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

Study design and animals

All animal procedures followed guidelines established by the National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals, Animal Research: Reporting of In Vivo Experiments (ARRIVE), and were approved by the Walter Reed Army Institute of Research (WRAIR) and University of Miami's Institutional Animal Care and Use Committees. Animals were randomized to experimental groups. Surgical procedures were performed under aseptic conditions. To identify which rodent strain best supported xenotransplantation, Fisher 344 and Sprague–Dawley (SD) with unilateral PBBI were produced. SD and Fisher 344 animals served as test strains to establish efficacy of a chemical immunosuppression regimen. The ATN SD “nude” rats served as positive control, as they lack T-cell-mediated immunity (see Table 1 for animal use). All animals were acquired from Envigo (Indianapolis, IN) at 2–3 months of age, and were operated on at an initial weight of ≥280 g. Following establishment of the transplant paradigm, behavioral assessments were evaluated in a separate study with immunosuppressed SD rats. The sample size calculations are described in the subsequent behavior testing section. Digitizing behavioral data from recorded video tapes, green fluorescent protein (GFP) cell number quantitation in histological sections using unbiased stereology was performed by investigators blinded to the study design and experimental groups. They were also not involved in surgeries, behavior testing, or planning of the experiments.

Anesthesia

Anesthesia was induced with isoflurane (2–5%) delivered in a mixture of 70% nitrogen and 30% oxygen. Body temperature was maintained at normothermia (37 ± 1°C) throughout all surgical procedures by means of a homeothermic heating system (Harvard Apparatus, South Natick, MA).

PBBI

The PBBI apparatus consists of a penetrating probe (Kadence Science, Lake Success, NY), a stereotaxic frame (Kopf, Tujunga, CA) equipped with a probe holder, and a hydraulic pressure-pulse generator (4B080; MITRE, MA). The penetrating probe is made of a 20G stainless steel tube with fixed perforations along one end that are sealed by a piece of airtight elastic tubing. The anesthetized rat was secured in a stereotaxic frame, and an incision was made along the midline to expose the dorsal surface of the skull. A cranial window was made at +4.5 mm anterior-posterior (AP) and +2mm medial-lateral (ML) from bregma on the right frontal pole. The multi-port balloon-tipped probe was inserted through the burr hole 12 mm into the brain at an angle 25 degrees lateral from midline and 50 degrees deep from the surface. The balloon was then rapidly expanded with water to 6.33 mm diameter for 40 ms by activating the HPD-1700 generator to induce a unilateral PBBI at 10% brain volume. This level of expansion produced a severe but survivable (< 10% mortality) temporary cavity mimicking a ballistic shockwave. The probe was retracted from the rat, the wound was closed with 9 mm wound clips, and the incision site was treated with topical antibiotic. ⁷⁷

Cell transplantation

Transplants were performed 7–10 days following injury. This time point was selected based on the expected abatement of the initial injury-induced inflammatory response and the scheduled immunosuppression regimen, and to avoid freezing or prolonged storage of cells. The anesthetized rat was secured in a stereotaxic frame, and the scalp was reopened along the midline to expose the skull surface. A Hamilton 8000 Gastight 10 μL micro syringe was backfilled and flushed with suspension media and then attached to a World Precision Instruments UMP3 micro syringe injector and micro4 controller. The syringe was filled with NSI-566 cells in suspension media (concentration of 100,000 cells/μL). ⁴⁷ A burr hole was made at −2.5 mm AP and +3 mm ML from bregma, ipsilateral to the injury. The micro syringe was lined up at −2.5 mm AP and +3 mm ML from bregma and advanced vertically 6 mm deep into the brain. Using the micro pump preset, 2 μL were injected at a rate of 1 μL/min. The needle was then retracted to 4 mm depth from the brain surface, and another 2 μL were injected. The transplant locations were within the perilesional zone (PLZ) of the injury, adjacent to the primary injury cavity. This PLZ was previously determined by autoradiographically mapping regional metabolic disruption and secondary cell death in this region following injury. ⁷⁸

Immune suppression

Tacrolimus was administered intraperitoneally (i.p.) at 3 mg/kg beginning 2 days prior to transplantation and continued daily for 2 weeks, then switched to 1 mg/kg/day for the rest of the survival period. Methylprednisolone was injected i.p. weekly beginning on the day of transplant at 10 mg/kg, continuing thereafter at 1 mg/kg throughout the course of survival. Mycophenolate mofetil in 5% dextrose was injected i.p. 30 mg/kg daily, only for the 1st week following transplantation. ^71,79,80 The animals were maintained on a 12 h/12 h light/dark cycle and given food ad libitum. They were provided an enhanced recovery diet following surgical procedures until baseline weight was re-established. Immune-deficient animals were handled under a laminar airflow hood, in a dedicated closed vivarium room, with sterile gloves for all long-term care procedures.

Behavior testing with the Morris water maze (MWM) test

Sample size calculations for behavioral outcome in this model were computed a priori with software “G*Power 3.1” with type I error α set at 0.05 with power (1-type II error β) 0.8 and estimated effect size (Cohen's d) d = 0.66. ⁷⁷ Learning and memory were assessed at 8 weeks following transplant using the MWM with video tracking system. A platform was placed in a pool (75 cm deep; 175 cm diameter filled to a depth of 60 cm with water maintained at temperature of 22°C) just under the water level at a fixed location 35 cm from the wall of the pool in the northeast quadrant. For each trial, the rat was released into the pool facing the wall from north, south, east, or west starting positions. The north and east start positions were designated short arms and the south and west positions were designated long arms. Start position of each trial was semi-random, alternating short and long, with each start point used once daily. Once released, the rat was allowed to swim until it reached the platform, or until a period of 60 sec had elapsed. If the rat failed to reach the target in the given period, it was manually placed on the platform. Once on the platform, the rat was allowed 10 sec rest before being returned to the cage for a 4–5 min inter-trial rest. Four trials (one at each start position) were performed daily over the course of the first 4 days. Latency to platform presented as the mean ± standard deviation in contrast to previously used methods with the TBI, PBBI models with cell transplantation, where the deficit persisted at least 10 weeks following injury. ^47,65,77,81 The EthoVision Video Tracking Software (Noldus EthoVision XT) was used to confirm latency to reach platform (which was manually recorded with a stopwatch immediately after the trial); path length and swim speed were traced from video using EthoVision. Animals with evidence of far greater than average transplant rejection (criteria such as surviving transplant cell counts at 8 weeks below one third of average cell survival, and the presence of immune cells) were excluded from behavior analyses.

Specimen collection and histology

Rats were perfused transcardially with 0.1 M phosphate-buffered saline (PBS), followed by cold 4% paraformaldehyde (PFA) in 0.1M phosphate buffer. Brains were dissected out and post-fixed in 4% PFA for 12 h, then transferred to a 30% sucrose solution for 24 h. Brains were frozen in M-1 embedding matrix using dry ice, and stored at −20°C before being sectioned on a cryostat at 40 μm thickness. Sections were stored free floating in 0.02% sodium azide in PBS prior to immunohistochemistry. Samples were assessed with the following primary antibodies: anti-GFP (Millipore MAB1083), NeuN (Millipore MAB377), doublecortin (DCX) (Millipore AB2253), glial fibrillary acidic protein (GFAP) (Dako Z0334), Olig2 (Millipore AB9610), HuNu (Millipore MAB1281), Ki67 (Millipore AB9260), nestin (Millipore ABD60), MBP (Covance, SMI 94), and Calbindin (Cell Signaling #2136X). Appropriate fluorescent secondary antibodies were used (Life Technologies) with 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI) prior to mounting and imaging.

Imaging and analysis

Volumetric GFP cell counts were generated using the physical fractionator method in StereoInvestigator (version 10.6 Stereo Investigator, MBF Bioscience, Williston, VT) on evenly spaced (0.2 mm apart) brain sections, and used to calculate estimated total cell survival. The percentage of NeuN and GFP double-positive cells in grafts was determined by analysis of raw image files with Imaris 6.0.0.1 software. High-resolution fluorescent images were generated on a confocal microscope (n = 3–5 sections/brain; 3 brains/group). Using Imaris analysis software (version 8.0.1), and based on NeuN-positive host hippocampal neurons, the fluorescence intensity and object diameter were estimated. This served as the threshold for NeuN signal; similarly, a GFP limit was set using the transplanted cells. Points of NeuN-positive signal were automatically counted by approximate diameter and intensity based on host neurons (using the Spots tool). Similarly, GFP-positive points were counted based on diameter and relative background subtraction. The co-occurrence of both signals as calculated by the Imaris software was used to estimate NeuN positivity of grafts at 8 and 16 week time points. The synaptophysin signal was similarly analyzed in Imaris by comparing the occurrence of synaptophysin-positive spots at a set signal threshold in sample sites from transplant location versus contralateral regions of host brain expressing no GFP signal. The incidence of synaptophysin signal was compared overall between GFP-negative and GFP-positive tissue.

Statistical analysis

Histopathological and behavioral end-points were compared by independent samples t tests or analysis of variance (ANOVA) followed by Fisher protected least significant difference (PLSD) post-hoc and paired t test analyses when appropriate (SigmaStat). All data are presented as the mean ± standard deviation, and p values <0.05 were considered significant. ^77,82 For all experiments, the trained investigators performing the behavioral and neuropathological assessments were blinded to the experimental groups. MWM results were compared using two way repeated measures ANOVA followed by Tukey's test. Data were graphed using GraphPad version 6.0.

Survival

Following PBBI expansion, ∼6mm lesion is produced, spanning the dorsal frontal cortex (+3.0 mm Bregma), anterior striatum, anterior-lateral edge of the caudate nucleus, corpus callosum, and anterior caudate-putamen, and terminating near the amygdala (−3.0 mm Bregma). The injury core and PLZ release a host of inflammatory cytokines including IL-1β, and thus contribute to secondary injury propagation. ^28,83 It was not known to what extent the toxic PBBI milieu would impede survival of a human cell transplant. Therefore, the human NSI-566 NSCs were transplanted into three different rat strains subjected to PBBI: 1) outbred SD standard laboratory rats, 2) relatively less immunocompetent Fischer 344 rats, and 3) immunocompromised SD ATN rats. The SD and Fischer animals were treated with an immunosuppression regimen as per the protocol previously described. ^71,79,80 ATN rats were treated with only methylprednisolone weekly, to reduce injury-induced inflammation. Rats were euthanized at specific time points to track graft survival (1, 5, 8, and 16 weeks following transplantation). Robust engraftment could be seen as far as 16 weeks post-transplantation, the last time pointed tested. In the first set of experiments, at 8 weeks, grafts were present in 15/16 SDs, 9/10 ATNs, and 4/6 Fischer 344 (28/32 total). The number of GFP-positive cells was quantitated using unbiased stereological counting, and supplemented with manual counts of confocal images when necessary. No significant difference between ATN and immunosuppressed SD or Fischer rats with PBBI was found in the number of surviving graft cells, which averaged 150% of the original transplanted cell numbers (SD = 6.5 × 10⁵, ATN = 6.2 × 10⁵, Fischer = 5.7 × 10⁵ cells). In next set of experiments for behavioral analysis, following stable immunosuppression of SD rats 3.7 × 10⁵ ± 1.1 × 10⁵ cells could be counted at 8 weeks post- transplantation. This suggests that the immunosuppression regimen is effective in supporting xenogeneic human cell graft survival for the majority of the animals (9/12), in the rodent PBBI model. The transplant size did not vary between 5 and 16 weeks post-transplantation (Fig. 1).

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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.

Differentiation

The following results were produced in immunosuppressed SD rats. The human origin of the GFP-positive xenograft was confirmed with HuNu, a marker for human cell nuclei (Fig. 2B, E, and H). At 1 week post-transplant, the cells appeared rounded, undifferentiated, and <5 μm in diameter (Fig. 2A–C). By 8 weeks, the cells had hypertrophied to >10 μm across with extended neuronal-like processes (Fig. 2 D–I). Processes extending from the central soma spread out to into the host tissue, lending support to the development of multipolar neurons. The process of transplanted NSC differentiation was evident based on the decreasing nestin-immunoreactivity over time. Nestin expression was high in transplanted cells at 2 weeks, but reduced by 8 weeks, and was almost absent by 16 weeks (Fig. 3A–L). A similar pattern was observed with nuclear Ki67, where expression at 2 weeks had diminished by 16 weeks (Fig. 3M–T). In brain sections (∼−4.0 mm bregma, 1 mm caudal to lesion) from a representative immunosuppressed SD rat shown in Figure 4 A, The DAPI-stained nucleus (Fig. 4B) indicated by a white arrow belongs to transplanted GFP-positive cells (Fig. 4C) that expressed the immature neuronal lineage marker DCX at 8 weeks (Fig. 4D). A 27 ± 12% fraction of GFP+ cells also expressed NeuN, a marker for mature neurons (1729 NeuN⁺ out of 6489 GFP⁺ cells) (Fig. 4E). The combined fluorescence of GFP and DCX (Fig. 4F) dilutes the GFP intensity (compare C with F). Overlay of DCX and NeuN (Fig. 4G) shows different localization of the two antigens. The cell appears yellow (Fig. 4H) because of the combined fluorescence of GFP and NeuN. Corresponding images for the 16 week post-transplantation time point show diffuse DCX expression because of the greater fragmentation into number of processes (Fig. 4D’). At this time, the fraction of NeuN-immunoreactivity of GFP+ cells increased to 43 ± 17% (2791 NeuN⁺ out of 6357 GFP⁺ cells), indicating prolonged neuronal differentiation (Fig. 4H’). Overlays confirm the co-expression of DCX with GFP (Fig. 4F’) and NeuN with GFP (Fig. 4H’), and lowered DCX in NeuN cells (Fig. 4G’) (compare G with G’). Further, by week 16 post- transplantation, Calbindin immunoreactivity could be observed in tranplant-derived cells with neuronal morphology (Fig. 4I–K and 4I’–K’). Robust expression of presynaptic protein synaptophysin could be seen in the transplant compared with the host (Fig. 4L), whereas there was no difference in the expression of the postsynaptic protein gephyrin (data not shown). Quantitative analysis of synaptophysin expression in GFP+ tissue and in contralateral GFP- host tissue indicated a significant increase in expression at the site of transplantation. (GFP+ synaptophysin = 350.6 ± 20.7 puncta/mm² n = 8, GFP- synaptophysin = 251.4 ± 21.9 puncta/mm² n = 9; p = 0.005) (Fig. 4M). Further orgthogonal analysis confirmed transplant- derived synpatophsin via demonstration of fluorescence overlap (Fig. 4N–P).

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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.

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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.)

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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.

The transplant cells did not express GFAP, a marker for astrocytes (Fig. 5A–C), or Olig2, a marker for oligodendrocytes as the human ubiquitin C promoter-driven GFP signal did not overlap with that from antibodies (Fig. 5D–F), as determined by evaluation of confocal imaging of transplant sites. Further, the GFP processes extending from the transplanted cells did not overlap with myelin basic protein from the host, indicating lack of transplant-derived myelination even at 16 weeks (Fig. 5G–I).

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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.)

Distribution

The GFP cell bodies could be seen up to 1 mm from the initial transplant sites at 8 weeks and even at 16 weeks, indicating little migration of the cells from injection sites. Cells extended processes up to ∼0.5–1 mm from the site of transplantation, by 8 weeks, and those on the edges of the site extended these processes farther into host tissue The transplant-derived processes, however, extended 5 mm rostral and 13 mm caudal to transplant site. Transplanted cells could be seen all along the injection tracts in the thalamus, cortex, and hippocampus and bordering the lateral ventricle. Examples of transplant cell morphology were consistent with pyramidal cells in the cortex (Fig. 6A), hippocampus (Fig. 6B), and both bipolar and multipolar interneuron-like cells in the thalamus (Fig. 6C). This suggests that transplant cells responded to regional cues to determine differentiation patterns. Distally, processes were seen in the ventral brainstem into the spinal cord, extending over 13 mm from the primary transplant site (Fig. 6D–H). These GFP processes extended around the ipsilateral thalamus into the internal capsule (Fig. 6 D, L, and N), along host white matter tracts, crossing the midline (Fig. 6J) and periaqueductal gray (Fig. 6M), and emerged along the contralateral white matter tracts (Fig. 6L and N). Shorter processes were observed throughout the cortex and parts of the hippocampus from the transplant tract.

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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.

Behavior

Significant impairment of spatial memory in SD rats with PBBI has been reported previously. ⁷⁷ The effect of transplants on the PBBI-induced deficit was assessed using the MWM at 8 weeks post-transplantation. This time point was deemed the minimum duration needed for differentiation and integration of human neural stem cells. ^2,79 For latency to platform, a two way repeated measures ANOVA (one factor repetition) revealed a statistically significant interaction between groups and acquisition days (F _2,84 = 2.689, p = 0.019). Experimental groups were sham (no PBBI, no cells, n = 12), vehicle (PBBI, no cells, n = 11), and transplant (PBBI, cell transplant, n = 9). The main effect was with acquisition days (F _2,84 = 34.505, p ≤ 0.001) but not experimental groups (F _2,84 = 1.861, p = 0.174). Next all pairwise multiple comparison procedures (Tukey test) showed that latency to platform was reduced significantly in all experimental groups from day 1 to day 4 of testing (sham p < 0.001, vehicle p = 0.011, transplant p < 0.001). This decrease in latency to platform was not uniform in all groups (sham = 63%, vehicle = 33%, and transplant = 72%). It plateaued from acquisition day 2 to day 4 in the vehicle group (PBBI+vehicle) while continuing to decline in both the sham and transplant groups (sham p = 0.011, vehicle p = 0.259, transplant p < 0.001). Latency was not significantly different between sham and treatment groups (p = 0.676) (Fig. 7A). However, a one way ANOVA conducted on the day 4 data revealed significant between-group differences (F _2,30 = 5.952 and p = 0.0067), with post-hoc analysis showing significant improvement on latency to the hidden platform versus PBBI+vehicle (Fig. 7B). The effect size (Hedge's g) for latency to platform at 8 weeks post-transplantation was −1.33 for PBBI+vehicle versus sham, whereas that for PBBI+vehicle vesus PBBI+transplant was −1.0.

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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.

For path length, with experimental groups sham (no PBBI, no cells, n = 12), vehicle (PBBI, no cells, n = 10), and transplant (PBBI, cell transplant, n = 9), two way repeated measures ANOVA (one factor repetition) did not reveal a statistically significant interaction between groups and acquisition days (F _2,81 = 2.022, p = 0.072). Albeit, the main effect was with acquisition days (F _2,81 = 20.319, p ≤ 0.001) but not experimental groups (F _2,81 = 1.736, p = 0.195) (Fig. 7C). One way ANOVA of the day 4 path length was also significant (F _{2, 28} = 6.791 and p = 0.0039) (Fig. 7D). The path length tracings of a representative animal of each group from four release points are shown in Figure 7E. The differences in swim speeds were not statistically significant (data not shown).