Changsheng Qu

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.

Collagen scaffolds populated with human marrow stromal cells reduce lesion volume and improve functional outcome after traumatic brain injury.

OBJECTIVETraumatic brain injury (TBI) causes extensive loss of cerebral parenchyma; however, no strategy for reconstruction has been clinically effective. Our group and others have used human marrow stromal cells (hMSCs) to treat rats subjected to TBI and found no significant changes in the lesion volume, although functional outcome was improved significantly. To identify new ways of delivering hMSCs into the injured brain and to maximize the therapeutic benefits of hMSC treatment, we investigated the use of collagen scaffolds implanted with hMSCs as a cell delivery system for treatment of TBI. METHODSCollagen scaffolds populated with 3 × 106 hMSCs were transplanted into the lesion cavity of the injured cortex 4 days after TBI, and the rats were euthanized 35 days after TBI. We measured sensorimotor function and spatial learning using an array of function tests, and the brain tissue was processed for histopathology analysis. CONCLUSIONThe data show that scaffolds populated by hMSCs improve spatial learning and sensorimotor function, reduce the lesion volume, and foster the migration of hMSCs into the lesion boundary zone after TBI in rats. hMSC-populated scaffolds may be a new way to reconstruct the injured brain and improve neurological function after TBI.

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.

Human Marrow Stromal Cell Treatment Provides Long-Lasting Benefit After Traumatic Brain Injury In Rats

OBJECTIVE:This study was designed to investigate the effects of human bone marrow stromal cell (hMSC) administration in rats for 3 months after traumatic brain injury (TBI). METHODS:Adult male Wistar rats (n = 60) were injured with controlled cortical impact and divided into four groups. The three treatment groups (n = 10 each) were injected with 2 × 106, 4 × 106, and 8 × 106 hMSCs, respectively, intravenously, whereas the control group (n = 30) received phosphate-buffered saline. All injections were performed 1 day after injury into the tail veins of rats. Neurological functional evaluation of animals was performed before and after injury by use of Neurological Severity Scores. Animals were sacrificed 3 months after TBI, and brain sections were stained by immunohistochemistry. RESULTS:Statistically significant improvement in functional outcome was observed in all three treatment groups compared with control values (P < 0.05). This benefit was visible 14 days after TBI and persisted until 3 months (end of trial). There was no difference in functional outcome among the three treatment groups. Histological analysis showed that hMSCs were present in the lesion boundary zone at 3 months with all three doses tested. CONCLUSION:hMSCs injected in rats after TBI survive until 3 months and provide long-lasting functional benefit. Functional improvement may be attributed to stimulation of endogenous neurorestorative functions such as neurogenesis and synaptogenesis.

SIMVASTATIN ATTENUATES MICROGLIAL CELLS AND ASTROCYTE ACTIVATION AND DECREASES INTERLEUKIN-1B LEVEL AFTER TRAUMATIC BRAIN INJURY

OBJECTIVEOur 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. METHODSControlled cortical impact injury was performed in adult male Wistar rats. The rats were randomly divided into 3 groups: sham, saline control group, and simvastatin treatment group. Simvastatin was administered orally starting at day 1 after TBI until animals were killed at days 1, 3, 7, 14, and 35 after treatment. Functional outcome was measured using modified neurological severity scores. Enzyme-linked immunosorbent assay and immunohistochemical staining were used to measure the expression of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α and to identify activated microglial cells and astrocytes. RESULTSAt days 1 and 3 after simvastatin or saline treatment, cytokine levels in the lesion boundary zone were significantly higher in the simvastatin- and saline-treated rats compared with the sham group, peaking at day 3. Simvastatin only reduced the level of IL-1β but not IL-6 and tumor necrosis factor-α, compared with the saline group. Also, simvastatin significantly reduced the number of activated microglial cells and astrocytes compared with the saline control animals. There was also a trend toward improvement of modified neurological severity score, reaching statistical significance (P = 0.003) toward the end of the trial. CONCLUSIONOur data demonstrate that TBI causes inflammatory reaction, including increased levels of IL-1β, IL-6, and tumor necrosis factor-α, as well as activated microglial cells. Simvastatin selectively reduces IL-1β expression and inhibits the activation of microglial cells and astrocytes after TBI, which might be one of the mechanisms underlying the therapeutic benefits of simvastatin treatment of 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.

Delayed administration of erythropoietin reducing hippocampal cell loss, enhancing angiogenesis and neurogenesis, and improving functional outcome following traumatic brain injury in rats: comparison of treatment with single and triple dose.

OBJECT This efficacy study was designed to investigate traumatic brain injury (TBI) in rats treated with delayed erythropoietin (EPO) administered in a single dose compared with a triple dose. METHODS Young adult male Wistar rats were randomly divided into the following groups: 1) sham group (6 animals); 2) TBI/saline group (6 animals); 3) TBI/EPOx1 group (6 animals); and 4) TBI/EPOx3 group (7 animals). Traumatic brain injury was induced by controlled cortical impact over the left parietal cortex. Erythropoietin (5000 U/kg) or saline was administered intraperitoneally on Day 1 (EPOx1 group) or on Days 1, 2, and 3 (EPOx3 group) postinjury. Neurological function was assessed using a modified neurological severity score, foot-fault, and Morris water maze tests. Animals were killed 35 days after injury and brain sections were stained for immunohistochemistry. RESULTS Compared with 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 improved histological and long-term functional outcomes in rats after TBI. The triple doses of delayed EPO treatment produced better histological and functional outcomes in rats, although a single dose provided substantial benefits compared with saline treatment.

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.

Treatment of traumatic brain injury with a combination therapy of marrow stromal cells and atorvastatin in rats.

OBJECTIVEThis study investigated the effects of a combination therapy of marrow stromal cells (MSCs) and statins (atorvastatin) after traumatic brain injury in rats. METHODSThirty-two female Wistar rats were injured by controlled cortical impact and divided into four groups. Group I was injected with MSCs (1 × 106) intravenously 24 hrs after traumatic brain injury. Group II was administered atorvastatin (0.5 mg/kg) orally for 14 days starting 24 hours after traumatic brain injury. Group III received MSCs (1 × 106) combined with atorvastatin (0.5 mg/kg). Group IV (control) was injected with saline. MSCs were harvested from the bone marrow of male rats to identify male donor cells within female recipient animals by localization of Y chromosomes. Functional analysis was performed using modified neurological severity scores and the Morris water maze test. Animals were sacrificed 35 days after injury and brain sections stained with immunohistochemistry. RESULTSNo functional improvement was seen in animals treated with MSCs or atorvastatin alone (Groups I and II). However, functional improvement was seen with both testing modalities (modified neurological severity scores and Morris water maze) in animals receiving combination therapy (Group III). Microscopic analysis showed that significantly more MSCs were present in animals receiving combination therapy than in those receiving MSCs alone. Also, significantly more endogenous cellular proliferation was seen in the hippocampus and injury boundary zone of the combination therapy group than in the monotherapy or control groups. CONCLUSIONWhen administered in combination with MSCs, atorvastatin increases MSC access and/or survival within the injured brain and enhances functional recovery compared with monotherapy.

Therapeutic effects of erythropoietin on histological and functional outcomes following traumatic brain injury in rats are independent of hematocrit

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 four 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 postinjury. 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 were 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.