Cell culture
Rat glioma C6 cells were purchased from the Beijing Institutes of Life Science, Chinese Academy of Sciences (Beijing, China) and cultured as monolayers in Gibco® RPMI 1640 medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing Gibco® 10% Fetal Bovine Serum (Thermo Fisher Scientific, Inc.) in a humidified incubator (NuAire, Inc., Caerphilly, UK) containing 5% CO2 at 37°C. Cells in the exponential phase of growth were used for the present study.
Rat C6 glioma model
All animal protocols in the present study were approved by the Ethics Committee of the Institutional Research Board of Harbin Medical University (Harbin, China; approval no. HMUIRB20150051). Male pathogen-free Wistar rats weighing 220–250 g (60 rats in total; age, 7–8 weeks) were purchased from the Animal Experiment Center of Harbin Medical University, and were kept for 24 h at room temperature and with free access to water and standard laboratory food prior to C6 cell injection. Injection of C6 cells was performed as previously described (9). Briefly, the rats were anesthetized with 10% chloral hydrate (3 ml/kg; CAS no. 302-17-0; Yangzhou Aoxin Chemical Factory, Yangzhou, China) and fixed in stereotaxic apparatus (Motorized Lab Standard Stereotaxic Instrument; catalog no. 51700, Stoelting Co., Wood Dale, IL, USA) for the facilitation of the injection. Following sterilization and a skin incision in the scalp of the rats, a hole was rendered in the skull 1.0 mm anterior to the anterior fontanel and 1.0 mm lateral to the sagittal suture. A syringe was inserted 5 mm into the cerebral cortex and 10 µl C6 glioma cell suspension (1×106 cells) was injected, as described previously (7). The duration of the procedure was 10 min. The syringe remained in the brain for 5 min followed by a slow extraction. The hole was sealed with bone wax (catalog no. 1029754; B. Braun Melsungen AG; Melsungen, Germany) and the scalp was sutured. Following sterilization, the rats were returned to their accommodation and monitored.
Study design
The presence of glioma was confirmed in the rats 7 days following injection with C6 cells using unenhanced and enhanced magnetic resonance imaging (MRI; Signa HDxt 3.0T; GE Healthcare Bio-Sciences, Pittsburgh, PA, USA), as previously described (10). T1-weighted coronal and axial images were acquired from unenhanced and enhanced scans. Enhanced scans were achieved using an intravenous injection of gadolinium-diethylene triamine pentaacetic acid (Gd-DTPA; 0.4 ml/kg; Magnevist®; Bayer AG, Leverkusen, Germany) through the tail vein of the rats. The rats with glioma were randomly assigned to 4 groups (n=15): Control group, the rats received no treatment; PDT group, the rats received PDT treatment on day 8 subsequent to injection with C6 cells; torasemide group, the rats received 5mg/kg torasemide intraperitoneally (Yoko Pharmaceutical Co., Ltd., Nanjing, China) on day 8 subsequent to injection for a duration of 3 days; and PDT + torasemide group, the rats received 5mg/kg torasemide intraperitoneally following PDT on day 8 subsequent to injection for a duration of 3 days. The rats in the control and PDT groups received saline at volumes equivalent to the torasemide received by the rats in the torasemide and PDT + torasemide groups. On day 21 subsequent to injection, 5 rats in each group were sacrificed. Glioma tissues were harvested and processed immediately for wet-to-dry weight (W/D) ratio, histological examination, immunohistochemistry and western blot analysis. The remaining 10 rats in each group were used to determine survival times.
PDT treatment
Hematoporphyrin monomethyl ether (HMME; Shanghai Fudan-Zhangjiang Bio-Pharmaceutical Co., Ltd., Shanghai, China) was administrated via the tail vein of the rats at a dose of 5 mg/kg on day 8 subsequent to C6 cell injection, and were kept in the dark for 3 h. Subsequently, the rats were anesthetized with 10% chloral hydrate (3 ml/kg) and fixed in a stereotaxic apparatus to expand the hole in the skull to 10 mm in diameter. The tumor was exposed by a microneurosurgical method 5 min later. The optical fiber of the PDT equipment (wavelength, 630 nm; DIOMED 630 PDT Laser; model, T2USA; Diomed Ltd., Cambridge, UK) was placed on the tumor surface and PDT was administered at 80 J/cm2 in a 30 mm2 region for 10 min, as previously described (8). Following PDT, the hole was sealed with bone wax and the scalp was sutured.
W/D ratio of peritumoral tissues
Regions of peritumoral tissue 1–4 mm in diameter were resected from glioma. The resected tissues were placed on an ice plate and were immediately weighed. Subsequently, the tissues were desiccated in a 105°C oven for 24 h until a stable dry weight was achieved. The W/D ratio was calculated for water content of the tumor.
Histological observation
Glioma tissues were harvested 21 days following C6 injection and were immediately fixed in 10% formalin (catalog no. 50-00-0; Dezhou Yun Xin Experimental Instrument Co., Ltd., Dezhou, China), and embedded in paraffin. Tissue sections 6 µm thick were cut and stained with hematoxylin and eosin (H&E; catalog no. C0105, Beyotime Institute of Biotechnology, Shanghai, China) for light microscopy (Eclipse 80i; Nikon Corporation, Tokyo, Japan).
Immunohistochemistry
Paraffinized peritumoral edema tissues were cut into 6-µm thick sections for immunohistochemistry. The sections were deparaffinized and hydrated followed by antigen retrieval; the tissues were incubated with 3% hydrogen peroxide (catalog no. 7722-84-1; Shanghai Ziyi Reagent Factory, Shanghai, China) for 10 min and subsequently citric acid buffer (catalog no. CW0128; Beijing Tianrui Technology Co., Ltd., Beijing, China) for 3 min. The tissue sections were incubated overnight at 4°C with the primary antibodies rabbit anti-rat NKCC-1 antibody (polyclonal; dilution, 1:500; catalog no. ab59791, Abcam, Cambridge, UK), rabbit anti-rat MMP2 antibody (polyclonal; dilution, 1:200; catalog no. ab38898; Abcam) and mouse anti-rat glial fibrillary acidic protein antibody (GFAP; polyclonal; dilution, 1:500; catalog no. ab7260; Abcam). Subsequently the tissues were washed with phosphate-buffered saline three times prior to the addition of the goat anti-rabbit secondary antibody (monoclonal; dilution, 1:2,000; catalog no. TA130024; OriGene Technologies, Inc., Beijing, China) for 30 min at 37°C. The positive index of a tissue section was the number of cells out of 50 cells that expressed stained cells in 5 fields of view. The tissue sections were blindly examined by a pathologist (Harbin Medical University).
Western blot analysis
Peritumoral edema tissue was ground in liquid nitrogen and dissolved in a RIPA Lysis Buffer (Beyotime Institute of Biotechnology), which contained the protease inhibitor phenylmethanesulfonyl fluoride. The total protein was extracted from the tissue, and the protein concentration was determined using the BCA Protein Assay Kit (catalog no. P0012; Beyotime Institute of Biotechnology) and stored at −80°C for additional analysis. Equal amounts of protein (>20 µg) from each group were loaded and resolved using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (0.45 µm; EMD Millipore, Boston, MA, USA). The membranes were blocked with 5% non-fat milk in Tris-buffered saline, and subsequently incubated with rabbit anti-rat MMP2 and rabbit anti-rat NKCC1 antibodies at 4°C overnight. The membranes were incubated with goat anti-rabbit secondary antibody (monoclonal; dilution, 1:2,000; catalog no. TA130024; OriGene Technologies, Inc.) for 2 h at room temperature. The optical density of bands was determined using ImageJ software (version 1.48; National Institutes of Health, Bethesda, MA, USA) and the data were quantified by normalization to the density of β-actin (OriGene Technologies, Inc.).
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Total RNA of each tissue sample was collected using the RNAsimple Total RNA kit (catalog no. DP419; Tiangen Biotech, Co., Ltd., Beijing, China), according to the manufacturer's protocol. A final volume of 20 µl cDNA was synthesized from 500 ng total RNA using Super M-MLV Reverse Transcriptase (BioTek China, Beijing, China) and the AffinityScript cDNA Synthesis kit (Agilent Technologies, Inc., Santa Clara, CA, USA), according to the manufacturer's protocol. The qPCR was performed on standardized quantities of cDNA using Exicycler™ 96 (Bioneer Corporation, Daejeon, Korea). The following primers (Shanghai Kehua Bio-engineering Co., Ltd., Shanghai, China) were used: MMP2, forward 5′-GTGGCAATGGAGATGGACA-3′ and reverse 5′-GGTCATAATCCTCGGTGGTG-3′; NKCC1, forward 5′-CCTGGGAGAGTTCCACGAT-3′ and reverse 5′-TTCGGCAGTGTATGTGACCA-3′; and β-actin, forward 5′-TCAGGTCATCACTATCGGCAAT-3′ and reverse 5′-AAAGAAAGGGTGTAAAACGCA-3′. All samples were run in duplicate. Relative mRNA expression levels were calculated as 2−∆∆Cq (11).
Statistical analysis
All data were represented as the mean ± standard deviation. Statistical analyses were performed using the SPSS version 10.0 software (SPSS, Inc., Chicago, IL, USA). The difference in results between the various groups was analyzed using one-way analysis of variance. Survival data were analyzed by Kaplan-Meier survival analysis, and log rank test was used to compare the four groups. P<0.05 was considered to indicate a statistically significant difference.