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1.  Simvastatin Attenuates Astrogliosis after Traumatic Brain Injury through the Modulation of EGFR in Lipid Rafts 
Journal of neurosurgery  2010;113(3):591-597.
Objective
Our previous studies demonstrated that simvastatin treatment promotes neuronal survival and reduces inflammatory cytokine release from astrocytes after traumatic brain injury (TBI) in rats. Since reactive astrocytes produce inflammation mediators, in the current study we investigated the effect of simvastatin on astrocyte activation after TBI and its underlying signaling mechanisms.
Methods
Saline or simvastatin (1 mg/kg) was orally administered to rats starting at Day 1 after TBI and then daily for 14 days. Rats were sacrificed at 1, 3, 7, 14 days after treatment. Brain sections and tissues were prepared for immunohistochemical staining and Western blot analysis, respectively. Cultured astrocytes were subjected to oxygen-glucose deprivation (OGD) and followed by immunocytochemical staining with GFAP/caveolin-1 and Western blot analysis. Lipid rafts were isolated from the cell lysate and Western blot was carried out to detect the changes in epidermal growth factor receptor (EGFR) expression and phosphorylation in the lipid rafts.
Results
Simvastatin significantly promoted neuronal survival after TBI and attenuated activation of astrocytes. Simvastatin modified the caveolin-1 expression in lipid rafts in astrocyte cell membrane, suppressed the phosphorylation of EGFR in lipid rafts of astrocytes after OGD, and inhibited the OGD-induced interleukin-1 (IL-1) production.
Conclusions
These data suggest that simvastatin reduces reactive astrogliosis and rescues neuronal cells after TBI. These beneficial effects of simvastatin may be mediated by inhibiting astrocyte activation after TBI through modifying the caveolin-1 expression in lipid rafts and the subsequent modulation of EGFR phosphorylation in lipid rafts.
doi:10.3171/2009.9.JNS09859
PMCID: PMC3007601  PMID: 19895202
simvastatin; EGFR; lipid rafts; astrocyte; traumatic brain injury
2.  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
3.  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
4.  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
5.  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 
Journal of neurotrauma  2007;24(7):1132-1146.
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.
doi:10.1089/neu.2007.0288
PMCID: PMC1971229  PMID: 17610353
neurogenesis; rat; spatial learning; statins
6.  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
7.  Simvastatin Combined with Antioxidant Attenuates the Cerebral Vascular Endothelial Inflammatory Response in a Rat Traumatic Brain Injury 
BioMed Research International  2014;2014:910260.
Traumatic brain injury (TBI) leads to important and deleterious neuroinflammation, as evidenced by indicators such as edema, cytokine production, induction of nitric oxide synthase, and leukocyte infiltration. After TBI, cerebral vascular endothelial cells play a crucial role in the pathogenesis of inflammation. In our previous study, we proved that simvastatin could attenuate cerebral vascular endothelial inflammatory response in a rat traumatic brain injury. This purpose of this study was to determine whether simvastatin combined with an antioxidant could produce the same effect or greater and to examine affected surrogate biomarkers for the neuroinflammation after traumatic brain injury in rat. In our study, cortical contusions were induced, and the effect of acute and continuous treatment of simvastatin and vitamin C on behavior and inflammation in adult rats following experimental TBI was evaluated. The results demonstrated that simvastatin combined with an antioxidant could provide neuroprotection and it may be attributed to a dampening of cerebral vascular endothelial inflammatory response.
doi:10.1155/2014/910260
PMCID: PMC4071852
8.  Long-Lasting Benefits after Treatment of Traumatic Brain Injury (TBI) in Rats with Combination Therapy of Marrow Stromal Cells (MSCs) and Simvastatin 
Journal of neurotrauma  2008;25(12):1441-1447.
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 0.5 mg of simvastatin combined with 2 × 106 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.
doi:10.1089/neu.2007.0495
PMCID: PMC2719878  PMID: 19072586
combination therapy; marrow stromal cells (MSCs); simvastatin; traumatic brain injury (TBI)
9.  Long-Lasting Benefits after Treatment of Traumatic Brain Injury (TBI) in Rats with Combination Therapy of Marrow Stromal Cells (MSCs) and Simvastatin 
Journal of Neurotrauma  2008;25(12):1441-1447.
Abstract
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 0.5 mg of simvastatin combined with 2×106 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.
doi:10.1089/neu.2007.0495
PMCID: PMC2719878  PMID: 19072586
combination therapy; marrow stromal cells (MSCs); simvastatin; traumatic brain injury (TBI)
10.  Increasing Ang1/Tie2 expression by simvastatin treatment induces vascular stabilization and neuroblast migration after stroke 
In this study, we tested the hypothesis that the Angiopoietin 1 (Ang1)/Tie2 pathway mediates simvastatin-induced vascular integrity and migration of neuroblasts after stroke. Rats were subjected to 2 hrs of middle cerebral artery occlusion (MCAo) and treated, starting 1 day after stroke with or without simvastatin (1 mg/kg, daily) for 7 days. Simvastatin treatment significantly decreased blood–brain barrier (BBB) leakage and concomitantly, increased Ang1, Tie2 and Occludin expression in the ischaemic border (IBZ) compared to the MCAo control group. Simvastatin also significantly increased doublecortin (DCX, a marker of migrating neuroblasts) expression in the IBZ compared to control MCAo rats. DCX was highly expressed around vessels. To further investigate the signalling pathway of simvastatin-induced vascular stabilization and angiogenesis, rat brain microvascular endothelial cell (RBMEC) culture was employed. The data show that simvastatin treatment of RBMEC increased Ang1 and Tie2 gene and protein expression and promoted phosphorylated-Tie2 activity. Simvastatin significantly increased endothelial capillary tube formation, an index of angiogenesis, compared to non-treated control. Inhibition of Ang1 or knockdown of Tie2 gene expression in endothelial cells significantly attenuated simvastatin-induced capillary tube formation. In addition, simvastatin significantly increased subventricular zone (SVZ) explant cell migration compared to non-treatment control. Inhibition of Ang1 significantly attenuated simvastatin-induced SVZ cell migration. Simvastatin treatment of stroke increases Ang1/Tie2 expression and thereby reduces BBB leakage and promotes vascular stabilization. Ang1/Tie2 expression induced by simvastatin treatment promotes neuroblast microvascular coupling after stroke.
doi:10.1111/j.1582-4934.2008.00380.x
PMCID: PMC3710660  PMID: 18544044
stroke; angiopoietin 1; Tie2; simvastatin; vascular stabilization
11.  Statins Protect the Blood Brain Barrier Acutely after Experimental Intracerebral Hemorrhage 
Objectives
The goal of this study was to measure the impact of simvastatin and atorvastatin treatment on blood brain barrier (BBB) integrity after experimental intracerebral hemorrhage (ICH).
Methods
Primary ICH was induced in 27 male Wistar rats by stereotactic injection of 100 µL of autologous blood into the striatum. Rats were divided into three groups (n= 9/group): 1) oral treatment (2 mg/kg) of atorvastatin, 2) oral treatment (2 mg/kg) simvastatin, or 3) phosphate buffered saline daily starting 24-hours post-ICH and continuing daily for the next 3 days. On the fourth day, the animals underwent magnetic resonance imaging (MRI) for measurements of T1sat (a marker for BBB integrity), T2 (edema), and cerebral blood flow (CBF). After MRI, the animals were sacrificed and immunohistology or Western blotting was performed.
Results
MRI data for animals receiving simvastatin treatment showed significantly reduced BBB dysfunction and improved CBF in the ICH rim compared to controls (P<0.05) 4 days after ICH. Simvastatin also significantly reduced edema (T2) in the rim at 4 days after ICH (P<0.05). Both statin-treated groups demonstrated increased occludin and endothelial barrier antigen levels within the vessel walls, indicating better preservation of BBB function (P<0.05) and increased number of blood vessels (P<0.05).
Conclusions
Simvastatin treatment administered acutely after ICH protects BBB integrity as measured by MRI and correlative immunohistochemistry. There was also evidence of improved CBF and reduced edema by MRI. Conversely, atorvastatin showed a non-significant trend by MRI measurement.
PMCID: PMC3583226  PMID: 23459792
Intracerebral hemorrhage; Atorvastatin; Occludin; Simvastatin; Blood brain barrier
12.  Prior Use of 3-Hydroxy-3-Methyl-Glutaryl-Coenzyme A Reductase Inhibitor, Simvastatin Fails to Improve Outcome after Experimental Intracerebral Hemorrhage 
Objective
Contrary to some clinical belief, there were quite a few studies regarding animal models of intracerebral hemorrhage (ICH) in vivo suggesting that prior use of statins may improve outcome after ICH. This study reports the effect of 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG CoA) reductase inhibitor, simvastatin given before experimental ICH.
Methods
Fifty-one rats were subjected to collagenase-induced ICH, subdivided in 3 groups according to simvastatin treatment modality, and behavioral tests were done. Hematoma volume, brain water content and hemispheric atrophy were analyzed. Immunohistochemical staining for microglia (OX-42) and endothelial nitric oxide synthase (eNOS) was performed and caspase-3 activity was also measured.
Results
Pre-simvastatin therapy decreased inflammatory reaction and perihematomal cell death, but resulted in no significant reduction of brain edema and no eNOS expression in the perihematomal region. Finally, prior use of simvastatin showed less significant improvement of neurological outcome after experimental ICH when compared to post-simvastatin therapy.
Conclusion
The present study suggests that statins therapy after ICH improves neurological outcome, but prior use of statins before ICH might provide only histological improvement, providing no significant impact on neurological outcome against ICH.
doi:10.3340/jkns.2011.50.5.403
PMCID: PMC3259458  PMID: 22259685
Inflammation; Intracerebral hemorrhage; Neuroprotection; Outcome; Rat; Statins
13.  Augmentation of neovascularization in murine hindlimb ischemia by combined therapy with simvastatin and bone marrow-derived mesenchymal stem cells transplantation 
Objectives
We postulated that combining high-dose simvastatin with bone marrow derived-mesenchymal stem cells (MSCs) delivery may give better prognosis in a mouse hindlimb ischemia model.
Methods
Mouse hindlimb ischemia model was established by ligating the right femoral artery. Animals were grouped (n = 10) to receive local injection of saline without cells (control and simvastatin groups) or with 5 × 106 MSCs (MSCs group).Animals received either simvastatin (20 mg/kg/d, simvastatin and combination groups) or saline(control and MSCs group) gavages for continual 21 days. The blood flow was assessed by laser Doppler imaging at day 0,10 and 21 after surgery, respectively. Ischemic muscle was harvested for immunohistological assessments and for VEGF protein detection using western blot assay at 21 days post-surgery. In vitro, MSCs viability was measured by MTT and flow cytometry following culture in serum-free medium for 24 h with or without simvastatin. Release of VEGF by MSCs incubated with different doses of simvastatin was assayed using ELISA.
Results
Combined treatment with simvastatin and MSCs induced a significant improvement in blood reperfusion, a notable increase in capillary density, a highest level of VEGF protein and a significant decrease in muscle cell apoptosis compared with other groups. In vitro, simvastatin inhibited MSCs apoptosis and increased VEGF release by MSCs.
Conclusions
Combination therapy with high-dose simvastatin and bone marrow-derived MSCs would augment functional neovascularization in a mouse model of hindlimb ischemia.
doi:10.1186/1423-0127-17-75
PMCID: PMC2946286  PMID: 20846454
14.  Improvement of Retinal Vascular Injury in Diabetic Rats by Statins Is Associated With the Inhibition of Mitochondrial Reactive Oxygen Species Pathway Mediated by Peroxisome Proliferator–Activated Receptor γ Coactivator 1α 
Diabetes  2010;59(9):2315-2325.
OBJECTIVE
Mitochondrial reactive oxygen species (ROS) plays a key role in diabetic retinopathy (DR) pathogenesis. However, whether simvastatin decreases diabetes-induced mitochondrial ROS production remains uncertain. The aim of this study was to clarify the beneficial effects and mechanism of action of simvastatin against diabetes-induced retinal vascular damage.
RESEARCH DESIGN AND METHODS
Diabetic rats and control animals were randomly assigned to receive simvastatin or vehicle for 24 weeks, and bovine retinal capillary endothelial cells (BRECs) were incubated with normal or high glucose with or without simvastatin. Vascular endothelial growth factor (VEGF) and peroxisome proliferator–activated receptor γ coactivator 1α (PGC-1α) in the rat retinas or BRECs were examined by Western blotting and real-time RT-PCR, and poly (ADP-ribose) polymerase (PARP), and p38 MAPK were examined by Western blotting. Mitochondrial membrane potential (Δψm) and ROS production were assayed using the potentiometric dye 5,5′,6,6′- Tetrachloro1,1′,3,3′-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1) or CM-H2DCFDA fluorescent probes.
RESULTS
Simvastatin significantly upregulated PGC-1α (P < 0.01), subsequently decreased Δψm (P < 0.05) and ROS generation (P < 0.01), inhibited PARP activation (P < 0.01), and further reduced VEGF expression (P < 0.01) and p38 MAPK activity (P < 0.01). Those changes were associated with the decrease of retinal vascular permeability, retinal capillary cells apoptosis, and formation of acellular capillaries.
CONCLUSIONS
Simvastatin decreases diabetes-induced mitochondrial ROS production and exerts protective effects against early retinal vascular damage in diabetic rats in association with the inhibition of mitochondrial ROS/PARP pathway mediated by PGC-1α. The understanding of the mechanisms of action of statins has important implications in the prevention and treatment of mitochondrial oxidative stress-related illness such as DR.
doi:10.2337/db10-0638
PMCID: PMC2927955  PMID: 20566666
15.  Simvastatin Blocks Blood-Brain Barrier Disruptions Induced by Elevated Cholesterol Both In Vivo and In Vitro 
Background. Hypercholesterolemia and disruptions of the blood brain barrier (BBB) have been implicated as underlying mechanisms in the pathogenesis of Alzheimer's disease (AD). Simvastatin therapy may be of benefit in treating AD; however, its mechanism has not been yet fully understood. Objective. To explore whether simvastatin could block disruption of BBB induced by cholesterol both in vivo and in vitro. Methods. New Zealand rabbits were fed cholesterol-enriched diet with or without simvastatin. Total cholesterol of serum and brain was measured. BBB dysfunction was evaluated. To further test the results in vivo, rat brain microvascular endothelial cells (RBMECs) were stimulated with cholesterol in the presence/absence of simvastatin in vitro. BBB disruption was evaluated. Results. Simvastatin blocked cholesterol-rich diet induced leakage of Evan's blue dye. Cholesterol content in the serum was affected by simvastatin, but not brain cholesterol. Simvastatin blocked high-cholesterol medium-induced decrease in TEER and increase in transendothelial FITC-labeled BSA Passage in RBMECs. Conclusions. The present study firstly shows that simvastatin improves disturbed BBB function both in vivo and in vitro. Our data provide that simvastatin may be useful for attenuating disturbed BBB mediated by hypercholesterolemia.
doi:10.1155/2012/109324
PMCID: PMC3296225  PMID: 22506129
16.  Expression of S100A6 in Rat Hippocampus after Traumatic Brain Injury Due to Lateral Head Acceleration 
In a rat model of traumatic brain injury (TBI), we investigated changes in cognitive function and S100A6 expression in the hippocampus. TBI-associated changes in this protein have not previously been reported. Rat S100A6 was studied via immunohistochemical staining, Western blot, and reverse transcription-polymerase chain reaction (RT-PCR) after either lateral head acceleration or sham. Reduced levels of S100A6 protein and mRNA were observed 1 h after TBI, followed by gradual increases over 6, 12, 24, and 72 h, and then a return to sham level at 14 day. Morris water maze (MWM) test was used to evaluate animal spatial cognition. TBI- and sham-rats showed an apparent learning curve, expressed as escape latency. Although TBI-rats displayed a relatively poorer cognitive ability than sham-rats, the disparity was not significant early post-injury. Marked cognitive deficits in TBI-rats were observed at 72 h post-injury compared with sham animals. TBI-rats showed decreased times in platform crossing in the daily MWM test; the performance at 72 h post-injury was the worst. In conclusion, a reduction in S100A6 may be one of the early events that lead to secondary cognitive decline after TBI, and its subsequent elevation is tightly linked with cognitive improvement. S100A6 may play important roles in neuronal degeneration and regeneration in TBI.
doi:10.3390/ijms15046378
PMCID: PMC4013634  PMID: 24739809
S100A6; hippocampus; cognitive deficits; traumatic brain injury (TBI); rats
17.  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
18.  Simvastatin Increases Notch Signaling Activity and Promotes Arteriogenesis After Stroke 
Background and Purpose
Notch signaling activity regulates arteriogenesis. Presenilin 1 (PS1) mediates Notch signaling activity via cleavage of Notch, liberating Notch intracellular domain (NICD). We tested the hypothesis that simvastatin enhances arteriogenesis after stroke by increasing PS1 activation of the Notch signaling pathway.
Methods
Rats were subjected to middle cerebral artery occlusion (MCAo) and treated with or without simvastatin (1 mg/kg) starting 24 hours after stroke and daily for 7 days; they were euthanized 14 days after stroke. Immunostaining, Western blot, and real-time polymerase chain reaction assays were performed.
Results
Simvastatin significantly increased arterial diameter, density, and vascular smooth muscle cell proliferation, and upregulated PS1, Notch1, and NICD expression in the ischemic border tissue and in the cerebral arteries compared with MCAo control rats, respectively. However, simvastatin did not increase arteriogenesis, PS1, and NICD expression in sham control animals. To investigate the mechanisms of simvastatin-induced arteriogenesis, primary cerebral artery cultures were used. Rats were subjected to MCAo and treated with or without simvastatin daily for 7 days. The cerebral arteries derived from these stroke rats were cultured in matrigel and treated with or without a γ40-secretase inhibitor II, which blocks Notch signaling activity, inhibiting NICD production. Arterial cell migration was measured. simvastatin treatment significantly increased arterial cell migration compared to control MCAo artery, whereas inhibition of Notch signaling activity by the γ40-secretase inhibitor II significantly attenuated simvastatin-induced arterial cell migration.
Conclusions
These data indicate that simvastatin increases arteriogenesis after stroke, and that simvastatin upregulation of PS1 expression and Notch signaling activity may facilitate an increase in arteriogenesis.
doi:10.1161/STROKEAHA.108.524116
PMCID: PMC2804086  PMID: 18927449
arteriogenesis; Notch signaling; presenilin 1; simvastatin; stroke
19.  Administration of Simvastatin after Kainic Acid-Induced Status Epilepticus Restrains Chronic Temporal Lobe Epilepsy 
PLoS ONE  2011;6(9):e24966.
In this study, we examined the effect of chronic administration of simvastatin immediately after status epilepticus (SE) on rat brain with temporal lobe epilepsy (TLE). First, we evaluated cytokines expression at 3 days post KA-lesion in hippocampus and found that simvastatin-treatment suppressed lesion-induced expression of interleukin (IL)-1β and tumor necrosis factor-α (TNF-α). Further, we quantified reactive astrocytosis using glial fibrillary acidic protein (GFAP) staining and neuron loss using Nissl staining in hippocampus at 4–6 months after KA-lesion. We found that simvastatin suppressed reactive astrocytosis demonstrated by a significant decrease in GFAP-positive cells, and attenuated loss of pyramidal neurons in CA3 and interneurons in dentate hilar (DH). We next assessed aberrant mossy fiber sprouting (MFS) that is known to contribute to recurrence of spontaneous seizure in epileptic brain. In contrast to the robust MFS observed in saline-treated animals, the extent of MFS was restrained by simvastatin in epileptic rats. Attenuated MFS was related to decreased neuronal loss in CA3 and DH, which is possibly a mechanism underlying decreased hippocampal susceptibility in animal treated with simvastatin. Electronic encephalography (EEG) was recorded during 4 to 6 months after KA-lesion. The frequency of abnormal spikes in rats with simvastatin-treatment decreased significantly compared to the saline group. In summary, simvastatin treatment suppressed cytokines expression and reactive astrocytosis and decreased the frequency of discharges of epileptic brain, which might be due to the inhibition of MFS in DH. Our study suggests that simvastatin administration might be a possible intervention and promising strategy for preventing SE exacerbating to chronic epilepsy.
doi:10.1371/journal.pone.0024966
PMCID: PMC3176286  PMID: 21949812
20.  Mechanically induced experimental knee osteoarthritis benefits from anti-inflammatory and immunomodulatory properties of simvastatin via inhibition of matrix metalloproteinase-3 
Background
We investigated the anti-inflammatory and immunomodulatory effect of simvastatin on articular cartilage via the inhibition of matrix metalloproteinase-3 (MMP-3), a matrix-degrading enzyme, in a mechanically induced experimental osteoarthritis (OA) animal model.
Materials and methods
Twenty-seven albino Wistar rats were divided in three groups of equal number. Unphysiologic loading of articular cartilage was simulated by transecting anterior cruciate ligaments of the right knees of 18 rats consisting of groups 1 and 2. Nine animals in group 2 received orally administered simvastatin 20 mg/kg per day by gavage for 8 weeks. Animals in group 3 were sham operated. All animals were sacrificed at postoperative 8 weeks. Effects of simvastatin on disease progression was evaluated by documenting OA changes in cartilage specimens using Osteoarthritis Research Society International (OARSI) OA cartilage histopathology assessment system scores combined with the percentage of MMP-3 expression in chondrocytes.
Results
Simvastatin treatment significantly down-regulated the percentage of MMP-3 expression in chondrocytes as assessed by immunohistochemistry methods. Suppression of this matrix-degrading enzyme by simvastatin also reduced OARSI scores, suggesting the potential for statins against OA progression.
Conclusions
Following knee trauma, OA initiates at the molecular level in a short period of time. Irreversible structural changes in cartilage that require demanding treatment strategies led us to focus on effective measures to prevent OA. Statins have immunomodulatory and anti-inflammatory properties independent from their serum-cholesterol-lowering effects. One of these widely used drugs, simvastatin, showed beneficial effects on OA progression and extent by reducing cartilage degradation in our experimental setting. If these results are confirmed by human trials, simvastatin might be considered by orthopedic surgeons as a disease-modifying drug during the early inflammatory phase of posttraumatic OA.
doi:10.1007/s10195-011-0154-y
PMCID: PMC3163794  PMID: 21863259
Simvastatin; Anterior cruciate ligament transection; Knee osteoarthritis; Matrix metalloproteinase-3
21.  Chemokine, vascular and therapeutic effects of combination Simvastatin and BMSC treatment of stroke 
Neurobiology of disease  2009;36(1):35-41.
We investigated the additive therapeutic effect that combination treatment of stroke with sub-therapeutic doses of Simvastatin, a HMG-CoA reductase inhibitor, and bone marrow stromal cells (BMSCs). Rats were administered Simvastatin (0.5 mg/kg), BMSCs (1×106) or combination Simvastatin with BMSCs starting at 24 hours after stroke. Combination treatment significantly improved neurological outcome, enhanced angiogenesis and arteriogenesis, and increased the number of engrafted-BMSCs in the ischemic brain. The number of engrafted-BMSCs and arteriogenesis were significantly correlated with functional outcome. Simvastatin significantly increased stromal cell-derived factor-1 (SDF1) expression in the ischemic brain and chemokine (CXC motif) receptor-4 (CXCR4) in BMSCs, and increased BMSC migration to RBMECs and astrocytes. Combination treatment of stroke upregulates the SDF1/CXCR4 axis and enhances BMSC migration into the ischemic brain, amplifies arteriogenesis and angiogenesis, and improves functional outcome after stroke.
doi:10.1016/j.nbd.2009.06.012
PMCID: PMC2748847  PMID: 19591934
bone marrow stromal cell; simvastatin; arteriogenesis; angiogenesis; stroke
22.  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
23.  Therapeutic Benefit of Treatment of Stroke With Simvastatin and Human Umbilical Cord Blood Cells: Neurogenesis, Synaptic Plasticity, and Axon Growth 
Cell transplantation  2012;21(5):845-856.
The therapeutic efficacy of cell-based therapy after stroke can be enhanced by making the host brain tissue more receptive to the administered cells, which thereby facilitates brain plasticity. We hypothesized that simvastatin increases human umbilical cord blood cell (HUCBC) migration into the ischemic brain and promotes brain plasticity and neurological functional outcome after stroke. Rats were subjected to 2-h middle cerebral artery occlusion (MCAo) and administered subtherapeutic doses of simvastatin (0.5 mg/kg, gavaged daily for 7 days), HUCBCs (1 × 106, one time injection via tail vein), or combination simvastatin with HUCBCs starting at 24 h after stroke. Combination treatment of stroke showed an interactive effect in improvement of neurological outcome compared with simvastatin or HUCBC monotherapy groups. In addition, combination treatment significantly increased brain-derived neurotrophic factor/TrkB expression and the number of engrafted HUCBCs in the ischemic brain compared with HUCBC monotherapy. The number of engrafted HUCBCs was significantly correlated with functional outcome (modified neurological severity score). Combination treatment significantly increased neurogenesis and synaptic plasticity in the ischemic brain, and promoted neuroblast migration in cultured subventricular zone explants. Using primary cultured neurons (PCNs), we found that combination treatment enhanced neurite outgrowth compared with nontreatment control, simvastatin or HUCBC supernatant monotherapy. Inhibition of TrkB significantly attenuated combination treatment-induced neurite outgrowth. Our data indicate that combination simvastatin and HUCBC treatment of stroke increases BDNF/TrkB expression, enhances HUCBC migration into the ischemic brain, amplifies endogenous neurogenesis, synaptic plasticity and axonal growth, and thereby improves functional outcome after stroke.
doi:10.3727/096368911X627417
PMCID: PMC3442771  PMID: 22405262
Simvastatin; Human umbilical cord blood cells (HUCBCs); Neurogenesis; Synaptic plasticity; Stroke
24.  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
25.  Changes of Pulmonary Pathology and Gene Expressions After Simvastatin Treatment in the Monocrotaline-Induced Pulmonary Hypertension Rat Model 
Korean Circulation Journal  2011;41(9):518-527.
Background and Objectives
Simvastatin's properties are suggestive of a potential pathophysiologic role in pulmonary hypertension. The objectives of this study were to investigate changes of pulmonary pathology and gene expressions, including endothelin (ET)-1, endothelin receptor A (ERA), inducible nitric oxide synthase (NOS2), endothelial nitric oxide synthase (NOS3), matrix metalloproteinase (MMP) 2, tissue inhibitor of matrix metalloproteinases (TIMP) and caspase 3, and to evaluate the effect of simvastatin on monocrotaline (M)-induced pulmonary hypertension.
Materials and Methods
Six week old male Sprague-Dawley rats were treated, as follows: control group, subcutaneous (sc) injection of saline; M group, sc injection of M (60 mg/kg); and simvastatin group, sc injection of M (60 mg/kg) plus 10 mg/kg/day simvastatin orally.
Results
On day 28, right ventricular hypertrophy (RVH) significantly decreased in the simvastatin group compared to the M group. Similarly, right ventricular pressure significantly decreased in the simvastatin group on day 28. From day 7, the ratio of medial thickening of the pulmonary artery was significantly increased in the M group, but there was no significant change in the simvastatin group. The number of muscular pulmonary arterioles was significantly reduced in the simvastatin group. On day 5, gene expressions of ET-1, ERA, NOS2, NOS3, MMP and TIMP significantly decreased in the simvastatin group.
Conclusion
Administration of simvastatin exerted weak inhibitory effects on RVH and on the number of muscular pulmonary arterioles, during the development of M-induced pulmonary hypertension in rats. Simvastatin decreased gene expressions on day 5.
doi:10.4070/kcj.2011.41.9.518
PMCID: PMC3193043  PMID: 22022327
Hypertension, pulmonary; Gene expression; Monocrotaline; Simvastatin

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