Intranasally Administered L-Myc-Immortalized Human Neural Stem Cells Migrate to Primary and Distal Sites of Damage after Cortical Impact and Enhance Spatial Learning

Abstract

As the success of stem cell-based therapies is contingent on efficient cell delivery to damaged areas, neural stem cells (NSCs) have promising therapeutic potential because they inherently migrate to sites of central nervous system (CNS) damage. To explore the possibility of NSC-based therapy after traumatic brain injury (TBI), isoflurane-anesthetized adult male rats received a controlled cortical impact (CCI) of moderate severity (2.8 mm deformation at 4 m/s) or sham injury (i.e., no cortical impact). Beginning 1-week post-injury, the rats were immunosuppressed and 1 × 10⁶ human NSCs (LM-NS008.GFP.fLuc) or vehicle (VEH) (2% human serum albumen) were administered intranasally (IN) on post-operative days 7, 9, 11, 13, 15, and 17. To evaluate the spatial distributions of the LM-NSC008 cells, half of the rats were euthanized on day 25, one day after completion of the cognitive task, and the other half were euthanized on day 46. 1 mm thick brain sections were optically cleared (CLARITY), and volumes were imaged by confocal microscopy. In addition, LM-NSC008 cell migration to the TBI site by immunohistochemistry for human-specific Nestin was observed at day 39. Acquisition of spatial learning was assessed in a well-established Morris water maze task on six successive days beginning on post-injury day 18. IN administration of LM-NSC008 cells after TBI (TBI + NSC) significantly facilitated spatial learning relative to TBI + VEH rats (p < 0.05) and had no effect on sham + NSC rats. Overall, these data indicate that IN-administered LM-NSC008 cells migrate to sites of TBI damage and that their presence correlates with cognitive improvement. Future studies will expand on these preliminary findings by evaluating other LM-NSC008 cell dosing paradigms and evaluating mechanisms by which LM-NSC008 cells contribute to cognitive recovery.

Figure 1

TBI-specific distribution of intranasally delivered LM-NSC008 cells in rats (day 25). (a) Confocal image (stitched at maximum intensity) of optically cleared coronal brain slice (1 mm thick). Rats that received either a TBI or sham injury and on days 7, 9, 11, 13, 15, and 17 post-surgery were IN-administered LM-NSC008 cells (1 × 10⁶ cells in 24 μL). (a–c) On day 25, the rats were euthanized and 1 mm brain slices were cleared using CLARITY (PACT) and imaged using confocal microscopy. Three coronal sections (S1, S2, and S3) are shown overlapping the TBI site. LM-NSC008 cells were lentivirally transduced to express eGFP.Ffluc protein for visualization (LM-NSC008 cells—green and red arrows). LM-NSC008 cells highlighted and quantified within boxes 1–7 (the pseudo-object was drawn at TBI site A∗).

Figure 2

TBI-specific distribution of intranasally delivered LM-NSC008 cells in rats (day 46). (a) Confocal image (stitched at maximum intensity) of optically cleared coronal brain slice (1 mm thick). Rats that received either a TBI or sham injury and on days 7, 9, 11, 13, 15, and 17 post-surgery were IN-administered LM-NSC008 cells (1 × 10⁶ cells in 24 μL). (a–d) On day 46, the rats were euthanized and 1 mm brain slices were cleared using CLARITY (PACT) and imaged using confocal microscopy. Coronal brain sections (S1, S2, S3, and S4) are shown overlapping the TBI site (LM-NSC008 cells—green and red arrows). LM-NSC008 cells highlighted in selected boxes 1–9 (the pseudo-object was drawn at TBI site A∗).

Figure 3

Quantification of distribution of LM-NSC008 cells at day 25 (R10) and day 46 (R30). (a, d) Distance LM-NSC008 cells migrated from the TBI site (A∗ in Figure 2 and this figure). (b, e) Distance quantified in combined sections (in mm) for LM-NSC008 cells migrated after intranasal administration for R10 (Figures 2(a)S1, 2(b)S2, and 2(c)S3) and R30 (S1, S2, S3, and S4). (c, f) Graphs represent migration dynamics of LM-NSC008 cells either from the TBI site or from the brain tissue boundary quantified at days 25 (blue curve) and 46 (orange curve). y-axis values are in millimeters (mm). Note there are an increased number of cells close to the TBI site and minimal migration at day 25 but there are fewer cells at the primary TBI site and greater migration at day 46. The data show that LM-NSC008 cells are migrating throughout the brain and into regions mediating the behaviors impacted by TBI, particularly during the later time which correlates with cognitive testing.

Figure 4

Immunohistochemistry staining using anti-human Nestin-specific antibodies to visualize LM-NSC008 cells at the TBI site on day 39 (female rats, coronal paraffin sections #64). (a) Bright field tile image of the rat brain with the CCI injury site —scale bar 1000 μm. (b-1, b-2) Parallel coronal brain sections with LM-NSC008 cells stained with DAB brown and highlighted with red arrows, 10x. (c) 20x magnification LM-NSC008 cells at the CCI injury site. Scale bars 100 μm and 50 μm, respectively.

Figure 5

Beginning 1 week after TBI or sham injury, adult male rats were IN-administered LM-NSC008 cells (1 × 10⁶ in 24 μL; TBI + NSC or sham + NSC) or VEH (2% human albumen serum; 24 μL; TBI + VEH or sham + VEH) on post-surgery days 7, 9, 11, 13, 15, and 17 and then were evaluated for the acquisition of spatial learning in a well-established Morris water maze. Mean (±S.E.M.) time(s) to locate the escape platform over days 18–23. ^∗p < 0.05 vs. TBI + VEH. ^#p < 0.05 vs. TBI + VEH and TBI + NSC. Shams did not differ from one another p > 0.05.