Monitoring bone marrow stem cells with a reporter gene system in experimental middle cerebral artery occlusion rat models

Abstract

This study was designed to investigate the feasibility of imaging bone marrow stem cells (BMSCs) in experimental middle cerebral artery occlusion (MCAO) rat models with a reporter gene-probe system, HSV1-tk-(131)I-2'-fluoro-2'-deoxy-1-β-d-arabinofuranosyl-5-iodouracil ((131)I-FIAU), and to choose the best strategies for stem cell injection, image acquisition, and imaging in vivo.

Methods: A recombinant adenovirus (Ad5-TIBE) carrying the herpes simplex virus type 1 thymidine kinase (TK) reporter gene (HSV1-tk) linked via the internal ribosome entry site to the brain-derived neurotrophic factor therapeutic gene was prepared. After transfection with Ad5-TIBE, BMSCs were introduced into MCAO rat models via local injection into the brain or via injection into the lateral ventricle, carotid artery, and tail vein. Normal rats were used as controls. Twenty-four hours after (131)I-FIAU injection, rats were sacrificed for biodistribution analysis. The expression of the TK gene was evaluated by real-time quantitative polymerase chain reaction and Western blot analysis. Autoradiography was used for ex vivo imaging. SPECT images were obtained in MCAO rat models.

Results: The percentage injected dose per gram (%ID/g) in infarcted brain tissue in rats receiving the injection into the brain was 0.124 ± 0.013; this value was significantly higher than those in rats receiving the injection into the ventricle (0.052 ± 0.004), carotid artery (0.061 ± 0.002), and tail vein (0.059 ± 0.005) as well as normal rats (0.005 ± 0.001). No differences were seen in the other cell transplantation groups. The %ID/g in infarcted brain tissue was higher than that in the contralateral brain tissue in all experimental rats but not in normal rats. The expression of the TK gene in rats receiving a local injection into the brain was superior to that in all of the other groups. TK messenger RNA and protein expression showed a positive correlation with the %ID/g in brain tissue. Greater radioactivity at the injection site than in the surrounding and contralateral brain tissues in all experimental rats was indicated through autoradiography. The ratio of counts in bilateral brain tissues reached its peak (6.63) 24 h after (131)I-FIAU injection. SPECT images showed that radioactivity accumulation in the brain was low but increased gradually over time.

Conclusion: The HSV1-tk-(131)I-FIAU reporter gene-probe system may be used to monitor BMSC activity in experimental MCAO rat models. Local injection of stem cells may provide an optimal means for cell transplantation, and imaging with (131)I-FIAU 24 h after injection provides peak target-to-nontarget count ratios.

Keywords: SPECT; autoradiography; biodistribution; middle cerebral artery occlusion; reporter gene.