Influence of Post-Traumatic Stress Disorder on Neuroinflammation and Cell Proliferation in a Rat Model of Traumatic Brain Injury

Long-term consequences of traumatic brain injury (TBI) are closely associated with the development of severe psychiatric disorders, such as post-traumatic stress disorder (PTSD), yet preclinical studies on pathological changes after combined TBI with PTSD are lacking. In the present in vivo study, we assessed chronic neuroinflammation, neuronal cell loss, cell proliferation and neuronal differentiation in specific brain regions of adult Sprague-Dawley male rats following controlled cortical impact model of moderate TBI with or without exposure to PTSD. Eight weeks post-TBI, stereology-based histological analyses revealed no significant differences between sham and PTSD alone treatment across all brain regions examined, whereas significant exacerbation of OX6-positive activated microglial cells in the striatum, thalamus, and cerebral peduncle, but not cerebellum, in animals that received TBI alone and combined TBI-PTSD compared with PTSD alone and sham treatment. Additional immunohistochemical results revealed a significant loss of CA3 pyramidal neurons in the hippocampus of TBI alone and TBI-PTSD compared to PTSD alone and sham treatment. Further examination of neurogenic niches revealed a significant downregulation of Ki67-positive proliferating cells, but not DCX-positive neuronally migrating cells in the neurogenic subgranular zone and subventricular zone for both TBI alone and TBI-PTSD compared to PTSD alone and sham treatment. Comparisons of levels of neuroinflammation and neurogenesis between TBI alone and TBI+PTSD revealed that PTSD did not exacerbate the neuropathological hallmarks of TBI. These results indicate a progressive deterioration of the TBI brain, which, under the conditions of the present approach, was not intensified by PTSD, at least within our time window and within the examined areas of the brain. Although the PTSD manipulation employed here did not exacerbate the pathological effects of TBI, the observed long-term inflammation and suppressed cell proliferation may evolve into more severe neurodegenerative diseases and psychiatric disorders currently being recognized in traumatized TBI patients.     

Brain ischemia, neurogenesis, and neurotrophic receptor expression in primates

Generation of new neurons persists in the normal adult mammalian brain, with neural stem/progenitor cells residing in at least two brain regions: the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus (DG). Adult neurogenesis is well documented in the rodent, and has also been demonstrated in vivo in nonhuman primates and humans. Brain injuries such as ischemia affect neurogenesis in adult rodents as both global and focal ischemic insults enhance the proliferation of progenitor cells residing in SGZ or SVZ. We addressed the issue whether an injury triggered activation of endogenous neuronal precursors also takes place in the adult primate brain. We found that the ischemic insult increased the number of progenitor cells in monkey SGZ and SVZ, and caused gliogenesis in the ischemia-prone hippocampal CA1 sector. To better understand the mechanisms regulating precursor cell division and differentiation in the primate, we analyzed the expression at protein level of a panel of potential regulatory molecules, including neurotrophic factors and their receptors. We found that a fraction of mitotic progenitors were positive for the neurotrophin receptor TrkB, while immature neurons expressed the neurotrophin receptor TrkA. Astroglia, ependymal cells and blood vessels in SVZ were positive for distinctive sets of ligands/receptors, which we characterized. Thus, a network of neurotrophic signals operating in an autocrine or paracrine manner may regulate neurogenesis in adult primate SVZ. We also analyzed microglial and astroglial proliferation in postischemic hippocampal CA1 sector. We found that proliferating postischemic microglia in adult monkey CA1 sector express the neurotrophin receptor TrkA, while activated astrocytes were labeled for nerve growth factor (NGF), ligand for TrkA, and the tyrosine kinase TrkB, a receptor for brain derived neurotrophic factor (BDNF). These results implicate NGF and BDNF as regulators of postischemic glial proliferation in adult primate hippocampus.     

Zinc chelation reduces traumatic brain injury-induced neurogenesis in the subgranular zone of the hippocampal dentate gyrus

Numerous studies have demonstrated that traumatic brain injury (TBI) increases hippocampal neurogenesis in the rodent brain. However, the mechanisms underlying increased neurogenesis after TBI remain unknown. Continuous neurogenesis occurs in the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG) in the adult brain. The mechanism that maintains active neurogenesis in the hippocampal area is not known. A high level of vesicular zinc is localized in the presynaptic terminals of the SGZ (mossy fiber). The mossy fiber of dentate granular cells contains high levels of chelatable zinc in their terminal vesicles, which can be released into the extracellular space during neuronal activity. Previously, our lab presented findings indicating that a possible correlation may exist between synaptic zinc localization and high rates of neurogenesis in this area after hypoglycemia or epilepsy. Using a weight drop animal model to mimic human TBI, we tested our hypothesis that zinc plays a key role in modulating hippocampal neurogenesis after TBI. Thus, we injected a zinc chelator, clioquinol (CQ, 30 mg/kg), into the intraperitoneal space to reduce brain zinc availability twice per day for 1 week. Neuronal death was evaluated with Fluoro Jade-B and NeuN staining to determine whether CQ has neuroprotective effects after TBI. The number of degenerating neurons (FJB (+)) and live neurons (NeuN (+)) was similar in vehicle and in CQ-treated rats at 1 week after TBI. Neurogenesis was evaluated using BrdU, Ki67 and doublecortin (DCX) immunostaining 1 week after TBI. The number of BrdU, Ki67 and DCX positive cell was increased after TBI. However, the number of BrdU, Ki67 and DCX positive cells was significantly decreased by CQ treatment. The present study shows that zinc chelation did not prevent neurodegeneration but did reduce TBI-induced progenitor cell proliferation and neurogenesis. Therefore, this study suggests that zinc has an essential role for modulating hippocampal neurogenesis after TBI.     

The HDAC inhibitor, sodium butyrate, stimulates neurogenesis in the ischemic brain

In the healthy adult brain, neurogenesis normally occurs in the subventricular zone (SVZ) and hippocampal dentate gyrus (DG). Cerebral ischemia enhances neurogenesis in neurogenic and non-neurogenic regions of the ischemic brain of adult rodents. This study demonstrated that post-insult treatment with a histone deacetylase inhibitor, sodium butyrate (SB), stimulated the incorporation of bromo-2'-deoxyuridine (BrdU) in the SVZ, DG, striatum, and frontal cortex in the ischemic brain of rats subjected to permanent cerebral ischemia. SB treatment also increased the number of cells expressing polysialic acid–neural cell adhesion molecule, nestin, glial fibrillary acidic protein, phospho-cAMP response element-binding protein (CREB), and brain-derived neurotrophic factor (BDNF) in various brain regions after cerebral ischemia. Furthermore, extensive co-localization of BrdU and polysialic acid–neural cell adhesion molecule was observed in multiple regions after ischemia, and SB treatment up-regulated protein levels of BDNF, phospho-CREB, and glial fibrillary acidic protein. Intraventricular injection of K252a, a tyrosine kinase B receptor antagonist, markedly reduced SB-induced cell proliferation detected by BrdU and Ki67 in the ipsilateral SVZ, DG, and other brain regions, blocked SB-induced nestin expression and CREB activation, and attenuated the long-lasting behavioral benefits of SB. Together, these results suggest that histone deacetylase inhibitor-induced cell proliferation, migration and differentiation require BDNF–tyrosine kinase B signaling and may contribute to long-term beneficial effects of SB after ischemic injury.     

Increased Amyloid Precursor Protein and Tau Expression Manifests as Key Secondary Cell Death in Chronic Traumatic Brain Injury

In testing the hypothesis of Alzheimer's disease (AD)‐like pathology in late stage traumatic brain injury (TBI), we evaluated AD pathological markers in late stage TBI model. Sprague–Dawley male rats were subjected to moderate controlled cortical impact (CCI) injury, and 6 months later euthanized and brain tissues harvested. Results from H&E staining revealed significant 33% and 10% reduction in the ipsilateral and contralateral hippocampal CA3 interneurons, increased MHCII‐activated inflammatory cells in many gray matter (8–20‐fold increase) and white matter (6–30‐fold increased) regions of both the ipsilateral and contralateral hemispheres, decreased cell cycle regulating protein marker by 1.6‐ and 1‐fold in the SVZ and a 2.3‐ and 1.5‐fold reductions in the ipsilateral and contralateral dentate gyrus, diminution of immature neuronal marker by two‐ and onefold in both the ipsilateral and contralateral SVZ and dentate gyrus, and amplified amyloid precursor protein (APP) distribution volumes in white matter including corpus callosum, fornix, and internal capsule (4–38‐fold increase), as well as in the cortical gray matter, such as the striatum hilus, SVZ, and dentate gyrus (6–40‐fold increase) in TBI animals compared to controls (P's < 0.001). Surrogate AD‐like phenotypic markers revealed a significant accumulation of phosphorylated tau (AT8) and oligomeric tau (T22) within the neuronal cell bodies in ipsilateral and contralateral cortex, and dentate gyrus relative to sham control, further supporting the rampant neurodegenerative pathology in TBI secondary cell death. These findings indicate that AD‐like pathological features may prove to be valuable markers and therapeutic targets for late stage TBI. J. Cell. Physiol. 232: 665–677, 2017. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.     

Genetic ablation of caveolin-1 increases neural stem cell proliferation in the subventricular zone (SVZ) of the adult mouse brain

Adult neural stem cells are self-renewing multipotent cells that have the potential to replace dysfunctional and/or dying neuronal cells at the site of brain injury or degeneration. Caveolins are well-known tumor-suppressor genes that were recently found to be involved in the regulation of stem cell proliferation. For instance, ablation of the caveolin-1 (Cav-1) gene in mice markedly increases the proliferation of intestinal and mammary stem cells. However, the roles of caveolins in the proliferation of adult neural stem cells still remain unknown. In this study, dual-label immunofluorescence analysis of the proliferation marker, Ki67, and the stem cell markers, nestin and Sox2, was performed on brains of 8 week-old wild-type (WT) and Cav-1 knockout (KO) mice. Our results demonstrate an increased number of Ki67-positive nuclei in the subventricular zone (SVZ) of Cav-1 KO brains. Importantly, our dual-label immunofluorescence analyses demonstrate increased co-localization of Ki67 with both nestin and Sox2 in the SVZ of Cav-1 KO brains. Remarkably similar results were also obtained with Cav-2 and Cav-3 KO mouse brains as well, with increased proliferation of adult neural stem cells. Thus, the SVZ of caveolin KO mouse brains displays an increased proliferation of adult neural stem cells. Caveolin proteins might represent new crucial regulators of adult neural stem cell proliferation.     

Ischemia-induced neurogenesis is preserved but reduced in the aged rodent brain

The adult mammalian brain retains the capacity for neurogenesis, by which new neurons may be generated to replace those lost through physiological or pathological processes. However, neurogenesis diminishes with aging, and this casts doubt on its feasibility as a therapeutic target for cell replacement therapy in stroke and neurodegenerative disorders, which disproportionately affect the aged brain. In previous studies, neurogenesis was stimulated by cerebral ischemia in young rodents, and the neurogenesis response of the aged rodent brain to physiological stimuli, such as hormonal manipulation and growth factors, was preserved. To investigate the effect of aging on ischemia-induced neurogenesis, transient (60 min) middle cerebral artery occlusion was induced in young adult (3-month) and aged (24-month) rats, who were also given bromodeoxyuridine to label newborn cells. As found in prior studies, basal neurogenesis in control, nonischemic rats was reduced with aging. Ischemia failed to stimulate neurogenesis in the dentate gyrus (DG) subgranular zone (SGZ), in contrast to results obtained previously after more prolonged (90-120 min) middle cerebral artery occlusion, but increased the number of BrdU-labeled cells in the forebrain subventricular zone (SVZ). This effect was less prominent in aged than in young adult rats, with fold-stimulation of BrdU incorporation reduced by approximately 20% and the total number of cells generated diminished by approximately 50%. BrdU-labeled cells in SVZ coexpressed neuronal lineage markers, consistent with newborn neurons. We conclude that ischemia-induced neurogenesis occurs in the aged brain, and that measures designed to augment this phenomenon might have therapeutic applications.     

Intravenous grafts of amniotic fluid-derived stem cells induce endogenous cell proliferation and attenuate behavioral deficits in ischemic stroke rats

We recently reported isolation of viable rat amniotic fluid-derived stem (AFS) cells [1]. Here, we tested the therapeutic benefits of AFS cells in a rodent model of ischemic stroke. Adult male Sprague-Dawley rats received a 60-minute middle cerebral artery occlusion (MCAo). Thirty-five days later, animals exhibiting significant motor deficits received intravenous transplants of rat AFS cells or vehicle. At days 60-63 post-MCAo, significant recovery of motor and cognitive function was seen in stroke animals transplanted with AFS cells compared to vehicle-infused stroke animals. Infarct volume, as revealed by hematoxylin and eosin (H&E) staining, was significantly reduced, coupled with significant increments in the cell proliferation marker, Ki67, and the neuronal marker, MAP2, in the dentate gyrus (DG) [2] and the subventricular zone (SVZ) of AFS cell-transplanted stroke animals compared to vehicle-infused stroke animals. A significantly higher number of double-labeled Ki67/MAP2-positive cells and a similar trend towards increased Ki67/MAP2 double-labeling were observed in the DG and SVZ of AFS cell-transplanted stroke animals, respectively, compared to vehicle-infused stroke animals. This study reports the therapeutic potential of AFS cell transplantation in stroke animals, possibly via enhancement of endogenous repair mechanisms.     

Scorpion Venom Heat-Resistant Peptide (SVHRP) Enhances Neurogenesis and Neurite Outgrowth of Immature Neurons in Adult Mice by Up-Regulating Brain-Derived Neurotrophic Factor (BDNF)

Scorpion venom heat-resistant peptide (SVHRP) is a component purified from Buthus martensii Karsch scorpion venom. Although scorpions and their venom have been used in Traditional Chinese Medicine (TCM) to treat chronic neurological disorders, the underlying mechanisms of these treatments remain unknown. We applied SVHRP in vitro and in vivo to understand its effects on the neurogenesis and maturation of adult immature neurons and explore associated molecular mechanisms. SVHRP administration increased the number of 5-bromo-2’-dexoxyuridine (BrdU)-positive cells, BrdU- positive/neuron-specific nuclear protein (NeuN)-positive neurons, and polysialylated-neural cell adhesion molecule (PSA-NCAM)-positive immature neurons in the subventricular zone (SVZ) and subgranular zone (SGZ) of hippocampus. Furthermore immature neurons incubated with SVHRP-pretreated astrocyte-conditioned medium exhibited significantly increased neurite length compared with those incubated with normal astrocyte-conditioned medium. This neurotrophic effect was further confirmed in vivo by detecting an increased average single area and whole area of immature neurons in the SGZ, SVZ and olfactory bulb (OB) in the adult mouse brain. In contrast to normal astrocyte-conditioned medium, higher concentrations of brain-derived neurotrophic factor (BDNF) but not nerve growth factor (NGF) or glial cell line-derived neurotrophic factor (GDNF) was detected in the conditioned medium of SVHRP-pretreated astrocytes, and blocking BDNF using anti-BDNF antibodies eliminated these SVHRP-dependent neurotrophic effects. In SVHRP treated mouse brain, more glial fibrillary acidic protein (GFAP)-positive cells were detected. Furthermore, immunohistochemistry revealed increased numbers of GFAP/BDNF double-positive cells, which agrees with the observed changes in the culture system. This paper describes novel effects of scorpion venom-originated peptide on the stem cells and suggests the potential therapeutic values of SVHRP.     

Evidence for reduced neurogenesis in the aging human hippocampus despite stable stem cell markers

Reduced neurogenesis in the aging mammalian hippocampus has been linked to cognitive deficits and increased risk of dementia. We utilized postmortem human hippocampal tissue from 26 subjects aged 18–88 years to investigate changes in expression of six genes representing different stages of neurogenesis across the healthy adult lifespan. Progressive and significant decreases in mRNA levels of the proliferation marker Ki67 (MKI67) and the immature neuronal marker doublecortin (DCX) were found in the healthy human hippocampus over the lifespan. In contrast, expression of genes for the stem cell marker glial fibrillary acidic protein delta and the neuronal progenitor marker eomesodermin was unchanged with age. These data are consistent with a persistence of the hippocampal stem cell population with age. Age‐associated expression of the proliferation and immature neuron markers MKI67 and DCX, respectively, was unrelated, suggesting that neurogenesis‐associated processes are independently altered at these points in the development from stem cell to neuron. These data are the first to demonstrate normal age‐related decreases at specific stages of adult human hippocampal neurogenesis.     

Vascular endothelial growth factor-B (VEGFB) stimulates neurogenesis: evidence from knockout mice and growth factor administration

Vascular endothelial growth factor-B (VEGFB) is an angiogenic and neuroprotective protein that reduces hypoxic and ischemic neuronal injury. To determine if VEGFB also regulates neurogenesis in the adult brain, we studied the effects of VEGFB administration in vitro and in vivo, as well as the effect of VEGFB gene knockout (KO) in mice, on bromodeoxyuridine (BrdU) incorporation and expression of immature neuronal markers in the subgranular zone (SGZ) of the hippocampal dentate gyrus and the forebrain subventricular zone (SVZ). Intracerebroventricular VEGFB administration increased BrdU incorporation into cells of neuronal lineage both in vitro and in vivo, and VEGFB-KO mice showed impaired neurogenesis, consistent with a neurogenesis-promoting effect of VEGFB. In addition, intraventricular administration of VEGFB restored neurogenesis to wild-type levels in VEGFB-KO mice. These results suggest a role for VEGFB in the regulation of adult neurogenesis, which could have therapeutic implications for diseases associated with central neuronal loss.     

Cellular organization of adult neurogenesis in the Common Marmoset

Adult neurogenesis within the subgranular zone (SGZ) of the hippocampal dentate gyrus and the subventricular zone (SVZ) of the lateral ventricle (LV) has been most intensely studied within the brains of rodents such as mice and rats. However, little is known about the cell types and processes involved in adult neurogenesis within primates such as the common marmoset (Callithrix jacchus). Moreover, substantial differences seem to exist between the neurogenic niche of the LV between rodents and humans. Here, we set out to use immunohistochemical and autogradiographic analysis to characterize the anatomy of the neurogenic niches and the expression of cell type-specific markers in those niches in the adult common marmoset brain. Moreover, we demonstrate significant differences in the activity of neurogenesis in the adult marmoset brain compared to the adult mouse brain. Finally, we provide evidence for ongoing proliferation of neuroblasts within both the SGZ and SVZ of the adult brain and further show that the age-dependent decline of neurogenesis in the hippocampus is associated with a decrease in neuroblast cells.     

Glycogen Synthase Kinase-3β Regulates Equilibrium Between Neurogenesis and Gliogenesis in Rat Model of Parkinson’s Disease: a Crosstalk with Wnt and Notch Signaling

Neurogenesis involves generation of functional newborn neurons from neural stem cells (NSCs). Insufficient formation or accelerated degeneration of newborn neurons may contribute to the severity of motor/nonmotor symptoms of Parkinson’s disease (PD). However, the functional role of adult neurogenesis in PD is yet not explored and whether glycogen synthase kinase-3β (GSK-3β) affects multiple steps of adult neurogenesis in PD is still unknown. We investigated the possible underlying molecular mechanism of impaired adult neurogenesis associated with PD. Herein, we show that single intra-medial forebrain bundle (MFB) injection of 6-hydroxydopamine (6-OHDA) efficiently induced long-term activation of GSK-3β and reduced NSC self-renewal, proliferation, neuronal migration, and neuronal differentiation accompanied with increased astrogenesis in subventricular zone (SVZ) and hippocampal dentate gyrus (DG). Indeed, 6-OHDA also delayed maturation of neuroblasts in the DG as witnessed by their reduced dendritic length and arborization. Using a pharmacological approach to inhibit GSK-3β activation by specific inhibitor SB216763, we show that GSK-3β inhibition enhances radial glial cells, NSC proliferation, self-renewal in the SVZ, and the subgranular zone (SGZ) in the rat PD model. Pharmacological inhibition of GSK-3β activity enhances neuroblast population in SVZ and SGZ and promotes migration of neuroblasts towards the rostral migratory stream and lesioned striatum from dorsal SVZ and lateral SVZ, respectively, in PD model. GSK-3β inhibition enhances dendritic arborization and survival of granular neurons and stimulates NSC differentiation towards the neuronal phenotype in DG of PD model. The aforementioned effects of GSK-3β involve a crosstalk between Wnt/β-catenin and Notch signaling pathways that are known to regulate NSC dynamics.     

Reduced Cerebral Oxygen Content in the DG and SVZ In Situ Promotes Neurogenesis in the Adult Rat Brain In Vivo

Neurogenesis in the adult brain occurs mainly within two neurogenic structures, the dentate gyrus (DG) of the hippocampus and the sub-ventricular zone (SVZ) of the forebrain. It has been reported that mild hypoxia promoted the proliferation of Neural Stem Cells (NSCs)in vitro. Our previous study further demonstrated that an external hypoxic environment stimulated neurogenesis in the adult rat brain in vivo. However, it remains unknown how external hypoxic environments affect the oxygen content in the brain and result in neurogenesis. Here we use an optical fiber luminescent oxygen sensor to detect the oxygen content in the adult rat brain in situ under normoxia and hypoxia. We found that the distribution of oxygen in cerebral regions is spatiotemporally heterogeneous. The Po₂ values in the ventricles (45∼50 Torr) and DG (approximately 10 Torr) were much higher than those of other parts of the brain, such as the cortex and thalamus (approximately 2 Torr). Interestingly, our in vivo studies showed that an external hypoxic environment could change the intrinsic oxygen content in brain tissues, notably reducing oxygen levels in both the DG and SVZ, the major sites of adult neurogenesis. Furthermore, the hypoxic environment also increased the expression of HIF-1α and VEGF, two factors that have been reported to regulate neurogenesis, within the DG and SVZ. Thus, we have demonstrated that reducing the oxygen content of the external environment decreased Po₂ levels in the DG and SVZ. This reduced oxygen level in the DG and SVZ might be the main mechanism triggering neurogenesis in the adult brain. More importantly, we speculate that varying oxygen levels may be the physiological basis of the regionally restricted neurogenesis in the adult brain.     

Clock genes regulate neurogenic transcription factors,including NeuroD1, and the neuronal differentiation of adult neural stem/progenitor cells

The circadian clock system plays multiple roles in our bodies, and clock genes are expressed in various brain regions, including the lateral subventricular zone (SVZ) where neural stem/progenitor cells (NSPCs) persist and postnatal neurogenesis continues. However, the functions of clock genes in adult NSPCs are not well understood. Here, we first investigated the expression patterns of Clock and Bmal1 in the SVZ by immunohistochemistry and then verified how the expression levels of 17 clock and clock-related genes changed during differentiation of cultured adult NSPCs using quantitative RT-PCR. Finally, we used RNAi to observe the effects of Clock and Bmal1 on neuronal differentiation. Our results revealed that Clock and Bmal1 were expressed in the SVZ and double-stained with the neural progenitor marker Nestin and neural stem marker GFAP. In cultured adult NSPCs, the clock genes changed their expression patterns during differentiation, and interestingly, Bmal1 started endogenous oscillation. Moreover, gene silencing of Clock or Bmal1 by RNAi decreased the percentages of neuronal marker Map2-positive cells and expression levels of NeuroD1 mRNA. These findings suggest that clock genes are involved in the neuronal differentiation of adult NSPCs and may extend our understanding of various neurological/psychological disorders linked to adult neurogenesis and circadian rhythm.     

Functional evaluation of neural stem cell differentiation by single cell calcium imaging

Neurogenesis in the adult mammalian brain occurs in two specific brain areas, the subventricular zone (SVZ) bordering the lateral ventricles and the subgranular zone (SGZ) of the hippocampus. Although these regions are prone to produce new neurons, cultured cells from these neurogenic niches tend to be mixed cultures, containing both neurons and glial cells. Several reports highlight the potential of the self-healing capacity of the brain following injury. Even though much knowledge has been produced on the neurogenesis itself, brain repairing strategies are still far away from patients cure. Here we review general concepts in the neurogenesis field, also addressing the methods available to study neural stem cell differentiation. A major problem faced by research groups and companies dedicated to brain regenerative medicine resides on the lack of good methods to functionally identify neural stem cell differentiation and novel drug targets. To address this issue, we developed a unique single cell calcium imaging-based method to functionally discriminate different cell types derived from SVZ neural stem cell cultures. The unique functional profile of each SVZ cell type was correlated at the single cell level with the immunodetection of specific phenotypic markers. This platform was raised on the basis of the functional response of neurons, oligodendrocytes and immature cells to depolarising agents, to thrombin and to histamine, respectively. We also outline key studies in which our new platform was extremely relevant in the context of drug discovery and development in the area of brain regenerative medicine.