Dose-dependent modulation of microglia activation in rats after penetrating traumatic brain injury (pTBI) by transplanted human neural stem cells.

Traumatic brain injury (TBI) often results in long-lasting patterns of neurological deficits including motor, sensory, and cognitive abnormalities. Cranial gunshot survivors are among the most disabled TBI patients and face a lifetime of disability with no approved strategies to protect or repair the brain after injury. Recent studies using a model of penetrating TBI (pTBI) have reported that human neural stem cells (hNSCs) transplantation can lead to dose and location-dependent neuroprotection. Evidence for regional patterns of microglial activation has also been reported after pTBI with evidence for microglial cell death by pyroptosis. Because of the importance of injury-induced microglial activation in the pathogenesis of TBI, we tested the hypothesis that dose-dependent hNSC mediated neuroprotection after pTBI was associated with reduced microglial activation in pericontusional cortical areas. To test this hypothesis, quantitative microglial/macrophage Iba1 immunohistochemistry and Sholl analysis was conducted to investigate the arborization patterns using four experimental groups including, (i) Sham operated (no injury) + low dose (0.16 million cells/rat), (ii) pTBI + vehicle (no cells), (iii) pTBI + low dose hNSCs (0.16 million/rat), and (iv) pTBI + high dose hNSCs (1.6 million cells/rat). At 3 months post-transplantation (transplants at one week after pTBI), the total number of intersections was significantly reduced in vehicle treated pTBI animals versus sham operated controls indicating increased microglia/macrophage activation. In contrast, hNSC transplantation led to a dose-dependent increase in the number of intersections compared to pTBI vehicle indicating less microglia/macrophage activation. The peak of Sholl intersections at 1 µm from the center of the microglia/macrophages ranged from ~6,500-14,000 intersections for sham operated, ~250-500 intersections for pTBI vehicle, ~550-1,000 intersections for pTBI low dose, and ~2,500-7,500 intersections for pTBI high dose. Plotting data along the rostrocaudal axis also showed that pericontusional cortical areas protected by hNSC transplantation had increased intersections compared to nontreated pTBI animals. These studies using a non-biased Sholl analysis demonstrated a dose-dependent reduction in inflammatory cell activation that may be associated with a neuroprotective effect driven by the cellular transplant in perilesional regions after pTBI.

Injury-Transplantation Interval-Dependent Amelioration of Axonal Degeneration and Motor Deficit in Rats with Penetrating Traumatic Brain Injury.

Penetrating traumatic brain injury (pTBI) is increasingly survivable, but permanently disabling as adult mammalian nervous system does not regenerate. Recently, our group demonstrated transplant location-dependent neuroprotection and safety of clinical trial-grade human neural stem cell (hNSC) transplantation in a rodent model of acute pTBI. To evaluate whether longer injury-transplantation intervals marked by chronic inflammation impede engraftment, 60 male Sprague-Dawley rats were randomized to three sets. Each set was divided equally into two groups: 1) with no injury (sham) or 2) pTBI. After either 1 week (groups 1 and 2), 2 weeks (groups 3 and 4), or 4 weeks after injury (groups 5 and 6), each animal received 0.5 million hNSCs perilesionally. A seventh group of pTBI animals treated with vehicle served as the negative control. All animals were allowed to survive 12 weeks with standard chemical immunosuppression. Motor capacity was assessed pre-transplant to establish injury-induced deficit and followed by testing at 8 and 12 weeks after transplantation. Animals were euthanized, perfused, and examined for lesion size, axonal degeneration, and engraftment. Compared to vehicle, transplanted groups showed a trend for reduced lesion size and axonal injury across intervals. Remote secondary axonal injury was significantly reduced in groups 2 and 4, but not in group 6. The majority of animals showed robust engraftment independent of the injury-transplant time interval. Modest amelioration of motor deficit paralleled the axonal injury trend. In aggregate, pTBI-induced remote secondary axonal injury was resolved by early, but not delayed, hNSC transplantation.

Assessing fetal human neural stem cells tumorigenicity potential in athymic rats with penetrating traumatic brain injury (pTBI).

Traumatic brain injuries (TBI) often produce disability in survivors due to unresolved inflammation and progressive neurodegeneration. The central nervous system in mammals is incapable of self-repair. Two decades of preclinical studies and clinical trials have provided insights into TBI pathophysiology that could be utilized to develop clinically relevant therapy. Our laboratory recently reported efficacy of clinical trial grade fetal human neural stem cells (hNSCs) in immunosuppressed rats with penetrating traumatic brain injury (pTBI). Next, in compliance with the United States Food and Drug Administration (USFDA) guidance, this study explores safety by assessing the tumorigenicity potential of intracranial hNSC transplants in athymic rats with pTBI. First, the maximum tolerated dose (MTD) was determined. Then, forty athymic pTBI rats were randomized to either: Group A. pTBI + vehicle or Group B. pTBI + hNSCs at MTD one week after injury with 6-months survival, sufficient time to uncover transplant associated tumorigenicity. A board-certified Pathologist examined hematoxylin-eosin (H&E), Ki67 immunostained brain and spinal cord, serial sections along with several abnormal peripheral masses for evidence of lesion, transplant, and oncogenesis. There was no evidence of transplant derived tumors or oncogenic tissue necrosis. Consistent with athymic literature, the lesion remained unchanged even after robust hNSC engraftment. This safety study supports the conclusion that hNSCs are safe for transplantation in pTBI. The differences in lesion expansion between immunosuppressed and athymic rats in the presence of hNSCs suggests an unexpected role for thymus derived cells in resolution of trauma induced inflammation.