Grafted Neural Stem Cells Show Lesion-Specific Migration in Radiation-Injured Rat Brains

Abstract

Neural stem cells (NSCs) exhibit preferential homing toward some types of brain lesion, but their migratory property during radiation brain injury (RBI) remains unexplored. Here, we use the superparamagnetic iron oxide (SPIO)-labeled magnetic resonance imaging (MRI) technology to determine the migration of transplanted NSCs in two partial RBI models in real time, created by administering 30-55 Gy of radiation to the right or posterior half of the adult rat brain. SPIO-labeled NSCs were stereotactically grafted into the uninjured side one week after RBI. The migration of SPIO-labeled NSCs in live radiation-injured brains was traced by MRI for up to 28 days after engraftment and quantified for their moving distances and speeds. A high labeling efficiency (>90%) was achieved by incubating NSCs with 100μg/ml of SPIO for 12-24 hours. Upon stereotactic transplantation into the healthy side of the brain, SPIO-labeled NSCs were distinctively detected as hypointense signals on T2-weighted images (T2WI), showed sustained survival for up to 4 weeks, and exhibited directional migration to the radiation-injured side of the brain with a speed of 86-127 μm/day. The moving kinetics of grafted NSCs displayed no difference in brains receiving a high (55 Gy) vs. moderate (45 Gy) dose of radiation, but was slower in the right RBI model than in the posterior RBI model. This study shows that NSCs can be effectively labeled by SPIO and traced in vivo by MRI, and that grafted NSCs exhibit directional migration toward RBI sites in a route-dependent but radiation dose-independent manner.

Keywords: graft; magnetic resonance imaging; neural stem cell; radiation brain injury; superparamagnetic iron oxide.

Fig. 1. Morphology, identification and SPIO-labeling of the NSCs. (A, B) primary (P0) and passaged (P2) NSCs under 100× magnification. Scale bars show 100 μm. (C) Photomicrographs of neurospheres positive marker nestin under 200× magnification. Scale bars show 50 μm. (D) NF-200 staining of neuron in the NSCs after neuron-induction under 400× magnification. Scale bars show 25 μm. (E) GFAP staining of astrocyte in the NSCs after astrocyte-induction under 400× magnification. Scale bars show 25 μm. (F) Measurement of 100 μg ml⁻¹ SPIO-labeled NSCs growth viability by MTT assay in different time points of two groups. (G–I) NSCs were labeled with 0, 20, 50, 100, or 200 μg ml⁻¹ of SPIO and MR imaged in the T1-weighted (G), T2-weighted (H) or T2* sequence (I).

Fig. 2. Rat models with radiation brain injury (RBI). Schematic diagrams and X-ray films of the right (A) and posterior (B) RBI models. The electron beam and irradiated regions are indicated by the blue arrows and red lines, respectively. Abbreviations: A, anterior; P, posterior; R, right; L, left; D, dorsal; V, ventral.

Fig. 3. MR images of the 45 Gy posterior RBI model. (A) T2-weighted images (T2WI) of rat brains that received 45 Gy of radiation on the posterior brain and SPIO-labeled NSC transplantation. (B) T2WI of rat brains that received 45 Gy of radiation on the posterior brain and non-labeled NSC transplantation. (C) T2WI of rat brains that received 45 Gy of radiation on the posterior brain and SPIO solution injection. The injected site and hypointense signals were indicated by red arrows and red lines, respectively. The RBI lesion was circled by yellow lines. The orientation of the films follows that of Fig. 2B.

Fig. 4. MR images of the 55 Gy posterior RBI model. (A) T2WI of rat brains that received 55 Gy of radiation on the posterior brain and SPIO-labeled NSC transplantation. (B) T2WI of rat brains that received 55 Gy of radiation on the posterior brain and non-labeled NSC transplantation. (C) T2WI of rat brains that received 55 Gy of radiation on the posterior brain and SPIO solution injection. The injected site and hypointense signals were indicated by red arrows and red lines, respectively. The RBI lesion was circled by yellow lines. The orientation of the films follows that of Fig. 2B.

Fig. 5. Quantification of NSC migration in the 45 Gy and 55 Gy posterior RBI models. (A) Distance of lesion-directed (A1) and lesion-opposite (A2) migration from 0 to 28 days after NSC transplantation in the 45 Gy posterior RBI model. (A3) Spreading distance of injected SPIO solution on the 0 and 28th day. (B) Distance of lesion-directed (B1) and lesion-opposite (B2) migration from 0 to 28 days after NSC transplantation in the 55 Gy posterior RBI model. (B3) Spreading distance of injected SPIO solution on the 0 and 28th day. (C) The net gain of distance between lesion-directed vs. lesion-opposite migration from 0 to 28 days in the 45 Gy (black bars) and 55 Gy (grey bars) models. (D) Calculation of the average speeds of migration by the slopes of the best-fit lines in the 45 Gy (D1) and 55 Gy (D2) models. Bars show mean (±sd); * and ** indicate p values < 0.01 and 0.001; n.s., not significant.

Fig. 6. MR images of the 30 Gy right RBI model. (A) T2WI of rat brains that received 30 Gy of radiation on the right brain and SPIO-labeled NSC transplantation. (B) T2WI of rat brains that received 30 Gy of radiation on the right brain and non-labeled NSC transplantation. (C) T2WI of rat brains that received 30 Gy of radiation on the right brain and SPIO solution injection. The injected site and hypointense signals were indicated by red arrows and red lines, respectively. The RBI lesion was circled by yellow lines. Abbreviations: R, right; L, left; D, dorsal; V, ventral.

Fig. 7. Quantification of NSC migration in the 30 Gy right RBI models. (A) Distance of lesion-directed (A1) and lesion-opposite (A2) migration from 0 to 28 days after NSC transplantation in the 30 Gy right RBI model. (A3) Spreading distance of injected SPIO solution on the 0 and 28th day. (B) The gain of distance between lesion-directed vs. lesion-opposite migration from 0 to 28 days in the 30 Gy right RBI (black bars) and 45 Gy posterior RBI (grey bars) models. (C) Calculation of the average speed of NSC migration in the right RBI model by the slope of the best-fit line.