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1.  Effects of Erythropoietin on Reducing Brain Damage and Improving Functional Outcome after traumatic brain injury in mice 
Journal of neurosurgery  2008;109(3):510-521.
Object
This study was designed to investigate the beneficial effects of recombinant human erythropoietin (rhEPO) treatment of traumatic brain injury (TBI) in mice.
Methods
Adult male C57BL/6 mice were divided into 3 groups: 1) saline group (TBI + saline, n = 13); 2) EPO group (TBI + rhEPO, n = 12); and 3) sham group (sham + rhEPO, n = 8). TBI was induced by controlled cortical impact. Bromodeoxyuridine (100 mg/kg) was injected daily for 10 days, starting 1 day after injury, for labeling proliferating cells. rhEPO was administered intraperitoneally at 6 hours, and at 3 and 7 days post-TBI (5000 U/kg body weight, total dosage = 15,000 U/kg). Neurological function was assessed using the Morris Water Maze and footfault tests. Animals were sacrificed 35 days after injury and brain sections stained for immunohistochemistry.
Results
TBI caused both tissue loss in the cortex and cell loss in the dentate gyrus (DG) and impaired sensorimotor function (footfaults) and spatial learning (Morris Water Maze). TBI alone stimulated cell proliferation and angiogenesis. As compared to saline treatment, rhEPO significantly reduced lesion volume in the cortex and cell loss in the DG after TBI and substantially improved sensorimotor function recovery and spatial learning performance. rhEPO enhanced neurogenesis in the injured cortex and the DG.
Conclusions
rhEPO initiated 6 hours post-TBI provides neuroprotection by decreasing lesion volume and cell loss as well as neurorestoration by enhancing neurogenesis, subsequently improving sensorimotor and spatial learning function. rhEPO is a promising neuroprotective and neurorestorative agent for TBI and warrants further investigation.
doi:10.3171/JNS/2008/109/9/0510
PMCID: PMC3977174  PMID: 18759585
erythropoietin; mouse; sensorimotor; spatial learning; traumatic brain injury
2.  Simvastatin Attenuates Axonal Injury after Experimental Traumatic Brain Injury and Promotes Neurite Outgrowth of Primary Cortical Neurons 
Brain research  2012;1486C:121-130.
The beneficial effects of simvastatin on experimental traumatic brain injury (TBI) have been demonstrated in previous studies. In this study, we investigated the effects of simvastatin on axonal injury and neurite outgrowth after experimental TBI and explored the underlying mechanisms. Wistar rats were subjected to controlled cortical impact or sham surgery. Saline or simvastatin was administered for 14 days. A modified neurological severity score (mNSS) test was performed to evaluate functional recovery. Immunohistochemistry studies using synaptophysin, neurofilament H (NF-H) and amyloid-β precursor protein (APP) were performed to examine synaptogenesis and axonal injury. Primary cortical neurons (PCNs) were subjected to oxygen glucose deprivation (OGD) followed by various treatments. Western blot analysis was utilized to assess the activation of phosphatidylinositol-3 kinase (PI-3K)/Akt/mammalian target of rapamycin (mTOR) and glycogen synthase kinase 3β (GSK- 3β)/adenomatous polyposis coli (APC) pathways. Simvastatin decreased the density of APP-positive profiles and increased the density of NF-H -positive profiles. Simvastatin reduced mNSS, which was correlated with the increase of axonal density. Simvastatin treatment stimulated the neurite outgrowth of PCNs after OGD, which was attenuated by LY294002 and enhanced by lithium chloride (LiCl). Simvastatin activated Akt and mTOR, inactivated GSK-3β and dephosphorylated APC in the injured PCNs. Our data suggest that simvastatin reduces axonal injury, enhances neurite outgrowth and promotes neurological functional recovery after experimental TBI. The beneficial effects of simvastatin on neurite outgrowth may be mediated through manipulation of the PI-3K/Akt/mTOR and PI-3K/GSK-3β/APC pathways.
doi:10.1016/j.brainres.2012.09.039
PMCID: PMC3496081  PMID: 23026078
axonal injury; glycogen synthase kinase 3β; neurite outgrowth; simvastatin; traumatic brain injury
3.  Effect of Simvastatin on Glioma Cell Proliferation, Migration and Apoptosis 
Neurosurgery  2009;65(6):1087-1097.
OBJECTIVE
In this study, we seek to investigate the effects of simvastatin on proliferation, migration and apoptosis in human U251 and U87 glioma cells and the underlying molecular mechanism.
METHODS
We used colony formation assay to test the cell proliferation, in vitro scratch assay to examine the cell migration, and caspase-3 activity assay, annexin V staining and cytochrome C release to evaluate the cell apoptosis. Lipid raft fractions were isolated from glioma cells. Total cholesterol content assay was used to test the change of cholesterol level in lipid raft fractions. Immunocytochemistry staining was performed to detect the changes of lipid rafts in cell membrane. Western blotting analysis was performed to examine the signal transduction both in cells and in lipid raft fractions.
RESULTS
Simvastatin inhibited proliferation and migration of U251 and U87 cells dose-dependently. Simvastatin induced an increase of caspase-3 activity, annexin V staining, and downregulated the PI3K/Akt pathway. Simvastatin also decreased cholesterol content in lipid raft fractions, suppressed caveolin-1 expression in the lipid rafts and induced Fas translocation into lipid rafts, suggesting that simvastatin may inhibit pro-survival PI3K/Akt pathway and trigger caspase-3-dependent apoptotic cell death through the modulation of lipid rafts.
CONCLUSION
These results suggest that modulation of lipid rafts, Fas translocation and PI3K/Akt/caspase-3 pathway are involved in the antitumor effect of simvastatin and it may have a potential role in cancer prevention and treatment.
doi:10.1227/01.NEU.0000360130.52812.1D
PMCID: PMC3711082  PMID: 19934968
apoptosis; glioma; lipid rafts; PI3K/Akt pathway; simvastatin
4.  Treatment of Traumatic Brain Injury in Mice with Marrow Stromal Cells 
Brain research  2008;1208:234-239.
Objective
This study was designed to investigate the potential beneficial effects of bone marrow stromal cell (MSC) treatment of traumatic brain injury (TBI) in mice.
Methods
Twelve female C57BL/6J mice (weight, 21–26 g) were injured with controlled cortical impact and divided into 2 groups (n = 6 each). The experimental group was injected with MSCs (0.3 × 106) intravenously one day after TBI, whereas the control group was injected with saline. MSCs were harvested from male mice, and male to female transplantation performed to identify male donor cells within female recipient animals. This was achieved by localizing Y chromosomes within the female mice. Neurological function was assessed using the Morris water maze and Foot Fault tests. All mice were sacrificed 35 days after TBI. Brain sections were stained using in situ hybridization and immunohistochemistry to identify MSCs as well as to analyze vascular density following MSC treatment.
Results
Both modalities of testing demonstrated significant improvement in neurological function in the MSC-treated group compared to the saline-treated control group (p < 0.05). Histologically, Y-chromosome labeled MSCs were easily identified in the injured brain, localized primarily around the lesion boundary zone. There was also significant increase in vascular density in the lesion boundary zone and hippocampus of MSC-treated mice compared to control mice.
Conclusion
This is the first study to show beneficial effects of MSC treatment after TBI in mice.
doi:10.1016/j.brainres.2008.02.042
PMCID: PMC3707111  PMID: 18384759
Traumatic brain injury (TBI); marrow stromal cells (MSCs); mice
5.  Simvastatin Increases Phosphorylation of Akt and its Downstream Signaling Targets and Suppresses Apoptosis after Traumatic Brain Injury 
Journal of neurosurgery  2008;109(4):691-698.
Object
Our previous studies found that simvastatin treatment of traumatic brain injury (TBI) in rats had beneficial effects on spatial learning functions. In the current study we wanted to determine whether simvastatin suppressed neuronal cell apoptosis after TBI, and if so, the underlying mechanisms of this process.
Methods
Saline or simvastatin (1 mg/kg) was administered orally to rats starting at Day 1 after TBI and then daily for 14 days. Modified neurological severity scores (NSS) were employed to evaluate the sensory motor functional recovery. Rats were sacrificed at 1, 3, 7, 14 and 35 days after treatment and brain tissue was harvested for TUNEL staining, caspase-3 activity assay and Western blot analysis. Simvastatin significantly decreased NSS from Days 7 to 35 after TBI, significantly reduced the number of TUNEL-positive cells at Day 3, suppressed the caspase-3 activity at Days 1 and 3 after TBI, and increased phosphorylation of Akt as well as FOXO1, IκB and eNOS, which are the downstream targets of the pro-survival Akt signaling protein.
Conclusions
These data suggested that simvastatin reduces the apoptosis in neuronal cells and improves the sensory motor function recovery after TBI. These beneficial effects of simvastatin may be mediated through activation of Akt, FOXO1 and NF-κB signaling pathways, which suppress the activation of caspase-3 and apoptotic cell death, and thereby lead to neuronal function recovery after TBI.
doi:10.3171/JNS/2008/109/10/0691
PMCID: PMC3703644  PMID: 18826357
simvastatin; apoptosis; Akt; FOXO1; IκB; traumatic brain injury
6.  Comparison of Neurite Density Measured by MRI and Histology after TBI 
PLoS ONE  2013;8(5):e63511.
Background
Functional recovery after brain injury in animals is improved by marrow stromal cells (MSC) which stimulate neurite reorganization. However, MRI measurement of neurite density changes after injury has not been performed. In this study, we investigate the feasibility of MRI measurement of neurite density in an animal model of traumatic brain injury (TBI) with and without MSC treatment.
Methods
Fifteen male Wistar rats, were treated with saline (n = 6) or MSCs (n = 9) and were sacrificed at 6 weeks after controlled cortical impact (CCI). Healthy non-CCI rats (n = 5), were also employed. Ex-vivo MRI scans were performed two days after the rats were sacrificed. Multiple-shell hybrid diffusion imaging encoding scheme and spherical harmonic expansion of a two-compartment water diffusion displacement model were used to extract neurite related parameters. Bielshowski and Luxol Fast blue was used for staining axons and myelin, respectively. Modified Morris water maze and neurological severity score (mNSS) test were performed for functional evaluation. The treatment effects, the correlations between neurite densities measured by MRI and histology, and the correlations between MRI and functional variables were calculated by repeated measures analysis of variance, the regression correlation analysis tests, and spearman correlation coefficients.
Results
Neurite densities exhibited a significant correlation (R2>0.80, p<1E−20) between MRI and immuno-histochemistry measurements with 95% lower bound of the intra-correlation coefficient (ICC) as 0.86. The conventional fractional anisotropy (FA) correlated moderately with histological neurite density (R2 = 0.59, P<1E−5) with 95% lower bound of ICC as 0.76. MRI data revealed increased neurite reorganization with MSC treatment compared with saline treatment, confirmed by histological data from the same animals. mNSS were significantly correlated with MRI neurite density in the hippocampus region.
Conclusions
The present studies demonstrated that neurite density can be estimated by MRI after TBI and MRI measurement of neurite density is a sensitive marker to MSC treatment response.
doi:10.1371/journal.pone.0063511
PMCID: PMC3661595  PMID: 23717439
7.  Impact of inhibition of erythropoietin treatment-mediated neurogenesis in the dentate gyrus of the hippocampus on restoration of spatial learning after traumatic brain injury 
Experimental Neurology  2012;235(1):336-344.
Our previous study demonstrates that delayed (initiated 24 hours post injury) erythropoietin (EPO) therapy for traumatic brain injury (TBI) significantly improves spatial learning. In this study, we investigated the impact of inhibition of EPO treatment-mediated neurogenesis on spatial learning after experimental TBI. Young male Wistar rats (318±7g) were subjected to unilateral controlled cortical impact injury. TBI rats received delayed EPO treatment (5,000 U/kg in saline) administered intraperitoneally once daily at 1, 2, and 3 days post injury and intracerebroventricular (icv) infusion of either a mitotic inhibitor cytosine-b-D-arabinofuranoside or vehicle (saline) for 14 days. Another 2 groups of TBI rats were treated intraperitoneally with saline and infused icv with either a mitotic inhibitor Ara-C or saline for 14 days. Animals receiving sham operation were infused icv with either Ara-C infusion or saline. Bromodeoxyuridine (BrdU) was administered to label dividing cells. Spatial learning was assessed using a modified Morris water maze test. Animals were sacrificed at 35 days after injury and brain sections stained for immunohistochemical analyses. As compared to the saline treatment, immunohistochemical analysis revealed that delayed EPO treatment significantly increased the number of BrdU-positive cells and new neurons co-stained with BrdU and NeuN (mature neuron marker) in the dentate gyrus in TBI rats. EPO treatment improved spatial learning after TBI. Ara-C infusion significantly abolished neurogenesis and spatial learning recovery after TBI and EPO treatment. Both EPO and Ara-C reduced the number of astrocytes and microglia/macrophages in the dentate gyrus after TBI. Our findings are highly suggestive for an important role of EPO-amplified dentate gyrus neurogenesis as one of the mechanisms underlying EPO therapeutic treatments after TBI, strongly indicating that strategies promoting endogenous neurogenesis may hold an important therapeutic potential for treatment of TBI.
doi:10.1016/j.expneurol.2012.02.015
PMCID: PMC3334417  PMID: 22414310
astrocytes; erythropoietin; microglia; neurogenesis; spatial learning; traumatic brain injury
8.  MRI of Neuronal Recovery after Low-Dose Methamphetamine Treatment of Traumatic Brain Injury in Rats 
PLoS ONE  2013;8(4):e61241.
We assessed the effects of low dose methamphetamine treatment of traumatic brain injury (TBI) in rats by employing MRI, immunohistology, and neurological functional tests. Young male Wistar rats were subjected to TBI using the controlled cortical impact model. The treated rats (n = 10) received an intravenous (iv) bolus dose of 0.42 mg/kg of methamphetamine at eight hours after the TBI followed by continuous iv infusion for 24 hrs. The control rats (n = 10) received the same volume of saline using the same protocol. MRI scans, including T2-weighted imaging (T2WI) and diffusion tensor imaging (DTI), were performed one day prior to TBI, and at 1 and 3 days post TBI, and then weekly for 6 weeks. The lesion volumes of TBI damaged cerebral tissue were demarcated by elevated values in T2 maps and were histologically identified by hematoxylin and eosin (H&E) staining. The fractional anisotropy (FA) values within regions-of-interest (ROI) were measured in FA maps deduced from DTI, and were directly compared with Bielschowsky’s silver and Luxol fast blue (BLFB) immunohistological staining. No therapeutic effect on lesion volumes was detected during 6 weeks after TBI. However, treatment significantly increased FA values in the recovery ROI compared with the control group at 5 and 6 weeks after TBI. Myelinated axons histologically measured using BLFB were significantly increased (p<0.001) in the treated group (25.84±1.41%) compared with the control group (17.05±2.95%). Significant correlations were detected between FA and BLFB measures in the recovery ROI (R = 0.54, p<0.02). Methamphetamine treatment significantly reduced modified neurological severity scores from 2 to 6 weeks (p<0.05) and foot-fault errors from 3 days to 6 weeks (p<0.05) after TBI. Thus, the FA data suggest that methamphetamine treatment improves white matter reorganization from 5 to 6 weeks after TBI in rats compared with saline treatment, which may contribute to the observed functional recovery.
doi:10.1371/journal.pone.0061241
PMCID: PMC3630155  PMID: 23637800
9.  Erythropoietin Mediates Neurobehavioral Recovery and Neurovascular Remodeling Following Traumatic Brain Injury in Rats by Increasing Expression of Vascular Endothelial Growth Factor 
Translational stroke research  2011;2(4):619-632.
Erythropoietin (EPO) improves functional recovery after traumatic brain injury (TBI). Here, we investigated the role of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) on EPO-induced therapeutic efficacy in rats after TBI. Young male Wistar rats were subjected to unilateral controlled cortical impact injury and then infused intracerebroventricularly with either a potent selective VEGFR2 inhibitor SU5416 or vehicle dimethyl sulfoxide. Animals from both groups received delayed EPO treatment (5,000 U/kg in saline) administered intraperitoneally daily at 1, 2, and 3 days post injury. TBI rats treated with saline administered intraperitoneally daily at 1, 2, and 3 days post injury served as EPO treatment controls. 5-bromo-2-deoxyuridine was administered to label dividing cells. Spatial learning and sensorimotor function were assessed using a modified Morris water maze test and modified neurological severity score, respectively. Animals were sacrificed at 4 days post injury for measurement of VEGF and VEGFR2 or 35 days post injury for evaluation of cell proliferation, angiogenesis and neurogenesis. EPO treatment promoted sensorimotor and cognitive functional recovery after TBI. EPO treatment increased brain VEGF expression and phosphorylation of VEGFR2. EPO significantly increased cell proliferation, angiogenesis and neurogenesis in the dentate gyrus after TBI. Compared to the vehicle, SU5416 infusion significantly inhibited phosphorylation of VEGFR2, cell proliferation, angiogenesis, and neurogenesis as well as abolished functional recovery in EPO-treated TBI rats. These findings indicate the VEGF/VEGFR2 activation plays an important role in EPO-mediated neurobehavioral recovery and neurovascular remodeling after TBI.
doi:10.1007/s12975-011-0120-2
PMCID: PMC3374663  PMID: 22707988
angiogenesis; erythropoietin; neurogenesis; traumatic brain injury; vascular endothelial growth factor
10.  Dose-dependent neurorestorative effects of delayed treatment of traumatic brain injury with recombinant human erythropoietin in rats 
Journal of neurosurgery  2011;115(3):550-560.
Object
Delayed (24 hours post injury) treatment with erythropoietin (EPO) improves functional recovery following experimental traumatic brain injury (TBI). In this study, we tested whether therapeutic effects of delayed EPO treatment for TBI are dose-dependent in an attempt to establish an optimal dose paradigm for the delayed EPO treatment.
Methods
Experimental TBI was performed in anesthetized young adult male Wistar rats using a controlled cortical impact device. Sham animals underwent the same surgical procedure without injury. The animals (8 rats/group) received 3 intraperitoneal injections of EPO (0, 1000, 3000, 5000 or 7000 U/kg body weight, at 24, 48 and 72 hours) after TBI. Sensorimotor and cognitive functions were assessed using a modified neurological severity score and foot fault test, and Morris water maze tests, respectively. Animals were sacrificed 35 days after injury and brain sections stained for immunohistochemical analyses.
Results
Compared to the saline treatment, EPO treatment at doses from1000 to 7000 U/kg did not alter lesion volume but significantly reduced hippocampal neuron loss, enhanced angiogenesis and neurogenesis in the injured cortex and hippocampus, and significantly improved sensorimotor function and spatial learning. The medium dose at 5000 U/kg exhibited a significant improvement in histological and functional outcomes compared with the lower or higher EPO dose groups.
Conclusions
These data demonstrate that delayed (24 hours post injury) treatment with EPO provides dose-dependent neurorestoration which may contribute to improved functional recovery after TBI, implying that application of an optimal dose of EPO is likely to increase successful preclinical and clinical trials for treatment of TBI.
doi:10.3171/2011.3.JNS101721
PMCID: PMC3166387  PMID: 21495821
angiogenesis; cell proliferation; erythropoietin; neurogenesis; rat; sensorimotor; spatial learning; traumatic brain injury
11.  Treatment of TBI with Collagen Scaffolds and Human Marrow Stromal Cells Increases the Expression of Tissue Plasminogen Activator 
Journal of Neurotrauma  2011;28(7):1199-1207.
Abstract
This study examines the effects of combination therapy of collagen scaffolds and human marrow stromal cells (hMSCs) on the expression of tissue plasminogen activator (tPA) after traumatic brain injury (TBI) in rats. Adult male Wistar rats (n=48) were injured with controlled cortical impact and treated either with scaffolds suffused with hMSCs (3×106) or hMSCs (3×106) alone transplanted into the lesion cavity 1 week after TBI. A control group was treated with saline. Neurological function was assessed using the Morris Water Maze test (MWM) and modified Neurological Severity Scores (mNSS). The rats were sacrificed 14 days after TBI and brain samples were processed for immunohistochemical analysis and quantitative Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) studies. Enhanced functional improvement was observed on both the mNSS and MWM tests in the scaffold+hMSC-treated group compared to the other two groups. Immunostaining with anti-human mitochondrial antibody (E5204) showed more hMSCs in the injury zone of the scaffold+hMSC group compared to the hMSC-alone group. Triple staining showed that more neurons were tPA-positive in the scaffold+hMSC group compared to the other two groups (p<0.05). Western blot analysis and qRT-PCR showed that scaffold+hMSC and hMSC-alone treatment enhanced the expression of tPA compared to controls (p<0.05), but tPA expression was significantly greater in the scaffold+hMSC group. The induction of tPA by hMSCs after TBI may be one of the mechanisms involved in promoting functional improvement after TBI.
doi:10.1089/neu.2010.1694
PMCID: PMC3283444  PMID: 21355820
collagen scaffolds; marrow stromal cells; tissue plasminogen activator; traumatic brain injury
12.  MRI evaluation of axonal reorganization after bone marrow stromal cell treatment of traumatic brain injury 
Nmr in Biomedicine  2011;24(9):1119-1128.
We treated traumatic brain injury (TBI) with human bone marrow stromal cells (hMSCs) and evaluated the effect of treatment on white matter reorganization using MRI. We subjected male Wistar rats (n = 17) to controlled cortical impact and either withheld treatment (controls; n = 9) or inserted collagen scaffolds containing hMSCs (n = 8). Six weeks later, the rats were sacrificed and MRI revealed selective migration of grafted neural progenitor cells towards the white matter reorganized boundary of the TBI-induced lesion. Histology confirmed that the white matter had been reorganized, associated with increased fractional anisotropy (FA; p <0.01) in the recovery regions relative to the injured core region in both treated and control groups. Treatment with hMSCs increased FA in the recovery regions, lowered T2 in the core region, decreased lesion volume and improved functional recovery relative to untreated controls. Immunoreactive staining showed axonal projections emanating from neurons and extruding from the corpus callosum into the ipsilateral cortex at the boundary of the lesion. Fiber tracking (FT) maps derived from diffusion tensor imaging confirmed the immunohistological data and provided information on axonal rewiring. The apparent kurtosis coefficient (AKC) detected additional axonal remodeling regions with crossing axons, confirmed by immunohistological staining, compared with FA. Our data demonstrate that AKC, FA, FTand T2 can be used to evaluate treatment-induced white matter recovery, which may facilitate restorative therapy in patients with TBI.
doi:10.1002/nbm.1667
PMCID: PMC3381889  PMID: 21432927
MRI; DTI; traumatic brain injury; rat brain
13.  Induction of angiogenesis and modulation of VEGFR-2 by simvastatin after traumatic brain injury 
Neurosurgery  2011;68(5):1363-1371.
Background
Our previous studies demonstrated that simvastatin reduced neuronal death, increased neurogenesis, and promoted functional recovery after TBI. Objective: To investigate the effect of simvastatin on angiogenesis after TBI, and the related signaling pathways.
Methods
Saline or simvastatin (1 mg/kg) was administered orally to rats starting at day 1 after TBI or sham surgery and then daily for 14 days. Rats were sacrificed at 3 and 14 days after treatment. Brain sections and tissues were prepared for immunohistochemical staining, ELISA, and Western blot analysis, respectively. Cultured rat brain microvascular endothelial cells (RBMVECs) were subjected to oxygen-glucose deprivation (OGD) followed by immunocytochemical staining with phallotoxins and vascular endothelial growth factor receptor-2 (VEGFR-2). Western blot analysis was carried out to examine the simvastatin-induced activation of the v-akt murine thymoma viral oncogene homolog (Akt) signaling pathway. The expression of VEGFR-2 was detected by ELISA.
Results
Simvastatin significantly increased the length of vascular perimeter, promoted the proliferation of endothelial cells, and improved the sensorimotor function after TBI. Simvastatin stimulated endothelial cell tube formation after OGD in vitro. VEGFR-2 expression in both brain tissues and cultured RBMVECs was enhanced after simvastatin treatment, which may be modulated by activation of Akt. Akt-dependent endothelial nitric oxide synthase (eNOS) phosphorylation was also induced by simvastatin in vivo and in vitro.
Conclusion
Simvastatin augments TBI-induced angiogenesis in the lesion boundary zone and hippocampus and improves functional recovery. Simvastatin also promotes angiogenesis in vitro. These beneficial effects on angiogenesis may be related to simvastatin-induced activation of the VEGFR-2/Akt/eNOS signaling pathway.
doi:10.1227/NEU.0b013e31820c06b9
PMCID: PMC3119744  PMID: 21307798
Angiogenesis; Simvastatin; Traumatic brain injury; VEGFR-2
14.  Erythropoietin promotes neurovascular remodeling and long-term functional recovery in rats following traumatic brain injury 
Brain research  2011;1384:140-150.
Erythropoietin (EPO) improves functional recovery after traumatic brain injury (TBI). This study was designed to investigate long-term (3 mo) effects of EPO on brain remodeling and functional recovery in rats after TBI. Young male Wistar rats were subjected to unilateral controlled cortical impact injury. TBI rats were divided into the following groups: 1) Saline group (n = 7); 2) EPO-6h group (n = 8); and 3) EPO-24h group (n = 8). EPO (5,000 U/kg in saline) was administered intraperitoneally at 6 h, and 1 and 2 days (EPO-6h group) or at 1, 2, and 3 days (EPO-24h group) post injury. Neurological function was assessed using a modified neurological severity score, footfault and Morris water maze tests. Animals were sacrificed at 3 mos after injury and brain sections stained for immunohistochemical analyses. Compared to the saline, EPO-6h treatment significantly reduced cortical lesion volume, while EPO-24h therapy did not affect the lesion volume (P<0.05). Both the EPO-6h and EPO-24h treatments significantly reduced hippocampal cell loss (P<0.05), promoted angiogenesis (P<0.05) and increased endogenous cellular proliferation (BrdU-positive cells) in the injury boundary zone and hippocampus (P<0.05) compared to saline controls. Significantly enhanced neurogenesis (BrdU/NeuN-positive cells) was seen in the dentate gyrus of both EPO groups compared to the saline group. Both EPO treatments significantly improved long-term sensorimotor and cognitive functional recovery after TBI. In conclusion, the beneficial effects of posttraumatic EPO treatment on injured brain persisted for at least 3 months. The long-term improvement in functional outcome may in part be related to the neurovascular remodeling induced by EPO.
doi:10.1016/j.brainres.2011.01.099
PMCID: PMC3065541  PMID: 21295557
angiogenesis; cell proliferation; erythropoietin; neurogenesis; functional recovery; traumatic brain injury
15.  Effects of posttraumatic carbamylated erythropoietin therapy on reducing lesion volume and hippocampal cell loss, enhancing angiogenesis and neurogenesis, and improving functional outcome in rats following traumatic brain injury 
Journal of neurosurgery  2010;114(2):549-559.
Object
Carbamylated erythropoietin (CEPO) is a modified erythropoietin molecule that does not affect hematocrit. In this study, we compared the efficacy of a single dose with triple dose of CEPO treatment of traumatic brain injury (TBI) in rats.
Methods
TBI was induced by controlled cortical impact over the left parietal cortex. CEPO (50 μg/kg) was administered intraperitoneally in rats with TBI at 6 hours (CEPO x 1 group) or 6, 24 and 48 hours (CEPO x 3 group) post injury. Neurological function was assessed using a modified neurological severity score, footfault and Morris water maze tests. Animals were sacrificed 35 days after injury and brain sections stained for immunohistochemistry to assess lesion volume, cell loss, cell proliferation, angiogenesis and neurogenesis after CEPO treatment.
Results
Compared to the vehicle treatment, single treatment of CEPO (6 hours) significantly reduced lesion volume and hippocampal cell loss, enhanced angiogenesis and neurogenesis in the injured cortex and hippocampus, and significantly improved sensorimotor functional recovery and spatial learning in rats after TBI. Importantly, triple dosing of CEPO (6, 24 and 48 hours) further reduced lesion volume and improved functional recovery and neurogenesis compared to the CEPO x 1 group.
Conclusions
Our results indicate that CEPO has considerable therapeutic potential in TBI and related pathologies and furthermore that repeated dosing in the sub-acute phase might have important pharmacological relevance.
doi:10.3171/2010.10.JNS10925
PMCID: PMC3057520  PMID: 21073254
angiogenesis; carbamylated erythropoietin; functional recovery; neurogenesis; traumatic brain injury
16.  The Treatment of TBI with Human Marrow Stromal Cells Impregnated into Collagen Scaffold: Functional Outcome and Gene Expression Profile 
Brain research  2010;1371:129-139.
We have previously demonstrated that human marrow stromal cells (hMSCs) embedded in collagen I scaffolds significantly enhance the restorative therapeutic effect of hMSCs after traumatic brain injury (TBI). In this study, we test the hypothesis that the collagen scaffold alters gene expression in hMSCs and that hMSCs impregnated into scaffolds increase the astrocytic expression of vascular endothelial growth factor (VEGF) in the injured brain. Following TBI induced by controlled cortical impact injury, scaffold with hMSCs (3.0 × 106), hMSCs-only and saline were implanted into the lesion cavity one week after brain injury (n = 8/each group). Morris water Maze and modified neurological severity scores were performed to evaluate the spatial learning and sensorimotor functions, respectively. Lesion volume and expression of VEGF were measured one week after different treatments. In vitro, total RNA from hMSCs was extracted one week after culture with or without collagen I scaffold for evaluation of gene microarrays. Furthermore, an RT-PCR study on a select subgroup of genes was performed to identify the changes of expression between the culturing hMSCs with collagen scaffolds and hMSCs only. The treatment of TBI with collagen scaffold impregnated with hMSCs significantly decreases the functional deficits from TBI within 7 days after treatment, and significantly enhances the VEGF expression of astrocytes in the injured brain compared to the hMSCs-only group. In vitro data indicate that collagen scaffolds stimulate hMSCs to express multiple factors which may contribute to hMSC survival, tissue repair and functional recovery after TBI.
doi:10.1016/j.brainres.2010.10.088
PMCID: PMC3022100  PMID: 21062621
endothelial vascular growth factor (VEGF); traumatic brain injury (TBI); marrow stromal cell; collagen scaffold; restorative therapy
17.  Sprouting of corticospinal tract axons from the contralateral hemisphere into the denervated side of the spinal cord is associated with functional recovery in adult rat after traumatic brain injury and erythropoietin treatment 
Brain research  2010;1353:249-257.
Erythropoietin (EPO) promotes functional recovery after traumatic brain injury (TBI). This study was designed to investigate whether EPO treatment promotes contralateral corticospinal tract (CST) plasticity in the spinal cord in rats after TBI. Biotinylated dextran amine (BDA) was injected into the right sensorimotor cortex to anterogradely label the CST. TBI was induced by controlled cortical impact over the left parietal cortex immediately after BDA injections. EPO (5,000 U/kg) or saline was administered intraperitoneally at Days 1, 2, and 3 post injury. Neurological function was assessed using a modified neurological severity score (mNSS) and footfault tests. Animals were sacrificed 35 days after injury and brain sections stained for histological analysis. Compared to the saline treatment, EPO treatment significantly improved sensorimotor functional outcome (lower mNSS and reduced footfaults) from Days 7 to 35 post injury. TBI alone significantly stimulated contralateral CST axon sprouting toward the denervated gray matter of the cervical and lumbar spinal cord; however, EPO treatment further significantly increased the axon sprouting in TBI rats although EPO treatment did not significantly affect axon sprouting in sham animals. The contralesional CST sprouting was highly and positively correlated with sensorimotor recovery after TBI. These data demonstrate that CST fibers originating from the contralesional intact cerebral hemisphere are capable of sprouting into the denervated spinal cord after TBI and EPO treatment, which may at least partially contribute to functional recovery.
doi:10.1016/j.brainres.2010.07.046
PMCID: PMC2933297  PMID: 20654589
axonal plasticity; erythropoietin; functional recovery; rats; traumatic brain injury
18.  Delayed administration of erythropoietin reduces hippocampal cell loss, enhances angiogenesis and neurogenesis, and improves functional outcome following traumatic brain injury in rats: comparison of treatment with single dose and triple dose 
Journal of neurosurgery  2010;113(3):598-608.
Object
This study was designed to investigate delayed erythropoietin (EPO) treatment for traumatic brain injury (TBI) in rats comparing efficacy of a single dose with triple doses.
Methods
Young adult male Wistar rats were randomly divided into the following groups: 1) Sham group (n = 6); 2) TBI + Saline group (n = 6); 3) TBI + EPOx1 group (n = 6); and 4) TBI + EPOx3 group (n = 7). TBI was induced by controlled cortical impact over the left parietal cortex. EPO (5,000 U/kg) or saline was administered intraperitoneally at 1 day (EPOx1 group) or at days 1, 2, and 3 (EPOx3 group) post injury. Neurological function was assessed using a modified neurological severity score (mNSS), footfault and Morris water maze tests. Animals were sacrificed 35 days after injury and brain sections stained for immunohistochemistry.
Results
Compared to the saline treatment, EPO treatment in both the EPOx1 and EPOx3 groups significantly reduced hippocampal cell loss, enhanced angiogenesis and neurogenesis in the injured cortex and hippocampus, and significantly improved neurological functional outcome. The EPOx3 group exhibited significantly improved functional and histological outcomes compared with the EPOx1 group.
Conclusions
These data demonstrate that delayed posttraumatic administration of EPO significantly improves histological and long-term functional outcomes in rats after TBI. The triple doses of delayed EPO treatment exhibit better histological and functional outcomes in rats although a single dose of EPO provides substantial benefits compared to saline treatment.
doi:10.3171/2009.9.JNS09844
PMCID: PMC2898921  PMID: 19817538
angiogenesis; cell proliferation; erythropoietin; neurogenesis; rat; sensorimotor; spatial learning; traumatic brain injury
19.  The Treatment of Traumatic Brain Injury with Velcade 
Journal of Neurotrauma  2010;27(9):1625-1634.
Abstract
Traumatic brain injury (TBI) elicits a strong inflammatory response that contributes to the acute pathological processes seen following TBI, including cerebral edema and disruption of the blood–brain barrier (BBB), in addition to longer-term neurological damage and cognitive impairment. Proteasome inhibitors reduce vascular thrombotic and inflammatory events and consequently protect vascular function. In the present study we evaluated the neuroprotective effect of Velcade® (bortezomib), a potent and selective inhibitor of proteasomes, which is in clinical use for the treatment of multiple myeloma. When administered within 2 h after TBI onset, Velcade reduced inflammatory responses, lesion volume, and neurological functional deficits, and enhanced neuronal survival. Western blot and ELISA showed that Velcade decreased the expression of NF-κB. These results suggest that in the experimental setting, Velcade is an effective neuroprotective agent for the treatment of TBI.
doi:10.1089/neu.2010.1359
PMCID: PMC2966855  PMID: 20649468
neuroprotection; rats; traumatic brain injury; Velcade
20.  Erythropoietin Improves Histological and Functional Outcomes after Traumatic Brain Injury in Mice in the Absence of the Neural Erythropoietin Receptor 
Journal of Neurotrauma  2010;27(1):205-215.
Abstract
Erythropoietin (EPO), essential for erythropoiesis, provides neuroprotection. The EPO receptor (EPOR) is expressed in both neural and non-neural cells in the brain. This study was designed to test the hypothesis that EPO provides beneficial therapeutic effects, even in the absence of the neural EPOR. In this study, EPOR-null mice were rescued with selective EpoR expression driven by the endogenous EpoR promoter in hematopoietic tissue, but not in the neural cells. Anesthetized young adult female EPOR-null and wild-type mice were subjected to traumatic brain injury (TBI) induced by controlled cortical impact. EPO (5000 U/kg) or saline was intraperitoneally administered at 6 h and 3 and 7 days post-injury. Sensorimotor and spatial learning functions were assessed. Expression of EPOR and its downstream signal proteins were evaluated by Western blot analysis. Our data demonstrated that EPO treatment significantly reduced cortical tissue damage and hippocampal cell loss, and improved spatial learning following TBI in both the wild-type and EPOR-null mice. EPO treatment significantly improved sensorimotor functional recovery, with better outcomes in the wild-type mice. EPO treatment upregulated anti-apoptotic proteins (p-Akt and Bcl-XL) in the ipsilateral hippocampus and cortex of the injured wild-type and EPOR-null mice. These data demonstrate that EPO significantly provides neuroprotection following TBI, even in the absence of EPOR in the neural cells, suggesting that its therapeutic benefits may be mediated through vascular protection.
doi:10.1089/neu.2009.1001
PMCID: PMC2824224  PMID: 19715391
erythropoietin receptor null; mouse; sensorimotor; spatial learning; traumatic brain injury
21.  Therapeutic effects of erythropoietin on histological and functional outcomes following traumatic brain injury in rats are independent of hematocrit 
Brain research  2009;1294:153-164.
Erythropoietin (EPO) provides neuroprotection and neurorestoration after traumatic brain injury (TBI). The EPO doses used for treatment of TBI significantly increase hematocrit, which may affect the efficacy of EPO therapy for TBI. The aim of this study was to investigate whether normalization of hematocrit would affect EPO efficacy for treatment of TBI. Young adult male Wistar rats were randomly divided into 4 groups: 1) Sham group (n=6); 2) TBI + saline group (n=6); 3) TBI + EPO group (n=6); and 4) TBI + EPO + hemodilution group (n=7). TBI was induced by controlled cortical impact over the left parietal cortex. EPO (5,000 U/kg) or saline was administered intraperitoneally at days 1, 2, and 3 post injury. Neurological function was assessed using a modified neurological severity score (mNSS), footfault and the Morris water maze (MWM) tests. Animals were sacrificed 35 days after injury and brain sections stained for immunohistochemistry. Compared to the saline treatment, EPO treatment significantly reduced hippocampal cell loss, enhanced angiogenesis and neurogenesis in the injured cortex and hippocampus, and significantly improved sensorimotor functional outcome (lowered mNSS and foot faults) and spatial learning (MWM test). Normovolemic hemodilution effectively normalized the hematocrit and did not significantly affect the histological and functional outcome of EPO therapy for TBI. These data for the first time demonstrate that increased hematocrit does not affect therapeutic effects of EPO on histological and long-term functional outcomes in rats after TBI and also suggest that neuroprotection and neurorestoration of EPO treatment are independent of hematocrit.
doi:10.1016/j.brainres.2009.07.077
PMCID: PMC2746872  PMID: 19646970
angiogenesis; erythropoietin; hemodilution; neurogenesis; traumatic brain injury
22.  Treatment of Traumatic Brain Injury in Mice with Bone Marrow Stromal Cell-Impregnated Collagen Scaffolds 
Journal of neurosurgery  2009;111(4):658-665.
Object
This study was designed to investigate new ways of delivering human marrow stromal cells (hMSCs) into the injured brain by impregnating them into collagen scaffolds to treat traumatic brain injury (TBI).
Methods
C57BL/6J mice were injured with controlled cortical impact (n = 8) and treated with 0.3 × 106 hMSCs impregnated into three-dimensional porous collagen scaffolds transplanted into the lesion cavity. Additional experimental groups (n = 8 mice/group) were treated with scaffolds implanted alone into the lesion cavity, and hMSCs administered alone intracerebrally or intravenously or saline injected into the lesion core. All treatments were performed 7 days after TBI. Spatial learning was measured using a modified Morris water maze test and brain tissue was processed for histopathological analysis.
Results
The results showed that hMSCs when delivered with scaffolds were more effective than hMSCs administered alone (intravenously or intracerebrally) in improving spatial learning, reducing lesion volume, and increasing vascular density after TBI.
Conclusion
Collagen scaffolds populated with hMSCs may be a new way to reconstruct injured brain and improve neurological function after TBI.
doi:10.3171/2009.4.JNS081681
PMCID: PMC2756329  PMID: 19425888
scaffolds; human marrow stromal cells; lesion volume; traumatic brain injury; collagen
23.  LONG-TERM BENEFITS AFTER TREATMENT OF TRAUMATIC BRAIN INJURY WITH SIMVASTATIN IN RATS 
Neurosurgery  2009;65(1):187-192.
OBJECTIVE
This study was designed to investigate the long-term effects of simvastatin treatment after traumatic brain injury (TBI) in rats.
METHODS
Adult female Wistar rats (n=24) were injured with controlled cortical impact and divided into three groups. The first two groups were treated with simvastatin 0.5 mg/kg or 1 mg/kg administered orally for 14 days starting one day after TBI. The third group (control) received phosphate-buffered saline (PBS) orally for 14 days. Neurological functional outcome was measured with modified neurological severity scores (mNSS) performed 1 day before TBI and after TBI on Days 1, 4, 7, 14 and biweekly thereafter. All animals were sacrificed 3 months after TBI. Brain tissues of half of the animals were processed for preparation of paraffin-embedded sections for immunohistological studies. The remaining half was frozen for ELISA studies for quantification of brain-derived neurotrophic factor (BDNF) in hippocampus and cortex.
RESULTS
Results showed that both doses of simvastatin significantly improved functional outcome compared to control with no difference between the two doses. Simvastatin treatment of 1 mg/kg increased the number of morphologically intact neurons in hippocampus with 0.5 mg/kg having no significant effect. ELISA studies showed that 0.5 mg/kg of simvastatin significantly increased BDNF levels within hippocampus with 1 mg/kg having no significant effect; neither dose had any effect on BDNF levels within the cortex.
CONCLUSION
Simvastatin treatment provides long-lasting functional improvement after TBI in rats. It also enhances neuronal survival in the hippocampus and increases BDNF levels in the hippocampus secondary to simvastatin treatment.
doi:10.1227/01.NEU.0000343540.24780.D6
PMCID: PMC2741617  PMID: 19574841
Simvastatin; Traumatic brain injury; Long term; Newly generated cells
24.  Simvastatin attenuates microglia, astrocyte activation and decreases IL-1β Level following traumatic brain injury 
Neurosurgery  2009;65(1):179-186.
Objective
Our previous studies demonstrated that simvastatin promotes neurological functional recovery after traumatic brain injury (TBI) in rat; however, the underlying mechanisms remain poorly understood. The purpose of this study was to investigate the anti-inflammatory effect of simvastatin by measuring the level of cytokines and activation of glial cells.
Methods
Controlled cortical impact injury was performed in adult male Wistar rats. The rats were randomly divided into three groups: sham, saline control group and simvastatin treatment group. Simvastatin was administered orally starting at day 1 after TBI until sacrifice. Animals were sacrificed at 1, 3, 7, 14, and 35 days after treatment. Functional outcome was measured using modified neurological severity scores (mNSS). ELISA and immunohistochemical staining were employed to measure the expression of IL-1β, IL-6 and TNF-α, and to identify activated microglia and astrocytes.
Results
At days 1 and 3 after simvastatin or saline treatment, cytokine levels in the lesion boundary zone were significantly higher in the simvastatin-treated rats and saline-treated rats compared to the sham group, peaking at day 3. Simvastatin only reduced the level of IL-1 β but not IL-6 and TNF-α compared with the saline group. Also, simvastatin reduced significantly the number of activated microglia and astrocytes compared to the saline control animals. There was also a trend towards improvement of mNSS score, reaching statistical significance (P=0.003) towards the end of the trial.
Conclusion
Our data demonstrate that TBI causes inflammatory reaction, including increased levels of IL-1β, IL-6 and TNF-α, as well as activated microglia. Simvastatin selectively reduces IL-1β expression and inhibits the activation of microglia and astrocytes after TBI, which may be one of the mechanisms underlying the therapeutic benefits of simvastatin treatment of TBI.
doi:10.1227/01.NEU.0000346272.76537.DC
PMCID: PMC2749520  PMID: 19574840
Astrocyte; Interleukin 1 beta; Microglia; Simvastatin; Traumatic brain injury
25.  Delayed transplantation of human marrow stromal cell-seeded scaffolds increases transcallosal neural fiber length, angiogenesis, and hippocampal neuronal survival and improves functional outcome after traumatic brain injury in rats 
Brain research  2009;1263:183-191.
Traumatic brain injury (TBI) is a major cause of death and disability worldwide; however, no effective treatment has been clinically identified. Our recent studies show that the combination of collagen scaffolds with human bone morrow stromal cells (hMSCs) for treatment of TBI improves functional outcome and reduces the lesion volume when this combination was applied at day 4 after TBI in rats. The mechanisms underlying these benefits remain unclear. Whether further delayed treatment with this combination will provide benefits has not been investigated. In the present study, we investigated whether the delayed (7 days post injury) transplantation would have beneficial effects on functional and histological outcome and sought to elucidate underlying mechanisms of therapeutic action. Collagen scaffolds seeded with 3 × 106 hMSCs, scaffolds alone, 3 × 106 hMSCs alone, or saline were transplanted into the lesion cavity of the injured cortex 7 days after TBI. Sensorimotor function and spatial learning were measured. Corticocortical labeling with 1, 1″-dioleyl-3, 3, 3″, 3″-tetramethylindocarbocyanine methanesulfonate (DiI) was performed at day 36 after TBI. The rats were sacrificed 43 days after TBI, and the brain tissue was processed for DiI-labeling fiber and immunohistochemical analyses. The present data show that delayed transplantation of hMSCs or scaffolds seeded with hMSCs improved spatial learning and sensorimotor function, enhanced angiogenesis in the injured cortex and the ipsilateral hippocampus and increased DiI-labeled neural fiber length in the injured cortex. hMSC-seeded scaffolds may be a new and effective way to improve neurological function after TBI.
doi:10.1016/j.brainres.2009.01.032
PMCID: PMC2737675  PMID: 19368838
Angiogenesis; neural fiber length; human bone marrow stromal cell; scaffolds; sensorimotor; spatial learning; traumatic brain injury

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