Grb2 Expression in Acute Spinal Cord Injury After Methylprednisolone Intrathecal Injection in Rats

Ethics Statement

All animal procedures were performed in accordance with the guidelines of the China Council for Animal Care and approved by the Central South University Animal Care Committee (Protocol No: 20200220005). The animals were housed in specific pathogen-free facilities at the Central South University. All surgeries and imaging were performed under anesthesia using ketamine, and all efforts were exerted to minimize suffering. Mice were humanely euthanized using CO₂ at a flow rate of 20% of the chamber volume/min following the guidelines of the China Council for Animal Care.

Animals

In total, 120 8- to 12-week-old male SD rats (Central South University Laboratory) weighing 300 to 400 g were used for this study. All animals were maintained in a standard housing environment with ad libitum access to food and water. Samples of spinal cord tissues were collected to search for biomarkers using iTRAQ coupled with 2D-LC-MS/MS. In all experiments, the animals were randomly and equally divided into the following 12 experimental groups (n = 10): MP-0, MP-3, MP-6, MP-8, MP-12, and MP-24 (MP intrathecal injection after ASCI at 0, 3, 6, 8, 12, and 24 hours); C-0, C-3, C-6, C-8, C-12, and C-24 (NS intrathecal injection after ASCI at 0, 3, 6, 8, 12, and 24 hours).

Surgery

The animals were anesthetized using an intraperitoneal injection of ketamine (100 mg/kg), atropine (10 mg/kg), and diazepam (5 mg/kg). After inducing a surgical plane of anesthesia (anesthesia endpoint was assessed by toe pinch and eye blink reflex), the surgical areas were shaved and disinfected. The animals were placed in a stereotaxic apparatus, and dorsal midline incisions were made from T6 to T12. After the thoracic vertebrae were carefully exposed, laminectomy was performed using a fine rongeur at the T8/10 level to expose the spinal cord. Following laminectomy, the spine was stabilized by clamping the transverse processes of one segment above and below the lesion site.3 A contusion was generated through the displacement-controlled Allen’s frame.4 The impactor tip was placed at the T9 center. A contusion was induced with displacement in the 2 groups. Afterward, MP and NS were intrathecally injected at 0, 3, 6, 8, 12, and 24 hours after ASCI. After surgery, the animals were kept in a temperature-controlled incubator until completely awake. Morphine (0.03 mg/kg) was subcutaneously administered to prevent postoperative pain.

Spinal Cord Tissue Collection

Spinal cord tissues were collected at 24 hours after MP and NS intrathecal injection. The T8/10 spinal cords (approximately 1 cm long and 500 mg) were dissected, the meninges were carefully removed, and the tissues were thoroughly rinsed with NS to remove blood. The spinal cord tissues were immediately frozen in liquid nitrogen and preserved at −80°C until analysis.

Separation and Identification of Differentially Expressed Proteins

Only 8 samples can be studied at a time using iTRAQ, and the samples should be simultaneously evaluated. Thus, the 6 control groups (C-0, C-3, C-6, C-8, C-12, and C-24) were analyzed using iTRAQ and 2D-LC-MS/MS, and the 4 groups with significant expression levels were selected. The 4 control groups and 4 MP groups were simultaneously analyzed.

iTRAQ Labeling and Strong Cation-Exchange Chromatography Fractionation

Spinal cord tissues were seeded in 6- or 8-well plates and transfected with 5 pmol miR-451 mimics using Lipofectamine 2000 (Invitrogen). After 48 hours of transfection, cells were harvested, and proteins were prepared and digested as previously described. The digested peptides were labeled with different iTRAQ reagents (Applied Biosystems), including 114, 115, 116, 117, 118, and 119. Subsequently, the labeled peptides were equally mixed and were fractionated using Poly SULFOETHYL ATM Strong Cation-Exchange column (200 × 4.6 mm, 5 μm particle size, and 200 Å pore size) using a high-performance liquid chromatography (HPLC) system (Shimadzu, Japan). The HPLC gradient consisted of 100% buffer A (10 mM KH₂PO₄, 25% acetonitrile, and pH 2.85) for 5 minutes; 0% to 20% buffer B (10 mM KH₂PO₄, 25% ACN, 500 mM KCL, and pH 3.0) for 15 minutes; 20% to 40% buffer B for 10 minutes; 40% to 100% buffer B for 5 minutes; and 100% buffer A for 10 minutes. The chromatograms were obtained at 218 nm. A total of 12 fractions were collected, desalted using Sep-Pak Vac C18 cartridges (Waters, Milford, MA), concentrated to dryness using vacuum centrifuge, and then reconstituted in 0.1% formic acid for 2D-LC-MS/MS analysis.

2D Liquid Chromatography Tandem Mass Spectrometry

LC-MS/MS analysis of the samples was performed on an AB SCIEX TripleTOF 5600 mass spectrometer (AB SCIEX, Framingham, MA, USA) coupled with an online micro flow HPLC system (Shimadzu, JAPAN). The HPLC system consisted of a trap column (200 µm × 2 cm, C18 material 5–10 µm, and 120 A°) and an analytical column (75 µm × 10 cm, C18 material 5 µm, and 120 A°). The peptides were separated with a nanobored C18 column with a picofrit nanospray tip (75 μm ID × 15 cm, and 5 μm particles; New Objectives, Wubrun, MA) at a constant flow rate of 20 μL/min. A splitter was used to obtain an effective flow rate of 0.2 μL/min. The MS spectra were acquired in the positive ion mode, and the peptides with +2 to +4 ions in the survey scan were targeted with a selected mass range of 300 to 2000 m/z. The 3 most abundantly charged peptides above a count threshold were selected for MS/MS and dynamically excluded for 30 seconds with ±30 mDa mass tolerance. The fragment intensity multiplier was set to 20, and the maximum accumulation time was 2 seconds. The peak areas of the iTRAQ reporter ions reflected the relative abundance of the proteins in the samples.

Bioinformatics Analysis

Mascot software (Matrix Science) version 2.3.02 was used to simultaneously identify and quantify proteins. In this version, only unique peptides used for protein quantification were selected. The spectra were combined into a single MGF (Mascot generic format) file after loading the raw data, and the MGF file was searched. The search parameters were as follows: trypsin was selected as the enzyme with one missed cleavage allowed; fixed modifications of carbamidomethylation at Cys, variable modifications of oxidation at Met; peptide tolerance was set to 10 ppm; and MS/MS tolerance was set to 0.05 Da. The peptide charge was set as Mr, and monoisotopic mass was selected. iTRAQ 6 or 8 plex was simultaneously selected for quantification during the search.

The search results were evaluated through additional filters before exporting the data. The filters for protein identification were set as follows: significance threshold P < 0.05 (with 95% confidence) and ion score or expected cut off less than 0.05 (with 95% confidence). For protein quantitation, the filters were set as follows: “median” was selected for the protein ratio type (http://www.matrixscience.com/help/quant_config_help.html); minimum precursor charge was set to +2 and minimum peptide was set to 2; and only unique peptides were used to quantify proteins. Median intensities were set to normal, and outliers were automatically removed. The peptide threshold was set as described above for identity.

Proteins with at least 1.2-fold changes between the treatment and control samples at P < 0.05 were determined as differentially expressed proteins. Quantitation was performed at the peptide level by following the procedures described in http://www.matrixscience.com/help/quant_statistics_help.html. Student’s t test was performed using Mascot 2.3.02 software.