Introduction

T raumatic brain injury (TBI) is a serious public health problem that causes disability and significant health care expenditures for those affected. While the clinical management of traumatic brain injury has greatly improved with the development of standardized approaches to care, there are currently no medical treatment adjuncts that have been shown effective in improving mortality or limiting disability following injury, which emphasizes the need for new therapeutic developments.

Recent evidence showed that neurogenesis continues in the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG) in adult mouse, rat, non-human primate, and human brain. 1– 5 newly generated cells can differentiate into functional mature neurons and integrate into neuronal networks, 6, 7 including those involved in cognitive function. 8– 10 we and other investigators have shown that ischemic brain injury stimulates the proliferation of neural stem/progenitor cells (nscs) located in the SVZ and SGZ of adult rodent brain.Hipoxia anoxia

the resulting newborn cells migrate into damaged brain regions, 11– 13 where they differentiate into mature neuronal cells. 11– 13 studies have reported that brain trauma also induces cell proliferation at the ipsilateral hippocampus and SVZ, 14– 17 which may persist for at least one year. 15 an animal study shows that a significant increase in newly generated cells was observed not only in the hippocampus, but also in the traumatized cerebral cortex, white matter structures, and some contralateral regions. 18 newborn doublecortin (DCX)-positive neuroblasts in the SVZ emigrate toward cerebral cortex lesions. 19 double-labeled studies have documented that many of these proliferating cells appear to differentiate into various adult cells, including astrocytes, oligodendrocytes, and neurons, 17, 20– 22 and that newly generated granule neurons are capable of extending projections along the hippocampal mossy fiber pathway in the acute post-traumatic period, 23 suggesting that these newborn cells can differentiate into neural lineages.Hipoxia anoxia taken together, these findings suggest that TBI induces neurogenesis in rodents. Although it remains unclear what relevance injury-induced neurogenesis may have in the recovery process following TBI, the demonstration of neurogenesis in damaged regions in adult brains and of the presence of proliferating cells with the ability to give rise to neurons in the damaged regions of brains after TBI have reinvigorated our hopes of rebuilding damaged tissues by endogenous neural cell replacement.

While much work has been conducted regarding the neurogenesis response to TBI in rodents, it remains largely unknown whether neurogenesis in adult human brain also responds to TBI in a similar manner. In the present study, we found that the expression of NSC protein markers, including DCX, TUC4, PSA-NCAM, SOX2, and neurod, was increased in the perilesional cortex of human brain after TBI compared to that of normal brain.Hipoxia anoxia confocal images showed that these NSC proteins were expressed in one single cell. In addition, we found that proliferative markers were expressed in cells located in the perilesional regions after TBI. Double or triple immunocytochemistry showed that NSC-positive cells expressed proliferative protein and that these cells were significantly increased after TBI, suggesting that TBI may also induce neurogenesis in human brain as it does in the animal TBI model.

Immunohistochemistry

Brain tissue was post-fixed in paraformaldehyde for 24 h, incubated with 30% sucrose for 3 days, and embedded in paraffin; 6-μm sections were cut on a microtome and stored at room temperature. Sections were deparaffinized with xylene and rehydrated with ethanol.Hipoxia anoxia to obtain more efficient immunostaining, brain sections were subjected to an antigen retrieval procedure according to the manufacturer’s instructions (vector laboratories, burlingame, CA). After heating under pressure for 2 min, samples were washed extensively in PBS. Endogenous peroxidase activity was blocked by 30 min incubation at room temperature in 1% H 2O 2. After several washes with PBS, sections were incubated in blocking solution (2% goat serum, 0.1% triton X-100, 1% bovine serum albumin in PBS) for 1 h at room temperature. Primary antibodies used were mouse monoclonal anti-ki67 antigen (1:50; novocastra, newcastle upon tyne, united kingdom), rabbit anti-ki67 antigen (1:100; zymed, south san francisco, CA), goat anti-minichromosome maintenance 2 (MCM2, 1:100; santa cruz biotechnology, santa cruz, CA), rabbit anti-cleaved caspase 3 (1:200; BD pharmingen, san diego, CA), affinity-purified goat anti-DCX (1:200; santa cruz biotechnology), mouse monoclonal anti-βiii-tubulin (TUJ1, 1:250; covance, berkeley, CA), rabbit polyclonal anti-TUC-4 (1:10,000; chemicon international, temecula, CA), mouse monoclonal anti-polysialylated neuronal cell adhesion molecule (PSA-NCAM, 1:500; chemicon), affinity-purified goat polyclonal anti-neuro D (1:100; santa cruz biotechnology), and rabbit anti-SOX2 (1:200; abcam, cambridge, MA).Hipoxia anoxia the markers for neural precursor cells have been evidenced to cross-react with proteins of the appropriate molecular weight in human brain tissues. 24 primary antibodies were added in blocking solution and incubated with sections at 4°C overnight. Sections were then washed with 1x PBS and incubated with biotinylated goat anti-rabbit or anti-goat antibody (1:200) (for polyclonal antibodies) or biotinylated horse anti-mouse antibody (1:200, for monoclonal antibodies) for 1 h at room temperature. Avidin–biotin complex (vector elite, vector laboratories) and a diaminobenzidine or nickel solution (vector laboratories) were used to obtain a visible reaction product. Controls for immunohistochemistry included pre-absorption and co-incubation of the antibodies with the corresponding antigens.Hipoxia anoxia sections were dehydrated, sealed, and coverslipped. A nikon microscope and a nikon digital color camera were used for examination and photography of the slides, respectively.

Double or triple immunostaining

Double or triple immunostaining was performed on brain sections as previously described. 12 the primary antibodies used, in addition to those listed above, were rabbit anti-musashi1 (1:500; chemicon) and mouse monoclonal anti-neun (1:200; chemicon). The secondary antibodies were alexa fluor 488-, 594-, or 647-conjugated donkey anti-mouse, anti-goat, or anti-rabbit igg (1:200–500; molecular probes, carlsbad, CA). Nuclei were counterstained with DAPI using prolong gold antifade reagent (molecular probes).Hipoxia anoxia fluorescence signals were detected using an LSM 510 NLO confocal scanning system mounted on an axiovert 200 inverted microscope (carl zeiss, jena, germany) equipped with a two-photon chameleon laser (coherent), and images were acquired using LSM 510 imaging software (carl zeiss). Two-, three-, or four-color images were scanned using argon, 543 hene, 633 hene, and chameleon (750–780 nm for DAPI) lasers. Selected images were viewed at high magnification, and 3-dimensional images were constructed using imars software. Controls included omitting either the primary or secondary antibody or pre-absorbing the primary antibody.

Results

To determine whether nscs in the adult human brain respond to TBI, we first performed immunocytochemistry for NSC protein markers in sections of cortical regions from 11 patients with TBI who underwent surgical intervention ( table 1).Hipoxia anoxia we found that DCX, a microtubule-associated protein expressed almost exclusively in immature neurons 25 and currently a “gold standard” in measuring neurogenesis, 26 was expressed in cells in the perilesional cortex of adult human brain after TBI. Some of the DCX-positive cells displayed morphological characteristics of migrating cells with a leading process and tail edge ( fig. 1A). TUC-4 (also known as TOAD-64 and CRMP-4), a protein expressed early in neuronal differentiation, 27 and PSA-NCAM, a marker for immature neuronal-committed progenitors that are at more mature stages than DCX, 28 were highly expressed in the cerebral cortex surrounding the damaged regions ( fig. 1B). In addition, we found that SOX2, a transcription factor expressed by self-renewing and multipotent stem cells of the embryonic neuroepithelium and considered a persistent marker for multipotential neural stem cells, 29 as well as neurod, a basic helix loop helix transcription factor expressed in immature, differentiating neuronal progenitor cells, 30, 31 were found in the nuclei of cells in the cortical regions after TBI ( fig. 1C), suggesting that they might be neural progenitor cells.Hipoxia anoxia since the tissue samples were from different cortex regions, we next asked whether there were regional differences in injury-induced neurogenesis. Of the 11 TBI patient samples, we found that the number of DCX-positive cells was between 3.66±0.577 (mean±SD) and 8.33±1.5; TUC4-positive cells were between 5.33±1.52 and 7.66±1.53; SOX2-positive cells were between 1.66±0.55 and 3.66±0.56; and PSA-NCA-positive cells were between 2.33±0.55 and 4.33±0.58. Interestingly, we also found that the DCX-, TUC-4-, PSA-NCA-, and SOX2-positive cells were present in the normal cortex ( fig. 2A–C); however, the number of these cells was significantly increased in the traumatic brain ( p0.05; fig. 2D).

Discussion

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The main finding of this study is that the expression of NSC proteins and cell proliferative markers is increased in peri-damaged brain regions after TBI in humans compared to that of normal brain. Two-photon laser scanning confocal images show that expression of NSC and proliferative proteins are observed together in the same cell. Quantification analysis indicates that the number of proliferative nscs is also significantly increased after TBI. Our data suggest that neurogenesis may be induced in human brain after TBI, and that the mature brain retains a degree of innate repair and regenerative potential to restore damaged neuronal populations through endogenous neurogenesis.

Many studies demonstrate that various brain injuries induce neurogenesis in a number of neurological disorders in humans, including huntington’s disease, 36 ischemic stroke, 37 alzheimer’s disease, 38 epilepsy, 39 and aneurysmal subarachnoid hemorrhage. 40 here, we further demonstrate that TBI may also induce neurogenesis in humans.Hipoxia anoxia to determine whether these newborn cells can replace damaged cells, the first question is whether these newly generated neurons are functional. Previous studies show that cognitive deficits are observed in animals and in humans after TBI. 41, 42 interestingly, cognitive deficits can be significantly self-recovery following all but the most severe episodes of TBI, and the cognitive functions of juveniles recover to a greater extent than adult patients following TBI. 41– 43 the mechanisms underlying this phenomenon remain unclear, although recent studies suggest that the cognitive recovery that occurs following TBI is associated with the integration of newly generated neurons. 42 the role of newborn cells in learning and memory has been tested using a range of hippocampus-dependent behaviors with mixed results.Hipoxia anoxia for example, studies show that trace eyeblink conditioning was affected in anti-proliferative drug-treated animals, and contextual fear conditioning was impaired following irradiation and genetic ablation of adult neurogenesis. In an effort to more precisely ablate specific populations of neural precursor cells, genetically engineered mice have become available that can regulate neurogenesis in a more direct and temporally controlled manner. We produced transgenic mice that express herpes simplex virus thymidine kinase (TK) under control of the DCX promoter. Treatment with the antiviral drug ganciclovir (GCV) depleted DCX-expressing and brdu-labeled cells from the SVZ and dentate gyrus and abolished neurogenesis and associated neuromigration induced by focal cerebral ischemia.Hipoxia anoxia GCV treatment of DCX-TK transgenic, but not WT mice, also increased infarct size and exacerbated post-ischemic sensorimotor behavioral deficits. These findings suggest that injury-induced neurogenesis contributes to brain injury outcome. 44

We found that neurogenesis mainly occurs in the peri-damaged brain regions after TBI in humans. However, it remains unclear whether these cells are born locally or from neurogenic regions. Normally, nscs reside in the postnatal SVZ and the SGZ of the dentate gyrus. The newborn cells migrate into the olfactory bulb via the rostral migratory stream, while the SGZ of the dentate gyrus provides new granular neurons throughout life. Following TBI, nscs in each of these areas become activated.Hipoxia anoxia previous studies have shown that increased SVZ neurogenesis with subsequent migration of newborn neurons into the damaged region has been described in a rat model of intracerebral hemorrhage and focal ischemia model. 2, 12, 45 therefore, it is possible that newborn cells in the SVZ can migrate into the boundary zone of the injured cortex after severe TBI. 46 however, little is known about cell migration after TBI. Recent studies show that stromal cell-derived factor-1α (SDF-1α) and its receptor, CXCR4, contribute to NSC migration after brain injury. 47 although SDF-1α expression did not increase after TBI, SDF-1α leaked from the injured area and diffused into the cortex after TBI. 48 little is known about which factors determine the magnitude and amplification of neurogenesis after TBI, but recent studies show that nerve growth factor and DCX expression correlates with improved outcome in children with severe TBI. 49

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Traumatic brain injury stimulates an increase in the proliferation of endogenous nscs, particularly in the neocortex where it does not normally occur, suggesting that the adult brain has the inherent potential to restore populations lost to injury and that it may become possible to manipulate endogenous multipotent precursors in situ to replace lost or damaged neurons. Our data show that NSC-protein markers are found in the cells located in normal cortex, but these cells barely express proliferative markers such as ki67, suggesting that, even if there are some nscs in the normal cortex in humans, the majority are in quiescent. Several studies have shown that a variety of pharmacologic agents associated with increasing neurogenesis lead to improved outcomes after TBI.Hipoxia anoxia these include growth factors, which are important mediators for neurogenesis. Specifically, basic fibroblast growth factor (FGF-2) and epidermal growth factor (EGF) have been shown to be a potent mitogenic factor for nscs both in vitro and in vivo. FGF-2 knockout mice exhibited diminished hippocampal DG neurogenesis in response to seizure or trauma, which can be reversed by administrating FGF-2. 5 TBI-induced proliferative response in the SVZ and hippocampus is linked to increased EGF expression. Compared to vehicle-infused animals, FGF-2- or EGF-infused animals had significantly more brdu-positive cells in the SVZ and hippocampus. In addition, animals treated with EGF or FGF-2 showed significant improvement in cognitive function. 50, 51 vascular endothelial growth factor (VEGF) is an angiogenic protein with neurotrophic and neuroprotective effects.Hipoxia anoxia recently, we found that VEGF also stimulate neurogenesis in vitro and in vivo. A study shows that VEGF significantly increases the number of proliferating cells in the SVZ and the perilesional cortex after TBI. Functional outcome was significantly better in mice treated with VEGF compared with vehicle-treated animals after TBI. 52, 53 erythropoietin (EPO), a naturally occurring cytokine, is most widely recognized for its role in stimulating the maturation, differentiation, and survival of hematopoietic progenitor cells. Michael chopp’s group found that rhepo administration significantly increases the number of brdu-labeled cells in both the contralateral and ipsilateral DG and promotes restoration of spatial memory after TBI. 54 notably, these growth factors not only stimulate neurogenesis, but also have a neuroprotective role.Hipoxia anoxia it therefore becomes difficult to attribute improvements in behavior to their effects on neurogenesis only. In order to demonstrate that functional recovery requires newly generated cells derived from NSC activation, more cell-specific genetic assays should be performed in the future.