The MicroRNA Expression Profiles of Human Temporal Lobe Epilepsy in HS ILAE Type 1.

Temporal lobe epilepsy (TLE) is associated with neurodegeneration, often leading to hippocampal sclerosis (HS). Type 1 HS, which is characterized by severe neuronal loss and gliosis predominantly in regions CA1 and CA4, is the most common subtype and is associated with the best prognosis according to the ILAE classification system. MiRNAs participate in the biological processes underlying many nervous system diseases, including epilepsy. However, the miRNA expression profile of HS ILAE type 1 is not completely understood. A total of 14 patients were identified as having the ILAE subtype, as determined by NeuN immunohistochemistry (ILAE type 1 = 7; no-HS = 7). Next-generation sequencing and reverse transcription polymerase chain reaction technology were used to validate the dysregulated miRNAs. Bioinformatics analysis of the predicted target genes was conducted using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses. In total, 1643 mature miRNAs were detected in this study, along with 5 miRNAs that were upregulated and 2 miRNAs that were downregulated in the type 1 group. Bioinformatics analysis showed that 1545 target genes were predicted using the miRDB and Targetscan databases and that these predicted genes showed enrichment in pathways associated with nucleic acid binding, intracellular and cellular macromolecule metabolic processes, and the PI3K-Akt signaling pathway. This study is the first to report the miRNA expression profile of HS ILAE type 1 compared with those of no-HS. These results provide new insights into the neuronal loss pathology of type 1 HS.

Tumor associated CD70 expression is involved in promoting tumor migration and macrophage infiltration in GBM.

Tumor migration/metastasis and immunosuppression are major obstacles in effective cancer therapy. Incidentally, these 2 hurdles usually coexist inside tumors, therefore making therapy significantly more complicated, as both oncogenic mechanisms must be addressed for successful therapeutic intervention. Our recent report highlights that the tumor expression of a TNF family member, CD70, is correlated with poor survival for primary gliomas. In this study, we investigated how CD70 expression by GBM affects the characteristics of tumor cells and the tumor microenvironment. We found that the ablation of CD70 in primary GBM decreased CD44 and SOX2 gene expression, and inhibited tumor migration, growth and the ability to attract monocyte-derived M2 macrophages in vitro. In the tumor microenvironment, CD70 was associated with immune cell infiltrates, such as T cells; myeloid-derived suppressor cells; and monocytes/macrophages based on the RNA-sequencing profile. The CD163+ macrophages were far more abundant than T cells were. This overwhelming level of macrophages was identified only in GBM and not in low-grade gliomas and normal brain specimens, implying their tumor association. CD70 was detected only on tumor cells, not on macrophages, and was highly correlated with CD163 gene expression in primary GBM. Additionally, the co-expression of the CD70 and CD163 genes was found to correlate with decreased survival for patients with primary GBM. Together, these data suggest that CD70 expression is involved in promoting tumor aggressiveness and immunosuppression via tumor-associated macrophage recruitment/activation. Our current efforts to target this molecule using chimeric antigen receptor T cells hold great potential for treating patients with GBM.

miR-656 inhibits glioma tumorigenesis through repression of BMPR1A.

Bone morphogenetic protein-2 (BMP-2), a member of the transforming growth factor-ß family, plays critical roles in cell differentiation, modeling and regeneration processes in several tissues. BMP-2 is also closely associated with various malignant tumors. microRNAs negatively and posttranscriptionally regulate gene expression and function as oncogenes or tumor suppressors. Herein, we report that miR-656 expression was significantly downregulated in glioma cell lines and tissues. We identified and confirmed that BMP receptor, type 1A (BMPR1A) is a direct target of miR-656. The expression of BMPR1A was negatively correlated with that of miR-656 in human glioma tissues. We further demonstrated that miR-656 suppressed glioma cell proliferation, neurosphere formation, migration and invasion with or without exogenous BMP-2. Engineered knockdown of BMPR1A diminished the antiproliferation effect of miR-656 in vitro. Moreover, the canonical BMP/Smad and non-canonical BMP/mitogen-activated protein kinase (MAPK) pathways were inhibited by miR-656 overexpression. Several cancer-related signaling molecules, including cyclin B, cyclin D1, matrix metalloproteinase-9, p21 and p27, were also involved in miR-656 function in glioma cells. The tumor-suppressing function of miR-656 was validated using an in vivo intracranial xenograft mouse model. Notably, ectopic expression of miR-656 markedly reduced tumor size and prolonged the survival of mice treated with or without BMP-2. These results elucidate the function of miR-656 in glioma progression and suggest a promising application for glioma treatment.

Correlations between hippocampal neurogenesis and metabolic indices in adult nonhuman primates.

Increased neurogenesis in feeding centers of the murine hypothalamus is associated with weight loss in diet-induced obese rodents (Kokoeva et al., 2005 and Matrisciano et al., 2010), but this relationship has not been examined in other species. Postmortem hippocampal neurogenesis rates and premortem metabolic parameters were statistically analyzed in 8 chow-fed colony-reared adult bonnet macaques. Dentate gyrus neurogenesis, reflected by the immature neuronal marker, doublecortin (DCX), and expression of the antiapoptotic gene factor, B-cell lymphoma 2 (BCL-2), but not the precursor proliferation mitotic marker, Ki67, was inversely correlated with body weight and crown-rump length. DCX and BCL-2 each correlated positively with blood glucose level and lipid ratio (total cholesterol/high-density lipoprotein). This study demonstrates that markers of dentate gyrus neuroplasticity correlate with metabolic parameters in primates.

Simvastatin-mediated upregulation of VEGF and BDNF, activation of the PI3K/Akt pathway, and increase of neurogenesis are associated with therapeutic improvement after traumatic brain injury.

This study was undertaken to evaluate the effect of simvastatin, a cholesterol-lowering agent, on the Akt-mediated signaling pathway and neurogenesis in the dentate gyrus (DG) of the hippocampus in rats after traumatic brain injury (TBI). Adult male Wistar rats were divided into three groups: (1) sham group (n = 8); (2) saline control group (n = 40); and (3) simvastatin-treated group (n = 40). Controlled cortical impact (CCI) injury was performed over the left parietal lobe. Simvastatin was administered orally at a dose of 1 mg/kg starting at day 1 after TBI and then daily for 14 days. Bromodeoxyuridine (BrdU) was injected intraperitoneally into rats. A modified Morris Water Maze (WM) task was performed between 31 and 35 days after treatment to test spatial memory (n = 8/group). Animals were sacrificed at 1, 3, 7, 14, and 35 days after treatment (n = 8/group/time point). Western blot was utilized to investigate the changes in the Akt-mediated signaling pathway. Enzyme-linked immunosorbent assay (ELISA) analyses were employed to measure vascular endothelial growth factor (VEGF) and brain-derived neurotrophin factor (BDNF) expression. Immunohistochemical and fluorescent staining were performed to detect the BrdU- and neuronal nuclei (NeuN)/BrdU-positive cells. Our data show that simvastatin treatment increases phosphorylation of v-akt murine thymoma viral oncogene homolog (Akt), glycogen synthase kinase-3beta (GSK-3beta), and cAMP response element-binding proteins (CREB); elevates the expression of BDNF and VEGF in the DG; increases cell proliferation and differentiation in the DG; and enhances the recovery of spatial learning. These data suggest that the neurorestorative effect of simvastatin may be mediated through activation of the Akt-mediated signaling pathway, subsequently upregulating expression of growth factors and inducing neurogenesis in the DG of the hippocampus, thereby leading to restoration of cognitive function after TBI in rats.

Long-lasting benefits after treatment of traumatic brain injury (TBI) in rats with combination therapy of marrow stromal cells (MSCs) and simvastatin.

This study was designed to investigate the beneficial effects of combination therapy of simvastatin and marrow stromal cells (MSCs) in improving functional outcome after traumatic brain injury (TBI) in rats. Adult female Wistar rats (n=72 and 8, per group) were injured with controlled cortical impact and treated either with monotherapy of MSCs or simvastatin or a combination therapy of these two agents. Different combination doses were tested, and nine groups of animals were studied. Neurological function was evaluated using Modified Neurological Severity Score (MNSS), and animals were sacrificed 3 months after injury. Coronal brain sections were stained with standard hematoxylin and eosin immunohistochemistry. Our results showed that, though functional improvement was seen with monotherapies of MSCs and simvastatin, the combination therapy when used in optimal doses was significantly better in improving functional outcome. This improvement was long lasting and persisted until the end of the trial (3 months). The optimum combination dose was 0.5mg of simvastatin combined with 2 x 10(6) MSCs. Post mortem analysis showed the presence of donor MSCs within the injured cortex. Endogenous cellular proliferation induced by the neurorestorative treatments was also observed in the lesion boundary zone. Our data show that MSCs and simvastatin have a synergistic effect in improving functional outcome after TBI.

Treatment of traumatic brain injury in mice with marrow stromal cells.

This study was designed to investigate the potential beneficial effects of bone marrow stromal cell (MSC) treatment of traumatic brain injury (TBI) in mice. 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 (0.3x10(6)) intravenously one day after TBI, whereas the control group was injected with saline. MSCs were harvested from male mice, and male to female transplantation was 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. 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 a significant increase in vascular density in the lesion boundary zone and hippocampus of MSC-treated mice compared to control mice. This is the first study to show beneficial effects of MSC treatment after TBI in mice.

Histological and functional outcomes after traumatic brain injury in mice null for the erythropoietin receptor in the central nervous system.

Erythropoietin (EPO) and its receptor (EPOR), essential for erythropoiesis, are expressed in the nervous system. Recombinant human EPO treatment promotes functional outcome after traumatic brain injury (TBI) and stroke, suggesting that the endogenous EPO/EPOR system plays an important role in neuroprotection and neurorestoration. This study was designed to investigate effects of the EPOR on histological and functional outcomes after TBI. Experimental TBI was induced in adult EPOR-null and wild-type mice by controlled cortical impact. Neurological function was assessed using the modified Morris Water Maze and footfault tests. Animals were sacrificed 35 days after injury and brain sections stained for immunohistochemistry. As compared to the wild-type injured mice, EPOR-null mice did not exhibit higher susceptibility to TBI as exemplified by tissue loss in the cortex, cell loss in the dentate gyrus, impaired spatial learning, angiogenesis and cell proliferation. We observed that less cortical neurogenesis occurred and that sensorimotor function (i.e., footfault) was more impaired in the EPOR-null mice after TBI. Co-accumulation of amyloid precursor protein (axonal injury marker) and calcium was observed in the ipsilateral thalamus in both EPOR-null and wild-type mice after TBI with more calcium deposits present in the wild-type mice. This study demonstrates for the first time that EPOR null in the nervous system aggravates sensorimotor deficits, impairs cortical neurogenesis and reduces thalamic calcium precipitation after TBI.

Effects of erythropoietin on reducing brain damage and improving functional outcome after traumatic brain injury in mice.

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) the saline group (TBI and saline [13 mice]); 2) EPO group (TBI and rhEPO [12]); and 3) sham group (sham and rhEPO [8]). Traumatic brain injury 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. Recombinant human erythropoietin 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 killed 35 days after injury, and brain sections were stained for immunohistochemical evaluation. RESULTS: Traumatic brain injury caused tissue loss in the cortex and cell loss in the dentate gyrus (DG) as well as impairment of sensorimotor function (footfault testing) and spatial learning (Morris water maze). Traumatic brain injury alone stimulated cell proliferation and angiogenesis. Compared with saline treatment, rhEPO significantly reduced lesion volume in the cortex and cell loss in the DG after TBI and substantially improved recovery of sensorimotor function and spatial learning performance. It enhanced neurogenesis in the injured cortex and the DG. CONCLUSIONS: Recombinant human erythropoietin initiated 6 hours post-TBI provided neuroprotection by decreasing lesion volume and cell loss as well as neurorestoration by enhancing neurogenesis, subsequently improving sensorimotor and spatial learning function. It is a promising neuroprotective and neurorestorative agent for TBI and warrants further investigation.

Increase in phosphorylation of Akt and its downstream signaling targets and suppression of apoptosis by simvastatin after traumatic brain injury.

OBJECT: In their previous studies, the authors found that simvastatin treatment of traumatic brain injury (TBI) in rats had beneficial effects on spatial learning functions. In the current study they wanted to determine whether simvastatin suppressed neuronal cell apoptosis after TBI, and if so, they wanted to examine the underlying mechanisms of this process. METHODS: Saline or simvastatin (1 mg/kg) was administered orally to rats starting on Day 1 after TBI and then daily for 14 days. Modified Neurological Severity Scores were used to evaluate the sensory motor functional recovery. Rats were killed at 1, 3, 7, 14, and 35 days after treatment, and brain tissue was harvested for terminal deoxynucleotidyl nick-end labeling (TUNEL) staining, caspase-3 activity assay, and Western blot analysis. RESULTS: Simvastatin significantly decreased the modified Neurological Severity Scores 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 Forkhead transcription factor 1, inhibitory-kappaB, and endothelial nitric oxide synthase, which are the downstream targets of the prosurvival 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, Forkhead transcription factor 1 and nuclear factor-kappaB signaling pathways, which suppress the activation of caspase-3 and apoptotic cell death, and thereby, lead to neuronal function recovery after TBI.

Early Life Stress Associated With Increased Striatal N-Acetyl-Aspartate: Cerebrospinal Fluid Corticotropin-Releasing Factor Concentrations, Hippocampal Volume, Body Mass and Behavioral Correlates.

INTRODUCTION: Using proton magnetic resonance spectroscopy imaging (1H-MRSI), the effects of early life stress (ELS) on nonhuman primate striatal neuronal integrity were examined as reflected by N-acetyl aspartate (NAA) concentrations. NAA measures were interrogated through examining their relationship to previously documented ELS markers -- cerebrospinal fluid (CSF) corticotropin-releasing factor (CRF) concentrations, hippocampal volume, body mass and behavioral timidity. Rodent models of depression exhibit increases in neurotrophic effects in the nucleus accumbens (NAcc). We hypothesized that rearing under conditions of ELS [Variable Foraging Demand: (VFD)] would produce persistent elevations of NAA concentrations (in absolute or ratio form) in ventral striatum/caudate nucleus (VS/CN) with altered correlation to ELS markers. METHODS: Eleven bonnet macaque males reared under VFD conditions and seven age-matched control subjects underwent 1H-MRSI during young adulthood. Voxels were placed over ventral striatum/caudate nucleus (VS/CN) to capture NAcc. Cisternal CSF CRF concentrations, hippocampal volume, body mass and response to a human intruder had been previously determined. RESULTS: VFD-reared monkeys exhibited significantly increased NAA/Cr concentrations in right VS/CN in comparison to normally-reared controls, controlling for multiple comparisons. In comparison to controls, VFD CSF CRF concentrations were directly associated with right VS/CN absolute NAA. Left hippocampal volume was inversely associated with left VS/CN N-acetyl aspartate/creatine (NAA/Cr) in VFD-reared but not in controls. Disruption of a normative inverse correlation between left VS/CN NAA and body mass was noted in VFD. Only non-VFD subjects exhibited a direct relationship between timidity response to an intruder and right VS/CN NAA. CONCLUSION: ELS produced persistent increases in VS/CN NAA, which demonstrated specific patterns of association (or lack thereof) to ELS markers in comparison to non-VFD subjects. The data are broadly consistent with a stable nonhuman primate phenotype of anxiety and mood disorder vulnerability whereby in vivo indicators of neuronal integrity, although reduced in hippocampus, are increased in striatum. The findings may provide a catalyst for further studies in humans and other species regarding a reciprocal hippocampal/NAcc relationship in affective disorders.

Targeting of microRNA-21-5p protects against seizure damage in a kainic acid-induced status epilepticus model via PTEN-mTOR.

OBJECTIVE: Studies have shown that microRNAs play a role in the development of epilepsy by regulating downstream target messenger (m)RNA. The present study aims to determine the changes associated with microRNA-21-5p (miR-21-5p) during epileptogenesis in a kainic acid rat model, and to assess whether the PTEN-mTOR pathway is a target of miR-21-5p. METHOD: Reverse transcription polymerase chain reaction (RT-PCR) was used to examine the quantitative expressions of miR-21-5p and PTEN, and Western blotting was used to test the activity of mTOR in the acute, latent, and chronic stages of epileptogenesis. The antagomir of miR-21-5p was injected into the intracerebroventricular space using a microsyringe. Neuronal death and epilepsy discharge were assessed by Nissl staining and electroencephalography (EEG), respectively. The Morris water maze (MWM) was used to assess the cognitive impairment in rats after status epilepticus (SE). RESULTS: Both miR-21-5p and mTOR were upregulated and PTEN was downregulated in rats during acute, latent, and chronic stages of epileptogenesis when compared with those of the control. After using antagomir miR-21-5p in vivo, miR-21-5p and mTOR decreased and the expression of PTEN increased compared with that in the SE model. The silencing of miR-21-5p diminished the number of abnormal spikes on EEG and decreased the number of neuron deletions on Nissl staining. The cognitive and memory impairment caused by epilepsy could also be improved after miR-21-5p knockdown in vivo. CONCLUSION: The results of the present study demonstrate that PTEN-mTOR is the target of miR-21-5p in a kainic acid model of epilepsy. The knockout of miR-21-5p decreases the neuronal damage in stages of epileptogenesis. The miR-21-5p/PTEN/mTOR axis may be a potential target for preventing and treating seizures and epileptic damage.

CD70, a novel target of CAR T-cell therapy for gliomas.

Background: Cancer immunotherapy represents a promising treatment approach for malignant gliomas but is hampered by the limited number of ubiquitously expressed tumor antigens and the profoundly immunosuppressive tumor microenvironment. We identified cluster of differentiation (CD)70 as a novel immunosuppressive ligand and glioma target. Methods: Normal tissues derived from 52 different organs and primary and recurrent low-grade gliomas (LGGs) and glioblastomas (GBMs) were thoroughly evaluated for CD70 gene and protein expression. The association between CD70 and patients' overall survival and its impact on T-cell death was also evaluated. Human and mouse CD70-specific chimeric antigen receptors (CARs) were tested respectively against human primary GBMs and murine glioma lines. The antitumor efficacies of these CARs were also examined in orthotopic xenograft and syngeneic models. Results: CD70 was not detected in peripheral and brain normal tissues but was constitutively overexpressed by isocitrate dehydrogenase (IDH) wild-type primary LGGs and GBMs in the mesenchymal subgroup and recurrent tumors. CD70 was also associated with poor survival in these subgroups, which may link to its direct involvement in glioma chemokine productions and selective induction of CD8+ T-cell death. To explore the potential for therapeutic targeting of this newly identified immunosuppressive axis in GBM tumors, we demonstrate that both human and mouse CD70-specific CAR T cells recognize primary CD70+ GBM tumors in vitro and mediate the regression of established GBM in xenograft and syngeneic models without illicit effect. Conclusion: These studies identify a previously uncharacterized and ubiquitously expressed immunosuppressive ligand CD70 in GBMs that also holds potential for serving as a novel CAR target for cancer immunotherapy in gliomas.

Statins increase neurogenesis in the dentate gyrus, reduce delayed neuronal death in the hippocampal CA3 region, and improve spatial learning in rat after traumatic brain injury.

Traumatic brain injury (TBI) remains a major public health problem globally. Presently, there is no way to restore cognitive deficits caused by TBI. In this study, we seek to evaluate the effect of statins (simvastatin and atorvastatin) on the spatial learning and neurogenesis in rats subjected to controlled cortical impact. Rats were treated with atorvastatin and simvastatin 1 day after TBI and daily for 14 days. Morris water maze tests were performed during weeks 2 and 5 after TBI. Bromodeoxyuridine (BrdU; 50 mg/kg) was intraperitoneally injected 1 day after TBI and daily for 14 days. Brain tissue was processed for immunohistochemical staining to identify newly generated cells and vessels. Our data show that (1) treatment of TBI with statins improves spatial learning on days 31-35 after onset of TBI; (2) in the non-neurogenic region of the hippocampal CA3 region, statin treatment reduces the neuronal loss after TBI, demonstrating the neuroprotective effect of statins; (3) in the neurogenic region of the dentate gyrus, treatment of TBI with statins enhances neurogenesis; (4) statin treatment augments TBI-induced angiogenesis; and (5) treatment with simvastatin at the same dose provides a therapeutic effect superior to treatment with atorvastatin. These results suggest that statins may be candidates for treatment of TBI.

Role of gender in outcome after traumatic brain injury and therapeutic effect of erythropoietin in mice.

The aim of this study was to investigate the role of gender in histological and functional outcome, angiogenesis, neurogenesis and therapeutic effects of recombinant human erythropoietin (rhEPO) in mice after traumatic brain injury (TBI). TBI caused both tissue loss in the cortex and cell loss in the dentate gyrus (DG) in the injured hemisphere at day 35 post TBI without a significant gender difference. After TBI, sensorimotor deficits were significantly larger in male mice compared to females, while similar spatial learning deficits were present in both genders. TBI alone significantly stimulated angiogenesis and neurogenesis in the cortex and in the DG of injured hemispheres in both genders. rhEPO at a dose of 5000 units/kg body weight administered intraperitoneally at 6 h, and 3 and 7 days after injury significantly reduced lesion volume and DG cell loss examined at day 35 after TBI as well as dramatically improved sensorimotor and spatial learning performance without an obvious gender proclivity. rhEPO significantly enhanced neurogenesis in the cortex and the DG of the ipsilateral hemisphere in male TBI mice. rhEPO did not affect angiogenesis in the ipsilateral cortex and DG in both genders after TBI. The present data demonstrate that posttraumatic administration of rhEPO improves histological and functional outcome in both genders, which may be mediated by reducing cortical tissue damage and DG cell loss in the ipsilateral hemisphere. In addition, the major gender propensity observed in the present study with mice after TBI without treatment is limited to sensorimotor deficits and cell proliferation.

Treatment of traumatic brain injury in rats with erythropoietin and carbamylated erythropoietin.

OBJECT: This study was designed to investigate the neuroprotective properties of recombinant erythropoietin (EPO) and carbamylated erythropoietin (CEPO) administered following traumatic brain injury (TBI) in rats. METHODS: Sixty adult male Wistar rats were injured with controlled cortical impact, and then EPO, CEPO, or a placebo (phosphate-buffered saline) was injected intraperitoneally. These injections were performed either 6 or 24 hours after TBI. To label newly regenerating cells, bromodeoxyuridine was injected intraperitoneally for 14 days after TBI. Blood samples were obtained on Days 1, 2, 3, 7, 14, and 35 to measure hematocrit. Spatial learning was tested using the Morris water maze. All rats were killed 35 days after TBI. Brain sections were immunostained as well as processed for the enzyme-linked immunosorbent assay to measure brain-derived neurotrophic factor (BDNF). RESULTS: A statistically significant improvement in spatial learning was seen in rats treated with either EPO or CEPO 6 or 24 hours after TBI (p < 0.05); there was no difference in the effects of EPO and CEPO. Also, these drugs were equally effective in increasing the number of newly proliferating cells within the dentate gyrus at both time points. A statistically significant increase in BDNF expression was seen in animals treated with both EPO derivatives at 6 or 24 hours after TBI. Systemic hematocrit was significantly increased at 48 hours and 1 and 2 weeks after treatment with EPO but not with CEPO. CONCLUSIONS: These data demonstrate that at the doses used, EPO and CEPO are equally effective in enhancing spatial learning and promoting neural plasticity after TBI.

Long-term recovery after bone marrow stromal cell treatment of traumatic brain injury in rats.

OBJECT: This study was designed to follow the effects of bone marrow stromal cell (BMSC) administration in rats after traumatic brain injury (TBI) for a 3-month period. METHODS: Forty adult female Wistar rats were injured by a controlled cortical impact and, 1 week later, were injected intravenously with one of three different doses of BMSCs (2 x 10(6), 4 x 10(6), or 8 x 10(6) cells per animal) obtained in male rats. Control rats received phosphate-buffered saline (PBS). Neurological function in these rats was studied using a neurological severity scale (NSS). The rats were killed 3 months after injury, and immunohistochemical stains were applied to brain samples to study the distribution of the BMSCs. Additional brain samples were analyzed by quantitative enzyme-linked immunosorbent assays to measure the expression of the growth factors brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF). Three months after injury, BMSCs were present in the injured brain and their number was significantly greater in animals that received 4 x 10(6) or 8 x 10(6) BMSCs than in animals that received 2 x 10(6) BMSCs. The cells were primarily distributed around the lesion boundary zone. Functional outcome was significantly better in rats that received 4 x 10(6) or 8 x 10(6) BMSCs, compared with control animals, although no improvement was seen in animals that received 2 x 10(6) BMSCs. All doses of BMSCs significantly increased the expression of BDNF but not that of NGF; however, this increase was significantly larger in animals that received 4 x 10(6) or 8 x 10(6) BMSCs than in controls or animals that received 2 x 10(6) BMSCs. CONCLUSIONS: In summary, when injected in rats after TBI, BMSCs are present in the brain 3 months later and significantly improve functional outcome.

Erythropoietin enhances neurogenesis and restores spatial memory in rats after traumatic brain injury.

Erythropoietin (EPO) is neuroprotective in models of stroke and traumatic brain injury (TBI) when administered prior to or within the first few hours after injury. We seek to demonstrate that EPO also has neurorestorative effects when administered late (i.e., 1 day) after TBI in the rat. Twelve rats were subjected to TBI. Six rats were treated with EPO daily for 14 days starting 1 day after injury, and an additional six rats were treated with saline. Bromodeoxyuridine (BrdU) was administered daily for 14 days. Memory tests using a Morris Water Maze were performed prior to and after injury and treatment. Animals were sacrificed at 15 days after TBI, and their brains were prepared for histological analysis of damage to the dentate gyrus (DG) and for evaluation of newly formed neurons using double labeling of BrdU and MAP-2. The data revealed a significant improvement in spatial memory and significant increase in the number of newly formed neurons with EPO treatment compared with control animals. These data suggest that EPO treatment initiated 1 day after TBI is neurorestorative by enhancing neurogenesis, as well as neuroprotective.

Effect of atorvastatin on spatial memory, neuronal survival, and vascular density in female rats after traumatic brain injury.

OBJECT: Atorvastatin administered after traumatic brain injury (TBI) induced by controlled cortical impact promotes functional improvement in male rats. Note, however, that parallel studies have not been performed in female rats. Therefore, the authors tested the effect of atorvastatin on TBI in female rats. METHODS: Atorvastatin (1 mg/kg/day) was orally administered for 7 consecutive days in female Wistar rats starting I day after TBI; control animals received saline. Modified neurological severity scores, the corner turn test, and the Morris water maze test were used to evaluate functional response to treatment. Rats were killed on Day 15 post-TBI, and brain tissue samples were processed for immunohistochemical staining. Atorvastatin administration after brain injury significantly promoted the restoration of spatial memory but did not reduce sensorimotor functional deficits. Treatment of TBI with atorvastatin increased neuronal survival in the CA3 region and the lesion boundary zone and prevented the loss of neuronal processes of damaged neurons in the hippocampal CA3 region but not in the lesion boundary zone on Day 15 after TBI. The protective effect of atorvastatin on the injured neurons perhaps is mediated by increasing the density of vessels in the lesion boundary zone and the hippocampus after TBI. CONCLUSIONS: . These data indicate that atorvastatin is beneficial in the treatment of TBI in female rats, although the effect may differ between sexes.

Intravenous administration of marrow stromal cells (MSCs) increases the expression of growth factors in rat brain after traumatic brain injury.

This study was designed to investigate the effects of intravenous administration of marrow stromal cells (MSCs) on the expression of growth factors in rat brain after traumatic brain injury (TBI). The fate of transplanted MSCs and expression of growth factors was examined by immunohistochemistry. In addition, the level of growth factors was measured quantitatively using enzyme linked immunosorbent assay (ELISA). Growth factors that were studied included nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and basic fibroblast growth factor (bFGF). For immunohistochemical studies, 12 male Wistar rats were subjected to TBI and then divided into three groups with the first group receiving no treatment, the second group receiving saline (placebo) and the third group receiving MSCs intravenously 1 day after TBI. The neurological function of rats was studied by using Rotarod motor test and modified neurological severity scores. The animals were sacrificed 15 days after TBI and brain sections stained by immunohistochemistry to study the distribution of MSCs as well as expression of growth factors NGF, BDNF, and bFGF. For quantitative analysis, a second set of male Wistar rats (n = 18) was subjected to TBI and then injected with either saline (n = 9) or MSCs (n = 9) 1 day after injury. These rats were sacrificed on days 2, 5, and 8 after TBI and brain extracts used to measure NGF, BDNF, and bFGF. We found that after transplantation, MSCs preferentially migrated into the injured hemisphere and there was a statistically significant improvement in the functional outcome of MSC-treated rats compared to control rats. NGF, BDNF, and bFGF were expressed in the injured brain of both treated as well as control rats; however, quantitative ELISA studies showed that expression of NGF and BDNF was significantly increased (p < 0.05) in the treated group. This study shows that intravenous administration of MSCs after TBI increases the expression of growth factors (NGF, BDNF), which possibly contributes to the improvement in functional outcome seen in these rats.