Monitoring Bone Marrow Stem Cells with a Reporter Gene System in Experimental Middle Cerebral Artery Occlusion Rat Models

Adenoviral Vector

An adenoviral vector (Ad5-TIBE) carrying the HSV1-tk, BDNF, and enhanced green fluorescent protein (EGFP) genes under the control of the cytomegalovirus promoter and Ad5-EGFP were constructed by Ming Hong Biology Co. Ltd. The total yields of Ad5-TIBE and Ad5-EGFP were 2 × 10¹⁰ and 3.6 × 10¹⁰ plaque-forming units/mL, respectively.

Experimental MCAO Models

This study was approved by the Institutional Animal Care and Use Committee at Huazhong University of Science and Technology. Adult male Sprague–Dawley rats weighing 250–300 g (supplied by the Experimental Animal Center of Tongji Medical Collage, Huazhong University of Science and Technology) were used to prepare the experimental MCAO models by a modification of the method of Kuge et al. (15). Animals were initially anesthetized intraperitoneally with 10% chloral hydrate at 0.3 mL/100 g of weight. Rat body temperature was maintained at 37°C with a heat lamp throughout the surgical procedure. The left common carotid artery, external carotid artery, and internal carotid artery were exposed under anesthesia. A 4-0 monofilament nylon suture with a tip rounded by flame heating was inserted from the left common carotid artery into the internal carotid artery and then advanced approximately 18 mm intracranially from the common carotid artery bifurcation. Sixty minutes after MCAO, rats were reanesthetized, and reperfusion was performed by withdrawal of the suture. All animals underwent behavioral testing 24 h after MCAO. A previously described (16) 5-point system was used to assess the severity of injury. Rats with scores ranging from 1 (failure to extend forepaw fully) to 3 (falling) points were used.

Cell Preparation and Transplantation

BMSCs were isolated and cultured from the bone marrow of Sprague–Dawley rats. Cells in the exponential growth phase were seeded at a density of 5 × 10⁵/well of 6-well culture plates. After reaching fusion, the cells were infected with Ad5-TIBE at a multiplicity of infection of 150 plaque-forming units per cell. The cells were exposed to the viral particles at 37°C for 2 h, the medium was removed, and the cells were washed once with phosphate-buffered saline (PBS) and recultured with normal medium for 36 h. Next, the cells were harvested for transplantation.

Previous studies showed that cell transplantation in the acute phase after MCAO can lead to more obvious functional improvements (17,18). Therefore, cell transplantation was performed 1 d after MCAO in this study. After the cells were prepared, a suspension containing 2 × 10⁶ cells was collected and centrifuged for 5 min at 1,000 rpm, and then the cells were suspended in 15 μL of PBS. A 25-μL microsyringe affixed to the stereotactic instrument was used to pump the suspension. The stereotactic coordinates for cell local injection were 1 mm before the bregma, 3 mm lateral to the midline, and 5 mm beneath the skull surface; the coordinates for ventricular injection were 1 mm after the bregma, 1.5 mm lateral to the midline, and 3.5 mm beneath the skull surface. A suspension of 2 × 10⁶ cells in 15 μL of PBS was injected into each rat that had undergone MCAO over a 5-min period on the stereotactic apparatus (Ruiwode Life Technology Co., Ltd.). However, for carotid artery and tail vein injections, a 0.5-mL cell suspension was prepared. All animals received penicillin (2 × 10⁵ U daily) intramuscularly as prophylaxis, and no immunosuppressant was used in any animal.

Radioiodination of 2′-Fluoro-2′-Deoxy-1-β-d-Arabinofuranosyl-Uracil

2′-Fluoro-2′-deoxy-1-β-d-arabinofuranosyl-uracil (Moravek Biochemicals) was labeled according to the method described by Lan et al. (19). The product was purified with methanol on a reverse-phase Sep-Pak C-18 column (Waters). The final product (¹³¹I-FIAU) was collected to determine biochemical purity with thin-layer chromatography in an acetoacetate–acetone–water (12:8:1, v/v) solution. The resulting product was incubated in PBS and human serum at 37°C for 12 and 24 h to verify stability.

Biodistribution

For biodistribution experiments, cells were transplanted into MCAO models via local injection (n = 5), ventricular injection (n = 5), carotid artery injection (n = 5), and tail vein injection (n = 5). Normal rats were used as a control group (n = 4). The animals were sacrificed 24 h after injection of 1.11 MBq of ¹³¹I-FIAU into each rat. Bilateral brain, thyroid, lung, heart, liver, stomach, pancreas, spleen, kidney, muscle, bone, blood, and small intestine tissues were extracted; their wet weights were measured, and γ-counts were determined. After correction for radioactive decay, the percentage injected dose per gram (%ID/g) and the ratio of the %ID/g in infarcted brain to the %ID/g in blood were calculated.

Real-Time Quantitative Polymerase Chain Reaction and Western Blotting

Total RNA was extracted from bilateral brain tissues, and complementary DNA was synthesized according to the instructions in a Maxima First Strand cDNA Synthesis Kit (Thermo Scientific). The primer sequences were 5′-CTCACCCTCATCTTCGACCG-3′ (forward) and 5′-CCTGCAGATACCGCACCGTA-3′(reverse). Amplification for 45 cycles was performed by use of a Real-time PCR Detection System (Applied Biosystems) with those primer sequences; the total reaction volume was 10 μL.

The brain tissue was removed immediately under anesthesia, ground completely, and centrifuged at 12,000 rpm and 4°C for 5 min. The supernatant was collected for protein quantitation by use of a bicinchoninic acid protein assay kit (Beyotime Biotechnology). Fifty micrograms of protein were loaded onto a sodium dodecyl sulfate–polyacrylamide gel for electrophoresis and electroblotted onto a polyvinylidene difluoride membrane (Invitrogen). The membrane was incubated with anti–thymidine kinase (TK) (1:500) overnight at 4°C, and β-actin was used as a control. AlphaEaseFC software, version 4.0 (Alpha Innotech), was used to verify the level of expression of the HSV1-tk gene. Each experiment was repeated in triplicate.

ARG

For ARG experiments, cells were transplanted into the brain via local injection. Twelve hours after cell transplantation, 1.11 MBq of ¹³¹I-FIAU were injected via the tail vein. Rats were divided into 4 groups as follows. In groups 1 and 2, the probes ¹³¹I-FIAU (group 1, n = 5) and ¹³¹I (group 2, n = 5) were injected into MCAO rat models with BMSCs transfected with Ad5-TIBE. In group 3, ¹³¹I-FIAU (n = 5) was injected into MCAO rat models transfected with Ad5-EGFP–modified BMSCs. In group 4, ¹³¹I-FIAU (n = 5) was injected into normal rats that had undergone transplantation with nontransfected BMSCs. Four rats randomly selected from each group were sacrificed at 2, 12, 24, and 48 h after injection. The brain tissue was removed immediately and prepared for frozen sectioning. After a series of 15-μm-thick sections were obtained, ARG was performed with a Cyclone Storage Phosphor System (PerkinElmer), and the counts in the images were analyzed with PerkinElmer OptiQuant image analysis software (PerkinElmer). The ratio of counts in bilateral brain tissues was calculated.

SPECT Imaging

As with the ARG experiments, cells were transplanted into the brain via local injection, and then 1.11 MBq of ¹³¹I-FIAU were injected. SPECT imaging was performed with rats in the prone position at 2, 8, and 24 h after probe injection. The matrix was 512 × 512, and the zoom was 2. Image acquisition was stopped when counts reached 120,000.

Statistical Analysis

Data are presented as mean ± SE and were analyzed with SPSS 11.5 software (SPSS Inc.). The statistical significance level was set at a P value of less than 0.05. For analysis of differences, we adopted the independent-samples t test for 2 groups and the Levene test, the least significant difference t test, and the Kruskal–Wallis test for multiple groups.