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, 27 (6), 489-507

Insulin-like Growth Factor-1 Receptor Dictates Beneficial Effects of Treadmill Training by Regulating Survival and Migration of Neural Stem Cell Grafts in the Injured Spinal Cord

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Insulin-like Growth Factor-1 Receptor Dictates Beneficial Effects of Treadmill Training by Regulating Survival and Migration of Neural Stem Cell Grafts in the Injured Spinal Cord

Dong Hoon Hwang et al. Exp Neurobiol.

Abstract

Survival and migration of transplanted neural stem cells (NSCs) are prerequisites for therapeutic benefits in spinal cord injury. We have shown that survival of NSC grafts declines after transplantation into the injured spinal cord, and that combining treadmill training (TMT) enhances NSC survival via insulin-like growth factor-1 (IGF-1). Here, we aimed to obtain genetic evidence that IGF-1 signaling in the transplanted NSCs determines the beneficial effects of TMT. We transplanted NSCs heterozygous (+/-) for Igf1r, the gene encoding IGF-1 receptor, into the mouse spinal cord after injury, with or without combining TMT. We analyzed the influence of genotype and TMT on locomotor recovery and survival and migration of NSC grafts. In vitro experiments were performed to examine the potential roles of IGF-1 signaling in the migratory ability of NSCs. Mice receiving +/- NSC grafts showed impaired locomotor recovery compared with those receiving wild-type (+/+) NSCs. Locomotor improvement by TMT was more pronounced with +/+ grafts. Deficiency of one allele of Igf1r significantly reduced survival and migration of the transplanted NSCs. Although TMT did not significantly influence NSC survival, it substantially enhanced the extent of migration for only +/+ NSCs. Cultured neurospheres exhibited dynamic motility with cytoplasmic protrusions, which was regulated by IGF-1 signaling. IGF-1 signaling in transplanted NSCs may be essential in regulating their survival and migration. Furthermore, TMT may promote NSC graft-mediated locomotor recovery via activation of IGF-1 signaling in transplanted NSCs. Dynamic NSC motility via IGF-1 signaling may be the cellular basis for the TMT-induced enhancement of migration.

Keywords: Insulin-like growth factor-1; Migration; Motility; Neural stem cell; Spinal cord injury; Treadmill training.

Figures

Fig. 1
Fig. 1. In vitro properties of Igf1r-heterozygous NSCs. (A) Representative images of neurospheres derived from NSCs wild-type (+/+) and heterozygous (+/−) for Igf1r gene. Since Igf1r-heterozygous line was crossed with DsRed.T3 strain, all neurospheres emit intense red fluorescence. Scale bars indicate 50 µm. Quantification graph comparing mean diameters of +/+ and +/− neuropsheres. N=4 independent cultures for each group. (B) Representative bright-field images at a lower magnification. Scale bars indicate 100 µm. Quantification graph comparing the number of +/+ and +/− neuropsheres. N=4 independent cultures for each group. (C, D) In vitro survival assays. NSCs were exposed to either 3-morpholinosydnonimine (SIN-1, 400 µM) (C) or H2O2 (1mM) (D) for 48 h with or without IGF-1 (20 ng/ml). The percentage of viable cells in the treatment groups was expressed as the percent value compared with that of control condition (without any treatment). N=4 independent cultures per group. ** and *** represent p<0.01 and p<0.001 compared to control condition, and ### represents p<0.001 comparing SIN-1 (C) or H2O2 (D) exposure groups with or without IGF-1 treatment by one-way ANOVA followed by Tukey's post hoc analysis at each time point.
Fig. 2
Fig. 2. Influence of IGF-1R in transplanted NSCs and treadmill training (TMT) on locomotor recovery. (A) Recovery of BMS (Basso Mouse Scale for locomotion) scores over the 8-week period after injury. Animals with transplanted NSCs wild-type (+/+) for Igf1r gene and combined treadmill training (TMT) together showed the best locomotor recovery. * and *** represent p<0.05 and p<0.001 by repeated-measures two-way ANOVA followed by post hoc Bonferroni analysis. N=9 animals for each group. (B, C) Ladder walk test at 4 (B) and 8 weeks (C) after injury. The number of hind-paw placement errors was counted at the 4- (B) and 8-week (C) time points. * represents p<0.05 by two-way ANOVA followed by post hoc Bonferroni analysis. N=9 animals for each group. (D~G) Catwalk automated footprint analysis assessed at 8 weeks after SCI. The graphs show (D) stride length (distance between the two consecutive hind-paw footprints), (E) base of support (width between the left and right hind-paws), (F) relative position (distance between the center pads of fore- and hind-paw prints), and (G) rotation angle (angle of the hind-paw axis relative to the horizontal plane). N=9 animals for each group.
Fig. 3
Fig. 3. Survival of NSC grafts in the injured spinal cord. (A, B) Representative images of longitudinal spinal cord sections at 4 (A) or 8 weeks (B) after injury. Spinal cord sections were immunostained with anti-RFP (red) and anti-glial fibrillary acidic protein (GFAP; green) antibodies to visualize the distribution of the transplanted NSCs in relation to the lesions. Scale bars indicate 1 mm. (C) Representative integrated drawing of stereologically counted RFP-positive NSCs at the 8-week time point. RFP-positive NSCs from 5 longitudinal sections 300 µm apart from each other were integrated in one image. The structural outlines of the spinal cord tissue in the integrated images were obtained from the most middle section in each animal. Scale bar indicates 1 mm. (D) Quantification graphs of the number of surviving NSCs at 4 and 8 weeks after injury. N=5 and 9 animals at 4 and 8 weeks, respectively, for each group. * and ** represent p<0.05 and p<0.01, respectively, by two-way ANOVA followed by post hoc Bonferroni analysis.
Fig. 4
Fig. 4. Phenotypic differentiation of NSCs after transplantation into the injured spinal cord. (A~D) Representative images of transplanted NSCs in the injured spinal cord from animals receiving Igf1r (+/+) NSC grafts with (C) or without (A) TMT, and from animals receiving Igf1r (+/−) NSC grafts with (D) or without (B) TMT. Transplanted cells were identified by immunoreactivity to red fluorescence protein (RFP). Neural cell phenotypes were determined by immunoreactivity (green) for neuronal marker DCX, mature oligodendrocyte marker APC-CC1, and astrocytic marker GFAP. RFP-positive NSCs (red) colocalized with neural cell markers are shown in yellow. Higher magnification and orthographic projection images of the boxed regions are shown at the bottom of each panel. All scale bars indicate 20 µm. (E) Quantification graphs of the number of differentiated NSCs at 8 weeks after injury. N=9 animals, for each group.
Fig. 5
Fig. 5. Treadmill training (TMT) enhances migration of NSCs in an IGF-1R-dependent manner. (A~D) Representative images of longitudinal spinal cord sections at 8 weeks after injury. Spinal cord sections were immunostained with anti-RFP (red) antibodies to visualize transplanted NSCs in the injured spinal cord. Boxed regions are magnified in (a~d). Scale bars indicate 500 µm. (a~d) Magnified images of the boxed regions in (A~D) showing NSC grafts that rostrally migrated. Scale bars indicate 200 µm. (a’~d’) Magnified images of the boxed regions in (a~d) showing migrating individual NSCs from the rostral grafts. Arrows indicated elongated cytoplasmic processes aligned with the direction of migration. Scale bars indicate 50 µm. (E) Quantification graphs comparing the extent of migration of NSCs at 4 and 8 weeks after injury. The migration extent was expressed as a migration index that was generated by dividing the longest longitudinal migratory distance by the longest transverse diameter of the RFP positive area. N=5 and 9 animals at 4 and 8 weeks, respectively, for each group. * and *** represent p<0.05 and p<0.001, respectively, by two-way ANOVA followed by post hoc Bonferroni analysis.
Fig. 6
Fig. 6. In vitro migration ability of NSCs. (A) Representative images of the bottom side of the Boyden chamber membrane separating upper and lower chambers. The membrane was stained to visualize NSCs that migrated to the bottom side. Scale bar indicates 50 µm. (B) Representative still images acquired at the designated time points of migratory NSCs in the scratch assay. Live imaging was performed to monitor migrating NSCs to the scratch created at the center of the culture plate. Scale bar indicates 200 µm. (C) Quantification graph of the Boyden chamber assay. *, **, and *** represent p<0.05, p<0.01, and p<0.001, respectively, by one-way ANOVA followed by post hoc Tukey's analysis. N=4 independent assays for each group. (D) Quantification graph of the scratch assay. Statistical analysis was done with the percent area of cell occupancy data obtained at 12-h time intervals. *** represents p<0.001 by repeated measures two-way ANOVA followed by post hoc Bonferroni analysis. N=3 independent assays for each group.
Fig. 7
Fig. 7. Spontaneous motility of cultured neurospheres. (A) Representative still images of cultured neurospheres obtained using a live cell imaging apparatus. Images were acquired at the time points designated at the upper right corners. 0 h indicates a start of image tracking for the next 12 h, not the absolute start of the culture. Grid lines were drawn to facilitate tracking positions of neurospheres. Colored dots indicate the marks of neurosphere centers that are traced to quantify distances of neurosphere movements. Neurospheres marked with dots of the same color in the images at different time points are the identical ones. Boxed area indicates the magnified regions in (B). Scale bar indicates 100 µm. (B) Magnified images at the regions boxed in (A). Images at a 1-h time interval (designated at the upper right corners) starting at the 6-h time point are presented. Arrowheads indicate the tips of cytoplasmic protrusions that are traced to quantify lengths of changes in cytoplasmic protrusions. The colors of arrowheads are the same as the ones for the center marks of the neurospheres in (A) which the protrusions belong to. (C) An exemplary drawing to illustrate motile paths of neurosphere centers tracked for 24 hours by an image analysis software. Different colors are used to illustrate motile paths of different individual neurospheres. Scale bar indicates 100 µm. (D, E) Quantification graphs of the moving distance (D) of the tracked neurospheres and the length of changes in cytoplasmic protrusions (E) growing from the tracked neurospheres. Each data point represents the distance moved or the length changed during the 30-minute period between two successive imaging sessions at every 30 minutes.
Fig. 8
Fig. 8. Potential signaling pathways implicated in the spontaneous motility of neurospheres. (A~D) Representative still images of cultured neurospheres obtained using a live cell imaging apparatus. Images were taken at the time points designated at the upper right corners. 0 h indicates a start of image tracking for the next 12 h, not the absolute start of the culture. Neurospheres were treat with control (CTL) PBS (A), a PI3K inhibitor LY294002 (10 µM) (B), an MEK/ERK inhibitor U0126 (10 µM) (C), and a ROCK inhibitor Y27632 (10 µg/ml) (D). Grid lines were drawn to facilitate tracking positions of neurospheres. Colored dots indicate the marks of neurosphere centers that are traced to quantify distances of neurosphere movements. Neurospheres marked with dots of the same color in the images at different time points are the identical ones. Scale bars indicate 100 µm. (E, F) Quantification graphs of the moving distance (E) of the tracked neurospheres and the length of changes in cytoplasmic protrusions (F) growing from the tracked neurospheres. Total cumulative distance or length traced for the entire culture duration was plotted. N=15 neurospheres tracked from three independent cultures. * and *** represent p<0.05 and p<0.001 compared to CTL condition by one-way ANOVA followed by Tukey's post hoc analysis.
Fig. 9
Fig. 9. Regulation of NSC motility by IGF-1/IGF-R signaling. (A~D) Representative still images of cultured neurospheres obtained using a live cell imaging apparatus. Images were taken at the designated time points. 0 h indicates a start of image tracking for the next 12 h, not the absolute start of the culture. Neurospheres were obtained from NSCs wild-type (+/+) (A~C) or heterozygous (+/−) (D) for Igf1r gene. +/+ neurospheres were treated with control (CTL) PBS (A), IGF-1 (50 ng/ml) (B), or an IGF-1R inhibitor picropodophyllin (PPP, 100 µM) (C). (+/−) neurospheres were treated with only CTL PBS (D). Grid lines are drawn to facilitate tracking positions of neurospheres. Colored dots indicate the marks of neurosphere centers that are traced to quantify distances of neurosphere movements. Neurospheres marked with dots of the same color in the images at different time points are the identical ones. Scale bars indicate 100 µm. (E, F) Quantification graphs of the moving distance (E) of the tracked neurospheres and the length of changes in cytoplasmic protrusions (F) growing from the tracked neurospheres. Total cumulative distance or length traced for the entire culture duration was plotted. N=15 neurospheres tracked from three independent cultures. *, **, and *** represent p<0.05, p<0.01, and p<0.001 compared to +/+ CTL condition by one-way ANOVA followed by Tukey's post hoc analysis.

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