Traumatic brain injury (TBI) is a burgeoning public health problem worldwide.1 Penetrating TBI (pTBI), particularly involving firearms, is an increasingly serious issue in the United States within the military2 and civilian contexts. Severe pTBI occurs when an object breaches the skull and dura, with direct damage to the brain parenchyma.3 Neurosurgical interventions (decompression and hematoma removal) manage primary injuries such as hemorrhage; laceration and contusion help reduce intracranial pressure. Timely neurosurgical intervention, improved neuroimaging, and acute trauma management have lowered the firearm fatality rate4 but left survivors with lifelong debilitating impairments.5 Secondary mechanisms such as hypoxia, hypotension, ischemia, and systemic inflammatory response also increase the mortality and morbidity.6,7 The goal in the hospital is to limit the impact of secondary injury, which would otherwise magnify the initial harm. Unfortunately, many drugs showing significant promise in preclinical studies did not translate through clinical trials to become therapeutics.8,9 Primary axotomy at the time of the injury triggers self-destructive mechanisms (e.g., Wallerian degeneration) in the severed distal portion and proximal soma.10 Incomplete clearance9,11,12 of damaged axonal fragments and accumulated β-amyloid precursors11,13 for several years14 provoke chronic inflammation,15–17 which contributes to delayed remote neurodegeneration and secondary axotomy.18 Thus, host immune response, although important for repair of injury, may cause further damage to healthy tissue.19 Secondary mechanisms are not readily amenable to medical treatments.20 In most cases of pTBI, a permanent cavity gliotic porencephalic cyst is evident in survivors,21,22 which expands because of inflammation driven by progressive tissue loss.9,23 Such long-term neuronal loss is especially important in humans2,11 because of limited pool of human neural stem cells (hNSCs) and consequent low neurogenic potential.24 In contrast to rodents, relative absence of reparative neurogenesis in primates25 impedes attempts to repair injured central nervous system (CNS) circuitry by boosting endogenous hNSC proliferation.26 However, because NSCs have the capacity to replace lost brain cells,27 stimulate recovery by repairing damaged CNS,28 stabilize TBI lesion,29 and modulate neuroinflammation,30,31 cell transplantation is considered as viable option. Numerous studies including those from our group have shown benefits of hNSC transplants in rodent models of severe focal TBI.32–34 Transplantation of human fetal NSCs (hNSCs) within 24 hours of experimental TBI reduced microglial activation and alleviated cognitive deficits.30,35 Recently, our laboratory demonstrated robust and durable engraftment of fetal origin hNSC from Neuralstem Inc. (NSI-566) after pTBI in rodents with amelioration of cognitive deficit.34 The U.S. Food and Drug Administration approved these NSCs for use in phase I/II clinical trials for other CNS disorders but not yet for TBI.28,36,37 The lack of effective Food and Drug Administration–approved TBI therapies and the limited potential of endogenous repair in human TBI provide a strong rationale to test neural stem cell (NSC) transplantation in severe pTBI.
In transplantation studies, directed at both focal TBI and SCI, authors have speculated that the limiting membrane surrounding the gliotic cavity may constitute a barrier to axonal regeneration and passage of putative trophic substances.38,39 To determine the best site, we hypothesized that hNSC transplant location (perilesional vs. intralesional), with respect to pTBI lesion, will influence grafted cell survival and endogenous tissue loss. To test this, 1 week after pTBI, hNSCs were transplanted within the lesion (intralesion) or area surrounding it (perilesion). At 12 weeks posttransplantation the following were measured: (1) histological quantification of stained brain sections for tissue loss, (2) transplanted cell engraftment as a primary outcome, and (3) motor deficit development as a secondary outcome.
MATERIALS AND METHODS
Study Design and Animal Procedures
All animal procedures followed guidelines established by the National Institutes of Health Guide for the Care and Use of Laboratory Animals, Animal Research: Reporting of In Vivo Experiments, and were approved by U.S. Army Medical Research and Material Command, Animal Care and Use Review Office, and University of Miami's Institutional Animal Care and Use Committees. Male Sprague-Dawley rats (no less than 280 g) were randomized to the following four groups. (1) pTBI treated with vehicle (media, no cells), (2) uninjured rats with cell transplants (sham+hNSCs), and PTBI groups with cell transplants either (3) into tissue surrounding the lesion (peri) or (4) into lesion core (intra). Unilateral pTBI was produced in rats under anesthesia using aseptic surgical procedures (see Table 1 for animal usage). Briefly, an incision was made along the midline of the skull. The pTBI probe was aligned at 50 degrees from vertical and 25 degrees from midline at a point 2 mm lateral and 4.5 mm rostral from bregma. A burr hole was made at the location, and the pTBI probe was inserted 12 mm into the brain via the burr hole. The probe was inflated to 6.33 mm diameter for 40 milliseconds and then retracted from the brain. The scalp was closed with 12 mm wound clips and cleaned again with chlorohexidine. Buprenorphine was administered subcutaneously (0.01 mg/kg).
A priori, desired effect size was set to 0.7 based on previous publications34,40 and using G*Power3.1 with power set at 0.80, α at 0.05; the sample size after accounting for 10% attrition was adjusted to n = 10 for the histopathology; and a sample size of n = 15 was needed for the motor outcome. Sample size calculations were authenticated with My Sample Size (https://www.mysamplesize.com/). Investigators blinded to the study design and experimental groups digitized brain sections images, manually counted green fluorescent protein (GFP)–positive cell numbers, and performed quantitation in histological sections using unbiased stereology.
One week following injury, animals were anesthetized again for transplantation. Following a midline scalp incision, the right frontal pole (−3.5 mm AP [anterio-posterior], −2 mm medio-lateral [ML] to bregma) was exposed. Using a gas tight 250-μL Hamilton syringe filled with 1 million GFP expressing NSI566 stem cells in media at 50,000 cells/μL cell deposited within lesion a single bolus. For injection into penumbra, the cell-filled microsyringe was aligned to +2.72 mm AP and +1.5 mm ML (from bregma), advanced ventrally to 6 mm depth for first cell drop, and raised to 4 mm below surface for second drop; then, two drops were deposited at +3.5 mm ML at two depths. Next, drops were at −2.28 mm AP with similar ML; thus, with eight cell drops, the corners of a stereotactically defined “5 mm box” (+2.72 to −2.28 mm bregma) were covered.34 All animals were immunosuppressed with tacrolimus, mycophenylate, and depomedrol as described earlier for 12 weeks.34 Perfusion, histology, and chemical stains were done using previously published standard protocols. Approximately 11 mm blocks of cerebrum between +3.72 mm and −6.78 mm bregma were processed into 35-μm-thick serial sections, for a total 14 series. Each series has 22 sections that are 0.5 mm apart and thus span both lesion and transplant. One such series, mounted on glass slides, was stained with hematoxylin and eosin. Glass slides with brain sections were scanned to produce images. Lesion volume analysis was done on these images of using modified version of CalLesion.41 The pTBI porencephalic cyst intersects with the lateral ventricle across the rostrocaudal axes of the brain, so as to be consistent with previous pTBI studies, lesion area was defined as the area of expanded ventricle plus lesion (porencephalic cyst) minus the area of contralateral ventricle expressed as percent of left hemisphere.40
Immunohistochemistry, Imaging, and Analysis
Immunohistochemistry was performed on the brain sections using a standard protocol described previously.15,34 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 then used to estimate the total cell survival as described earlier.34 Cortical thickness measurements and three-dimensional (3D) renderings were produced using Neurolucida as described earlier.34
Footfault Test, a Motor Assessment
The animals' ability to integrate motor responses was assessed using the footfault test validated previously.42 The rats were placed onto a suspended wire mesh (40 × 150 cm; height, 50 cm; mesh size, 5 cm), and the correct and incorrect placements (footfaults) of the affected forelimb were recorded for 60 seconds per trial from a recorded video (four trials per session).
Presence of graft was used as an inclusion criterion for behavior analysis, as the hypothesis depends on successful engraftment of transplant. One animal from each transplant group had to be excluded because of poor engraftment, defined as a presence of less than 1% or less of input cells.39 End-points were compared by analysis of variance (ANOVA) followed by post hoc and paired t test analyses when appropriate (Sigma Stat, San Jose, CA). All data are presented as the mean ± SEM, and p values of <0.05 were considered significant.40,43
The experimental design and details shown in Table 1 state the sources of variation in the study. Unilateral pTBI as described elsewhere44 produces progressive tissue loss.40 Representative sections from each experimental group (Fig. 1A) show that, when compared with sham, the vehicle-treated animals had an enlarged ventricle with a large porencephalic cyst, a thinner cerebral cortex, and substantial subcortical tissue loss. In both pTBI+hNSCs groups, 90% of the intralesion and perilesion animals had an engraftment of greater than 1% of the input cells. In the sham group, only 60% of the animals had successful hNSC engraftment, and transplanted cells appeared to persist as a thin sliver of tissue without visible damage to the normal brain parenchyma or migration of cells from the transplantation site. Engraftment of hNSCs in the sham group was thus significantly less than that in the pTBI+hNSCs groups. Cortical thinning as seen in vehicle-treated rats was prominent in the intralesional group but less so in the perilesional group (Fig. 1A). Lesion size reduction in both pTBI+hNSCs groups compared with vehicle can be seen at multiple levels across the brain (Fig. 1B). A two-way ANOVA of lesion size across bregma levels showed significant interaction (F39,448 = 2.81, p < 0.0001) between location and treatment and statistically significant differences at multiple bregma levels (F13,448 = 14.19, p < 0.0001) and between vehicle and transplant groups (F3,448 = 145.3, p < 0.0001). These results indicate a strong injury effect of pTBI and robust therapeutic effect of NSC treatment, respectively. A scatter plot showed the presence of a larger lesion in the vehicle-treated group, consistent with the progressive tissue loss seen in this model (significant injury effect). Critically, there was a significant reduction in lesion volume only in the perilesion transplantation group (vehicle vs. perilesion, p = 0.0024; intralesion vs. perilesion, p = 0.0069) (Fig. 1C).
In the current study, the presence of transplanted NSCs was associated with reduced lesion size in injured animals. Based on this observation, a more detailed quantitative examination of the transplant attributes including the number of GFP fluorescent cells as a measure of total hNSC engraftment and GFP colocalized with anti-neuronal nuclei antibody (NeuN)-positive cell counts as a measure of hNSC neuronal differentiation (NeuN positivity) was performed using unbiased stereology. The extent of hNSC engraftment volume and neuronal differentiation was greater in the injured groups compared with the uninjured transplanted group (Fig. 2A–D), but there were no significant differences between the perilesional and intralesional groups with respect to GFP-positive cell engraftment or graft derived NeuN-positive neuronal numbers (Fig. 2D). Therefore, we sought to determine quantity of host tissue sparing evident from a whole brain image to test for putative neuroprotective effect (Fig. 3A). Quantitation of brain sections with Neurolucida revealed increased cortical tissue sparing (Fig. 3B). To test if apparent cortical sparing tissue included motor cortex (identified using a standard rat brain atlas) and to quantitate the effect, modified version of CalLesion (Fig. 3C) was used. A two-way ANOVA of the spared motor cortical tissue revealed that between +3.72 mm and −0.28 mm bregma (region spanning rat motor cortex) there was significant reduction in lesion size (red shape in Fig. 3B) with significant tissue sparing (Fig. 3C, cyan region), specifically, the ipsilateral motor cortex (Fig. 3C, light green). One-way ANOVA revealed statistically significant differences in spared tissue between the two transplant groups; the sparing of the motor cortex was significantly better with perilesional transplantation. Concomitant reduction in lesion area and significant increase in cortical tissue sparing (F3,32 = 15.63) (Fig. 3D) specifically in motor cortex (F3,32 = 125) (Fig. 3E) suggest that transplantation of hNSCs reduced the progression of pTBI induced cortical tissue loss, which may be associated with the functional improvement (Figs. 2 and 3E). The highest sparing of cortical tissue and motor cortex is in the perilesional group, while the least is in the intralesional with the vehicle group in between.
Using the greater than 1% presence of grafted hNSCs as inclusion criteria, behavioral assessments made at 12 weeks posttransplantation were analyzed to correlate the histological effects of injury and contribution of the hNSCs to spared motor cortical tissue to motor function. One animal from each transplant group was excluded because of poor engraftment (<1% of the input number of cells); footfault videos of three more animals were not available because of equipment failures. The analysis thus included sham+hNSCs (n = 8), PTBI+vehicle (n = 10), PTBI+hNSCs intralesion (n = 7), and PTBI+hNSCs perilesion (n = 8). At 13 weeks postinjury, mean ± SEM front footfault values for the aforementioned four groups were 7.704 ± 1.828, 25.6 ± 2.386, 16.95 ± 1.429, and 17.33 ± 1.522. Footfaults were lower in the sham group and significantly higher in the vehicle-treated group as expected (Fig. 4). The injury effect size (mean difference/pooled standard deviation (sham vs. vehicle)) at 1 week (−4.1) was stable and persisted at 13 weeks postinjury (−3.9) with a p value of <0.0001. The footfault performance of the transplant groups was between these two extremes. One-way ANOVA showed statistically significant difference between groups in mean ± SEM footfaults with F4,38 = 13.95 and p < 0.0001. The mean difference from vehicle was 8.646 (intralesion) and 8.275 (perilesion), and p value was 0.0470 (intralesion) and 0.0490 (perilesion), respectively.
In this study, we tested the hypothesis whether transplant location–dependent effect would be observed in focal brain injury. The results suggest that the location of transplanted cells, with respect to lesion, played a critical role sparing motor cortex and improving longitudinal motor performance. It has been previously reported that the pTBI lesion grows by two-fold between weeks 1 and 10 postinjury in the brain regions, 3.72 mm to −0.28 mm from bregma.40 Such preclinical data are consistent with clinical observations of progressive tissue loss following TBI in humans10,23 and, in particular, following cranial gunshot wound.21,22 In the current study, the vehicle-treated pTBI group showed tissue damage similar to what has previously been reported40 (Fig. 1A–D). The NSC-mediated reductions in lesion size were greater in animals that received perilesional than intralesional NSC transplants (Figs. 1 and 2A). Despite differences in treatment initiation and survival durations, lesion reduction was comparable with that of pharmacological interventions, human amniotic cell transplants, or amniotic cell culture supernatants.45 Unlike pharmacological agents, which required repeated administration, transplantation of NSC at a single time-point appears to confer enduring benefits, ameliorating delayed secondary brain tissue loss. The hNSC transplant 1 week after injury primarily neuroprotective, that is, stabilized the lesion across the brain, prevented expansion into regions remote from the focal injury core consistent with the role of endogenous NSCs in TBI.29 This is in contrast to pharmacological agents, which thus far failed to confer such neuroprotection.19 Cell death in this pTBI model peaks in injured cortex and striatum between 24 hours and 72 hours, respectively, with delayed cell death in locations remote from injury such as the thalamus occurring around day 7. Additional tissue destruction (both gray and central white matter of the cerebral hemispheres, with ventriculomegaly) continues even up to 10 weeks postinjury.40,44 The pTBI model in this study recapitulated focal pTBI pathology as seen in humans.21,22 In this study, transplantation of hNSCs in the perilesional zone/penumbra mitigated secondary injuries up to 3 months postinjury (the longest time point tested in this model for any intervention). Nevertheless, the benefits seen here may not primarily be because of new axons, dendrites, or synapses between transplanted cells and host because such effects would need longer survival times to be established.28 Longer duration studies, for example, in gyrencephalic brains, may be needed to clearly establish cell replacement effects. Addition of proneurogenic compounds could accelerate and direct development of such connectivity.46
Could immunosuppressants have contributed to these observed beneficial effects? The immunosuppression regimen used here has been optimized for NSI-566RSC survival in rats, primates, and humans.28 There is no information on effect of mycophenylate or tacrolimus on TBI10; however, in vitro studies suggest that they could modulate neuroinflammation.10,47 Short duration combined use of mycophenylate and tacrolimus may mitigate some TBI secondary damage.47,48 Acute treatment with tacrolimus reduced incidence of nonconvulsive seizures but not cortical atrophy.49 The robust cortical neuroprotection in the perilesional group, but not in identically immunosuppressed intralesional group (both compared with vehicle), is consistent with a lack of beneficial effect of the immunosuppressants alone. Statistically significant differences in motor cortical neuroprotection observed (Fig. 3D) also argue against beneficial effects to immunosuppressant regimen alone.
Engraftment of hNSCs was three times greater in pTBI injured than in sham rats with putatively normal brains (Fig. 3); this effect was much greater for perilesional transplants than intralesional transplanted animals. Thus, the mean survival of transplanted cells when placed in an “8-point box” configuration was near 95% of the million cells transplanted (Fig. 2C), suggesting that the post-pTBI tissue neurotrophic milieu is similar for both transplant locations with both mechanistic and therapeutic implications. Perilesional multisite transplantation had a better effect on host tissue.
Limitations of the study are as follows: (1) absence of image-guided verification of (a) injury location and (b) presence of successful transplantation; better control of both could have reduced the variations in data. (2) No insights into the mechanistic aspects of host-hNSC interactions were explored, so it is not clear if decreased proteinopathy or neuroinflammation contributes to the tissue sparing. (3) Contribution of transplant-derived human neurons, axons, and synapses to motor deficit amelioration remains to be established. Longer study duration, sufficient to demonstrate maximal neuronal differentiation and axonal/synapse formations, could help establish cause and effect relationship between transplant-derived neuronal differentiation and deficit amelioration. (4) Inadvertent reduction in animal numbers weakens confidence in the behavior amelioration.
Collectively, our data support the use of these NSI-566 human fetal neural origin cells in focal severe TBI, and the study shows that a perilesional transplantation strategy is preferable to intralesional cell deposition. Despite robust engraftment in both sites, the neuroprotection extent was better when the cells were placed around lesion. Although further mechanistic and safety studies are warranted, these studies support the rationale for a clinical trial in this indication and support a perilesional stereotactic implantation strategy in humans with TBI.
R.M.B., D.A.S., and S.G. formulated the hypothesis, designed experiments, and obtained funding and regulatory approvals for the study. M.S.S. and Z.H. performed surgeries. A.M. performed behavior analysis, optimized methods for histology quantitation, and edited the article. L.S.Q., L.D., S.W.L., J.J., and G.R.G. performed histological quantitation and generated illustrations. L.S.Q., L.D., K.N.R., and C.B.A. performed immunohistochemistry. J.J. and M.S.S. used stereology to quantitate cells and render 3D representations. S.G. contributed with microscopy. M.S., Z.H., A.M., S.W.L. R.M.B., D.A.S., and S.G. contributed to the article writing.
We thank Dr. Thomas G Hazel, and Dr. Karl K Johe, Neuralstem, Inc. for the hNSCs (GFP labeled NSI-566 RSC), technical help from Dr Hyun-Joo Park for CalLesion, Student volunteer Isa Ciocca and Ms. Jare of The University of Miami Writing Center for checking the manuscript for grammar.
This research is funded by the U.S. Department of Defense W81XWH-16-2-0008, BA150111 CDMRP JPC-6, and approved by United State Army Medical Research and Material Command, Combat Casualty Care Research Program, Fort Detrick, MD.
For all authors, no conflicts are declared. K.N.R. presented at New England Science Symposium, Harvard, in 2018 and is recipient of Asclepius Laboratories Stem Cell and Regenerative Medicine Award.
This material has been reviewed by the Walter Reed Army Institute of Research. There is no objection to its presentation and/or publication. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting views of the Department of Defense. Research was conducted in an Association for Assessment and Accreditation of Laboratory Animal Care-accredited facility, under an Institutional Animal Care & Use Committee-approved protocol, in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, NRC Publication, 2011 edition.
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