTCATCAUGGCAT-3′). After 48h after transfection, the relative activity was determined with a Dual-Luciferase Reporter Assay Kit (Promega Corp.,WI, USA).
Oxygen-glucose deprivation and reperfusion (OGD/R) model
As we previously performed, the OGD/R model was established in 70–80% confluent VSC4.1 neurons to mimic the IR insult in vivo[5]. After twice washes and replacement the medium with glucose-free Hank’s Balanced Salt Solution (HBSS), the neurons were kept in an anaerobic chamber (95% N2 and 5% CO2) at 37 °C for 6h. Then the medium was changed into initial medium and air condition for another 18h to induce reoxygenation. The control neurons were cultured in normal and atmosphere for 24h without depriving oxygen and glucose.
VSC4.1 motor neuron cultureand treatments
VSC4.1 motor neurons were purchased from Huatuo Biotechnology Co., Ltd (Shanghai, China). According to the manufacturer’s instructions, the cells were grown in 75-cm2 flasks containing 6ml culture medium (89% Eagle"s minimum essential medium (EMEM) with supplemented with 10% foetal bovine serum (FBS) and 1% penicillin/streptomycin) at 37 °C with 5% CO2 in humidified air. The culture medium was replaced twice weekly.
For in vitro experiment, the neurons were pretreated with the synthetic miR and specific inhibitors 24h before underwent OGD/R insults [5]. As we previously, after seeded at a concentration of 4×105 per well, the miR-137-3p mimic (50 nmol/L) or NC (50 nmol/L) was cotransfected with 5 μL Lipofectamine 3000, whereas for inhibitor experiments, the Roscovitine (10 µM, Cdk5 inhibitor, Sigma-Aldrich Co., China) or Z-IETD-FMK (50 µM, caspase-8 inhibitor, R&D Systems,United States) was added into culture medium alone. The concentration of each treatment and the in vitro effects were determined by PCR in preliminary experiments.
Detection of CAPN-2 activity
The tensin homolog (PTEN) is a selective substrate for CAPN-2. PTEN is degraded as a result of CAPN-2 activation and is widely used for quantitative analysis of neuronal CAPN-2 activity in vivo and in vitro [19, 32]. As previously described, CAPN-2 enzymatic activity was assessed by the fold change in the mean fluorescence intensity (MFI) of PTEN (Santa Cruz, CA, USA). The increase in CAPN-2 activity was defined as the MFI in the treated group subtracted from the MFI in the control group. Total CAPN-2 activity was the MFI in the treated group summed with the MFI in the control group.
Detection of cytosolic [Ca2 +]
The intracellular [Ca2 +] in VSC4.1 neurons was measured with the Ca2+-sensitive indicator fura‐2/-acetoxymethyl ester (AM) (Molecular Probes, CA, USA) [14]. After each treatment, the neurons were loaded with 5 μM fura-2-AM for 30 min at 37°C in the dark. After being diluted to 1 × 106 cells/ml with the same Ca2 + buffer solution, Fura-2-AM was excited at wavelengths of 340 and 380 nm. The relative changes in intracellular [Ca2+] were determined by the fluorescence ratio (R) at 340/380 with the following formula: [Ca2+]=Kd×β×(R−Rmin)/(Rmax−R)[14]. We used the Calcium Calibration Buffer Kit with Magnesium (Molecular Probes, CA, USA) and determined that the Kd, a cell‐specific constant, for VSC4.1 neurons was 0.264 μM.
Lactate dehydrogenase (LDH) assay
The LDH released from VSC4.1 neurons was detected by a commercial LDH Assay Kit (Abcam, CA, USA). According to the manufacturer’s instructions, 50 µL medium was collected at 24h after each treatment and measured at the absorbance of 450 nm.
Detection of Caspase-8 activity
The caspase-8 activity was detected by the caspase‐8 assay kit (Abcam, CA, USA), which is based on the spectrophotometric detection of p‐nitroaniline (pNA) moiety after it is cleaved from the labeled substrate Ac-IETD by caspase-8. The sample were measured in triplicate at the absorbance at 405 nm.
Detection of p25/Cdk5and p35/Cdk5 activities by ELISA
The commercialized ELISA kits (Runyu Biological Technology Co., Shanghai,China) were used to measure the p25/Cdk5 and p35/Cdk5 activities in VSC 4.1 neurons. According to the manufactures instructions, the activities in supernatants were measured at 450 nm after each treatment. Each sample were performed in triplicate and the average was presented as ng/L.
Detection of neuronal apoptosis by flow cytometry
The apoptotic neurons were by detected by BD FACSCalibur flow cytometry (BD Bioscience, MA, USA) with excitation at 488 nm and emission at 530 nm [5]. Briefly, the 1×105 neurons were first stained with 10 µl Annexin V-fluorescein isothiocyanate (FITC) at 37 ℃ for 15 min and then counterstained with 5 µl propidium iodide (PI) for 30 min in the dark. The fluorescence was excitation at 488 nm and emission at 530 nm. Each sample was prepared in triplicate.
Quantitative RT-PCR
Total RNA was extracted from L4–6 segments of spinal cords or VSC4.1 neurons by the TRIzol/chloroform method or the miRNeasy FFPE kit (Qiagen, Hilden, Germany) [5]. RNA (500 ng) was reversely transcribed to cDNA by cDNA SuperMix (TaKaRa, China) or a MicroRNA Reverse Transcription Kit (Applied Biosystems, USA). The quantification of miR-137-3p and CAPN-2 were carried out with TaqMan MicroRNA Assays Kit or power SYBR green PCR master mix (Takara, China) on an Applied Biosystems 7500 RT-PCR System (Applied Biosystems, CA, USA). β-actin or U6 were used as an internal control. Each sample was measured in triplicate by the 2−ΔΔCT method.. The primers used in this study were as follows: miR-137-3p (forward: 5′-ACACTCATTATTGCTTA-3′; reverse: 5′-CTACGCGTATTGAGAGTAC-3′); CAPN-1 (forward: 5′-CTCCGGGGCAGGAGTAGGCA-3′; reverse: 5′-CTCCGGGGCAGGAGTAGGCA-3′); CAPN-2 (forward: 5′-CTCCGGGGCAGGAGTAGGCA-3′; reverse: 5′-AACTGGCTGTGGGGCTCCCA-3′); U6 (forward: 5′-CTCGCTTCGGCAGCACA-3′; reverse: 5′-AACGCTTCACGAATTTGCGT-3′) and β-actin (forward: 5′-GGAGATTACTGCCCTGGCTCCTA-3′; reverse: 5′-GACTCATCGTACTCCTGCTTGCTG-3′).
Double immunofluorescence (IF)
As previously described, for in vivo samples, the 20-μm-thick sectioned spinal cord were blocked with 10% bovine serum albumin (BSA) for 1 h. Then, the sections were incubated with the primary mouse anti-calpain-2 antibody (SantaCruz, sc-373967, 1:300, Dallas, USA) and the antibodies specific marker for neurons (rabbit anti-NeuN, Abcam, ab177487, 1:500), for astrocytes (rabbit anti-GFAP, Abcam, ab7260, 1:500) and for microglial cells (rabbit anti-Iba-1, Abcam, ab178847,1:400) overnight at 4 °C. Then the sections were incubated with Alexa 594-conjugated donkey anti-mouse IgG (1:500, Life Technologies, CA, USA) and Alexa 488-conjugated donkey anti-rabbit IgG (1:500, Life Technologies, CA, USA) for 2 h at room temperature.
For in vitro samples, after be fixed with 4% formaldehyde for 20 min at 4 °C, the neurons were permeabilized with 0.1% TritonX-100 for 10 min and blocked by 3% donkey serum for 1 h at room temperature. Then the neurons were incubated with primary rabbit anti-p35 antibody (Abcam, ab64960, 1:300, CA, USA), primary rabbit anti-TPPP/p25 antibody (Abcam, ab92305, 1:300, CA, USA), mouse anti-calpain-2 antibody, mouse anti-caspase-8 p18 antibody (SantaCruz, sc-393776,1:400, Dallas, USA) overnight at 4 °C, followed by Alexa-conjugated secondary antibodies (1:500, Life Technologies, CA, USA) for 1h at room temperature in the dark. For cell counting, the nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI, Beyotime Biotechnology, China) for 10 min at room temperature. The image were captured with a Leica TCS SP2 fluorescence microscope (Leica Microsystems, IL, USA). The integrated fluorescent density was measured with LEICA IM50 software (Nussloch, Germany)
Western blotting
The total proteins from L4–6 spinal cords or VSC4.1 neurons were extracted and purified with a protein extraction kit (KangChen, China) [4,6]. After determined by a BCA protein assay kit (Beyotime, China), equal protein concentration were loaded onto 10% SDS-PAGE gel and transferred to PVDF membranes. The membrane was incubated with 5% skim milk for 1 h to avoid nonspecific binding and probed with anti-calpain-1 antibody (SantaCruz, sc-271313, 1:400, Dallas, USA) , anti-calpain-2 antibody (1:500), anti-p35 (1:400), anti-TPPP/p25 antibody (1:500), anti-PTEN antibody (SantaCruz, sc-7974, 1:400, Dallas, USA), anti-Cdk5-antibody (SantaCruz, sc-6247, 1:300, Dallas, USA), anti-caspase-8 p18 antibody, anti-caspase-3 antibody (Abcam, ab184787, 1:500, CA, USA) or β-actin (SantaCruz, sc-47778, 1:2000, Dallas, USA) overnight at 4 °C. After washes, the membrane were incubated with peroxidase-conjugated secondary antibodies (Beyotime, A0192, 1:10,00, China) for 2 h at room temperature. The blots were detected by an ECL kit (Beyotime, China) and quantified by Quantity One software (Bio-Rad Laboratories, Italy).
Statistical analysis
The data were expressed as the mean±standard deviation (SD) and analyzed using SPSS 19.0 software (SPSS, Chicago,USA). Statistical comparisons between two groups were assessed by t tests or Mann-Whitney tests, whereas comparisons among three or more groups were performed one- or two-way ANOVA followed by the Tukey-Kramer test. A P value <0.05 was considered statistically significant.