Amelioration of Penetrating Ballistic-Like Brain Injury Induced Cognitive Deficits after Neuronal Differentiation of Transplanted Human Neural Stem Cells

Abstract

Penetrating traumatic brain injury (PTBI) is one of the major cause of death and disability worldwide. Previous studies with penetrating ballistic-like brain injury (PBBI), a PTBI rat model revealed widespread perilesional neurodegeneration, similar to that seen in humans following gunshot wound to the head, which is unmitigated by any available therapies to date. Therefore, we evaluated human neural stem cell (hNSC) engraftment to putatively exploit the potential of cell therapy that has been seen in other central nervous system injury models. Toward this objective, green fluorescent protein (GFP) labeled hNSC (400,000 per animal) were transplanted in immunosuppressed Sprague-Dawley (SD), Fisher, and athymic (ATN) PBBI rats 1 week after injury. Tacrolimus (3 mg/kg 2 days prior to transplantation, then 1 mg/kg/day), methylprednisolone (10 mg/kg on the day of transplant, 1 mg/kg/week thereafter), and mycophenolate mofetil (30 mg/kg/day) for 7 days following transplantation were used to confer immunosuppression. Engraftment in SD and ATN was comparable at 8 weeks post-transplantation. Evaluation of hNSC differentiation and distribution revealed increased neuronal differentiation of transplanted cells with time. At 16 weeks post-transplantation, neither cell proliferation nor glial lineage markers were detected. Transplanted cell morphology was similar to that of neighboring host neurons, and there was relatively little migration of cells from the peritransplant site. By 16 weeks, GFP-positive processes extended both rostrocaudally and bilaterally into parenchyma, spreading along host white matter tracts, traversing the internal capsule, and extending ∼13 mm caudally from transplantation site reaching into the brainstem. In a Morris water maze test at 8 weeks post-transplantation, animals with transplants had shorter latency to platform than vehicle-treated animals. However, weak injury-induced cognitive deficits in the control group at the delayed time point confounded benefits of durable engraftment and neuronal differentiation. Therefore, these results justify further studies to progress towards clinical translation of hNSC therapy for PTBI.

Keywords: PBBI; TBI; behavior deficit; cell transplantation; hNSC; neuronal differentiation.

FIG. 1.

Representative brain sections containing green fluorescent protein (GFP) human neural stem cell grafts (hNSCs) at 5, 8, and 16 weeks post-transplantation in immunosuppressed Sprague–Dawley (SD) rats with penetrating ballistic-like brain injury (PBBI). Adjacent to PBBI lesion, persistent engraftment (GFP fluorescence) of hNSCs with little migration from transplant site is evident. Scale bar 1 mm.

FIG. 2.

Morphological changes with time in vivo of human neural stem cell (hNSC)-green fluorescent protein (GFP). At 1 week post-transplantation, cells exhibit round undifferentiated neural stem cell morphology devoid of processes (A). HuNu immunoreactivity (red) confirms human origin of cells (B). Fluorescence overlay of GFP with HuNu renders hNSC yellow, which is absent in the host (top right corner of C). By week 8, GFP cells have differentiated neural cellular morphology (D–F). The cell in the white square at a higher magnification (insets). The morphology persists at week 16 (G–I). Scale bar 10 μm.

FIG. 3.

DAPI fluorescence reflects the nuclear density at time points indicated above the image (A, E, I, M, D, Q). GFP fluorescence is also relatively similar (B, F, J, N, R) while immunoreactivity to Nestin, neural stem cell marker (red) is diminished over time (C, G, K), similarly anti-Ki67 immunoreactivity diminished with time (O, S). The bottom panel shows the combined fluorescence of GFP and Nestin (D, H, L) or GFP and Ki67 (P, T). (Scale bar 10 μm.)

FIG. 4.

Increasing neuronal differentiation of green fluorescent protein (GFP) human neural stem cell grafts (hNSCs) with time in vivo is evident from 8 week images (A–H) and corresponding 16 week images (A’–H’), in A, the image of the whole hemisphere at 8 weeks post-transplantation shows overlap of 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI), GFP, doublecortin (DCX), and NeuN fluorescence. The black square in A is shown at a higher magnification in B–H. The DAPI-stained nucleus indicated by a white arrow in B is GFP (C), strongly DCX⁺ (D) with weak NeuN immunoreactivity (E). Overlay of GFP with DCX (F), DCX with NeuN (G), and GFP with NeuN (H) confirms neuronal differentiation of transplanted hNSC. Corresponding 16 week images (A’–H’) show weak, diffuse DCX (D’) and stronger NeuN (E’) that is confirmed by fluorescence overlay (F’–H’). Scale bar 10 μm. Neuronal marker expression at 16 weeks. Transplant-derived neurons show immunoreactivity to a mature neuronal marker, Calbindin (I–K). DAPI-stained whole hemisphere image shows GFP transplant (I) that is Calbindin positive (Cal⁺) (J). Overlay renders transplant yellow (K). Scale bar 1 mm. The white square (in I–K) near the corpus callosum is shown at a higher magnification to highlight a single GFP cell bearing neuronal morphology that is Cal⁺ (I’–K’). Scale bar 10 μm. DAPI and anti-synaptophysin antibody stained whole hemisphere with 16 week transplant shows synpatophysin immunoreactivity in gray matter and transplant but not white matter (L). Quantitation of synaptophysin immunoreactivity (red puncta in L) revealed greater synaptophysin puncta in transplant (green) than in host (red) (M). The region in the white box is shown at a higher magnification as an orthogonal view with two representative synaptophysin puncta, one each for transplant (orange arrow) and host (red arrow) (N). A zoomed-in view shows the presence of GFP signal in transplant (orange arrow) but not host (red arrow) synaptophysin puncta. Quantitation of fluorescence is shown in P. Scale bar 10 μm. The scale bar for A, A’, and I–L is 1 μm.

FIG. 5.

The first, second, and third columns show GFP (A, D, G), antibody (B, E ,H) and combined fluorescence (C, F, I) signal respectively. At 16 weeks post-transplantation, transplant cells do not express astrocytic marker, GFAP as evident by absence of overlap between GFP and GFAP (red) fluorescence (C). GFP positive transplant cells do not express oligodendrocytic precursor marker, Olig2 (F) or mature oligodendrocyte marker myelin basic protein (MBP) (I). (Scale bar 10 μm.)

FIG. 6.

Distribution of green fluorescent protein (GFP) cells and processes. Examples of transplant-derived cells and processes in the host parenchyma. Pyramidal-shaped cell in the cortex (A), hippocampus cornus ammonis 1 (CA1) (B), and thalamus (C) revealed by GFP and 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI) (blue) fluorescence. Cells in the thalamus appear to extend processes along the white matter tracts of the internal capsule (IC) (dotted outline in D). A sagittal section shows the brainstem and cerebellum in E. Higher magnification of the boxes F and G show the presence of GFP fibers in the corresponding higher resolution image on the right. The tip of a GFP process with a growth cone morphology is shown in H. μm bar is 10 μm for A–C, 1000 μm for D, and 10 μm for F–H. Confocal image of penetrating ballistic-like brain injury (PBBI) brain section with GFP+ human neural stem cell grafts (NSCs) at week 8 post-transplantation shows the distribution of transplant and GFP processes (image at center). White boxes in the image are shown at a higher magnification in a counterclockwise arrangement. The GFP+ transplant-derived processes bilaterally wrap the thalamus. GFP processes from the transplant (I) cross the posterior commissure (J) contralateral thalamic surface (contralateral thalamus, K) with the lateral ventricle (LV), culminating ventrally by the internal capsule (L). GFP cells and processes can be seen in the periaqueductal gray (M) and ipsilateral internal capsule (N). μm bar is 30 μm in K–N.

FIG. 7.

(A) The mean latency ± standard deviation for Morris Water Maze (MWM) behavioral outcome 8 weeks following transplantation reveals the beneficial effect of transplantation. Results across four acquisition days are compared between sham (no penetrating ballistic-like brain injury [PBBI], no cells), vehicle (PBBI, no cells), and transplant (PBBI, cell transplant) groups. Non-uniform latency to platform can be seen in all experimental groups from day 1 to day 4 of testing. By day 4, latency to reach platform was significantly lower in the sham group (blue) than in the vehicle group (red) (p < 0.01). Latency to reach the platform was also lower in the transplant (green) group than in the vehicle group (p < 0.05). Latency was not significantly different between the transplant and sham groups on any of the test days. There were no significant differences in latency on days 1–3 between any groups. The sham group reduced latency significantly from day 1 to day 2 and from day 3 to day 4 (p < 0.01). Vehicle group did not reduce latency significantly between concurrent days, but improved from day 1 to day 3 (p < 0.05). The transplant group reduced latency significantly across each concurrent day of testing (D1->D2->D3->D4, p < 0.05 each) (A). A one way analysis of variance (ANOVA) for just the acquisition day 4 data revealed significant differences between sham and PBBI+ vehicle or PBBI+vehicle and PBBI+transplant (**p < 0.001) (B). Path lengths did not differ significantly on a two way ANOVA (C). One way ANOVA of just the acquisition day 4 path length was significant for sham (blue) versus vehicle (red) (**p < 0.001) as well as vehicle (red) versus transplant (green) (*p < 0.05) (D). The path length tracings of a representative animal of each group from four release points (North = N, South = S, East = E, West = W) are shown in E.