Dongmei Yang

Simvastatin and Atorvastatin Improve Neurological Outcome After Experimental Intracerebral Hemorrhage

Background and Purpose— This study investigates the effects of statin treatment on experimental intracerebral hemorrhage (ICH) using behavioral, histological, and MRI measures of recovery. Methods— Primary ICH was induced in rats. Simvastatin (2 mg/kg), atorvastatin (2 mg/kg), or phosphate-buffered saline (n=6 per group) was given daily for 1 week. MRI studies were performed 2 to 3 days before ICH, and at 1 to 2 hours and 1, 2, 7, 14, and 28 days after ICH. The ICH evolution was assessed via hematoma volume measurements using susceptibility-weighted imaging (SWI) and tissue loss using T2 maps and hematoxylin and eosin (H&E) histology. Neurobehavioral tests were done before ICH and at various time points post-ICH. Additional histological measures were performed with doublecortin neuronal nuclei and bromodeoxyuridine stainings. Results— Initial ICH volumes determined by SWI were similar across all groups. Simvastatin significantly reduced hematoma volume at 4 weeks (P=0.002 versus control with acute volumes as baseline), whereas that for atorvastatin was marginal (P=0.09). MRI estimates of tissue loss (% of contralateral hemisphere) for treated rats were significantly lower (P=0.0003 and 0.001, respectively) than for control at 4 weeks. Similar results were obtained for H&E histology (P=0.0003 and 0.02, respectively). Tissue loss estimates between MRI and histology were well correlated (R2=0.764, P<0.0001). Significant improvement in neurological function was seen 2 to 4 weeks post-ICH with increased neurogenesis observed. Conclusions— Simvastatin and atorvastatin significantly improved neurological recovery, decreased tissue loss, and increased neurogenesis when administered for 1 week after ICH.

Mannitol enhances delivery of marrow stromal cells to the brain after experimental intracerebral hemorrhage.

Previous studies show that intravascular injection of human bone marrow stromal cells (hBMSCs) significantly improves neurological functional recovery in a rat model of intracerebral hemorrhage (ICH). In the present study, we tested the hypothesis that mannitol improves the efficiency of intraarterial MSC delivery (i.e., fewer injected cells required for therapeutic efficacy) after ICH. There were four post-ICH groups (N=9): group 1, negative control with only intraarterial injection of 1 million human fibroblasts in phosphate-buffered saline (PBS); group 2, intravenous injection of mannitol alone in PBS (1.5 g/kg); group 3, intraarterial injection of 1 million hBMSCs alone in PBS; and group 4, intravenous injection of mannitol (1.5 g/kg) in PBS followed by intraarterial injection of 1 million hBMSCs in PBS. Group 4 exhibited significantly improved neurological functional outcome as assessed by neurological severity score (NSS) and corner test scores. Immunohistochemical staining of group 4 suggested increased synaptogenesis, proliferating immature neurons, and neuronal migration. The number of hBMSCs recruited to the injured region increased strikingly in group 4. Tissue loss was notably reduced in group 4. In summary, the beneficial effects of intraarterial infusion of MSCs are amplified with intravenous injection of mannitol. Preadministration of mannitol significantly increases the number of hBMSCs located in the ICH region, improves histochemical parameters of neural regeneration, and reduces the anatomical and pathological consequences of ICH.

Localization of bone marrow stromal cells to the injury site after intracerebral hemorrhage in rats

OBJECT Previous studies demonstrated that intravascular injection of bone marrow stromal cells (BMSCs) significantly improved neurological functional recovery in a rat model of intracerebral hemorrhage (ICH). To further investigate the fate of transplanted cells, we examined the effect of male rat BMSCs administered to female rats after ICH. METHODS Twenty-seven female Wistar rats were subjected to ICH surgery. At 24 hours after ICH, these rats were randomly divided into 3 groups and injected intravenously with 1 ml phosphate-buffered saline or 0.5 million or 1 million male rat BMSCs in phosphate-buffered saline. To evaluate the neurological functional outcome, each rat was subjected to a series of behavioral tests (modified neurological severity score and corner turn test) at 1, 7, and 14 days after ICH. The rats were anesthetized intraperitoneally and killed, and the brain tissues were processed at Day 14 after ICH. Immunohistochemistry and in situ hybridization were used to identify cell-specific markers. RESULTS The male rat BMSCs significantly improved the neurological functional outcome and also significantly diminished tissue loss when intravenously transplanted into the rats after ICH. Immunoassay for bromodeoxyuridine (BrdU) and neuronal markers demonstrated a significant increase in the number of BrdU-positive cells, which indicated endogenous neurogenesis, and a significant increase in the number of cells positive for immature neuronal markers. In situ hybridization showed that more BMSCs resided around the hematoma of the rats treated with the 1-million-cell dose compared with the 0.5-million-cell-dose group. In addition, a subfraction of Y chromosome-positive cells were co-immunostained with the neuronal marker microtubule-associated protein-2 or the astrocytic marker glial fibrillary acidic protein. CONCLUSIONS Male rat BMSCs improve neurological outcome and increase histochemical parameters of neurogenesis when administered to female rats after ICH. This study has shown that the intravenously administered male rat BMSCs enter the brain, migrate to the perihematomal area, and express parenchymal markers.

Erythropoietin promotes neurological recovery after intracerebral haemorrhage in rats.

Background Attention has turned to neurorestorative therapies, including erythropoietin, for experimental ischaemic stroke and head injury. Treatments for intracerebral haemorrhage need to be developed, as this represents a particularly devastating and common form of neurological injury. Aim The aim of this study is to investigate the therapeutic potential of erythropoietin after intracerebral haemorrhage in rats and to measure its effects on mechanisms of recovery and neurogenesis. Methods Intracerebral haemorrhage was induced in 24 Wistar male rats by intrastriatal infusion of autologous blood. Recombinant human erythropoietin (5000 or 10000 U/kg BW/day) or saline was administered starting 1 day after intracerebral haemorrhage and continued daily for 1 week (n = 8 for each group). To label proliferating cells, 5‘-bromo-2’ deoxyuridine was injected daily for 13 days after intracerebral haemorrhage. All animals survived for 2 weeks after intracerebral haemorrhage. Functional outcome, area of tissue loss and immunohistochemical staining were measured at 14 days after intracerebral haemorrhage. Global test or anova was used to test the erythropoietin dose effect. Results Rats receiving recombinant human erythropoietin after intracerebral haemorrhage exhibited significant improvement in modified neurological severity score and corner test at 14 days (P<0·05). Increased expression of phenotypes of synaptogenesis and proliferating immature neurons were shown by immunohistochemical staining. Only the group receiving a lower dose of recombinant human erythropoietin had significantly less tissue loss compared with the control group (P<0·05). In rats treated with recombinant human erythropoietin, double staining for 5‘-bromo-2’ deoxyuridine and TUJ1 revealed a subpopulation of cells that express an immature neuronal marker while still dividing. Conclusions Erythropoietin improves neurological outcome and increases histochemical parameters of neurogenesis when given after intracerebral haemorrhage in rats. Intriguingly, only the lower dose of recombinant human erythropoietin was effective in reducing tissue loss in the region of intracerebral haemorrhage.

Vascular recovery promoted by atorvastatin and simvastatin after experimental intracerebral hemorrhage: magnetic resonance imaging and histological study

Statins, 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitors, provide neuroprotection with beneficial effects on the nervous and vascular systems by improving or restoring endothelial function, decreasing oxidative stress and vascular inflammation, and enhancing angiogenesis and neurogenesis.10,23 In previous studies we found that atorvastatin and simvastatin enhanced functional outcome, and that atorvastatin enhanced angiogenesis and synaptogenesis after ischemic stroke.3 Similar beneficial effects of simvastatin and atorvastatin have been suggested in relation to hemorrhagic stroke since ischemic stroke and hemorrhagic stroke have a number of common injury-mechanisms.19,26 Contrast mechanisms in magnetic resonance imaging (MRI) are based on a wide variety of physical parameters, and MRI has been successfully applied to experimental ischemic stroke.27 MRI has a high sensitivity for assessing the temporal evolution of ICH and may be useful in evaluating the impact of therapeutic interventions after experimental ICH in animal models16,20, and eventually in humans. To date, little is known about the longitudinal multiparametric MRI assessment of therapeutic intervention on the experimental model of ICH. In the present study, we used longitudinal multiparametric MRI and immunohistochemistry to assess the therapeutic effects after ICH in rats. We hypothesized that 1) statins reduce edema and vascular injury while promoting angiogenesis and synaptogenesis, and 2) multiparametric MRI can assess the temporal evolution of brain tissue injury and repair and the effects of therapeutic intervention after experimental ICH.OBJECT Longitudinal multiparametric MR imaging and histological studies were performed on simvastatin- or atorvastatin-treated rats to evaluate vascular repair mechanisms after experimental intracerebral hemorrhage (ICH). METHODS Primary ICH was induced in adult Wistar rats by direct infusion of 100 μl of autologous blood into the striatal region adjacent to the subventricular zone. Atorvastatin (2 mg/kg), simvastatin (2 mg/kg), or phosphate-buffered saline was given orally at 24 hours post-ICH and continued daily for 7 days. The temporal evolution of ICH in each group was assessed by MR imaging measurements of T2, T1(sat), and cerebral blood flow in brain areas corresponding to the bulk of the hemorrhage (core) and edematous border (rim). Rats were killed after the final MR imaging examination at 28 days, and histological studies were performed. A small group of sham-operated animals was also studied. Neurobehavioral testing was performed in all animals. Analysis of variance methods were used to compare results from the treatment and control groups, with significance inferred at p ≤ 0.05. RESULTS Using histological indices, animals treated with simvastatin and atorvastatin had significantly increased angiogenesis and synaptogenesis in the hematoma rim compared with the control group (p ≤ 0.05). The statin-treated animals exhibited significantly increased cerebral blood flow in the hematoma rim at 4 weeks, while blood-brain barrier permeability (T1(sat)) and edema (T2) in the corresponding regions were reduced. Both statin-treated groups showed significant neurological improvement from 2 weeks post-ICH onward. CONCLUSIONS The results of the present study demonstrate that simvastatin and atorvastatin significantly improve the recovery of rats from ICH, possibly via angiogenesis and synaptic plasticity. In addition, in vivo multiparametric MR imaging measurements over time can be effectively applied to the experimental ICH model for longitudinal assessment of the therapeutic intervention.

Therapeutic effect of human umbilical tissue-derived cell treatment in Rats with experimental intracerebral hemorrhage

BACKGROUND AND PURPOSE The present study examines whether human umbilical tissue-derived cells (hUTC) have a neuro-restorative effect and improve functional recovery after intracerebral hemorrhage (ICH) in rats. METHODS Primary ICH was induced in male Wistar rats by stereotactic injection of 100μL of autologous blood into the striatal region adjacent to the subventricular zone. Briefly, the rats were randomly divided into six groups, each group was intravenously injected either with 2mL phosphate-buffered saline (PBS) or 3million hUTC in PBS at 1, 3 or 7days after ICH (n=8/group). To evaluate neurological functional outcome, each animal was subjected to the modified neurological severity score (mNSS) and corner turn tests at different time points after ICH. At four weeks post treatment, each group was anesthetized intraperitoneally, sacrificed, and brain tissues were processed histologically. Immunohistochemistry was employed to measure vascularity (vWF), neurogenesis (BrdU TUJ1, DCX and NeuN), synaptogenesis (synaptophysin) and apoptosis (TUNEL). RESULTS The hUTC-treated animals showed significantly improved neurological functional outcomes as assessed by mNSS and corner turn tests at 14, 21 and 28days post-injection in each treatment group (P<0.05) as compared to the PBS controls. Animals treated with hUTC were seen to have significantly increased cell proliferation, vascularity and synaptogenesis, as well as reduced apoptosis in the hematoma rim compared to the corresponding control group (P<0.05). CONCLUSIONS Intravenously infused hUTC have a beneficial effect after experimental ICH by functional and histochemical measurements of neural cell proliferation and synaptogenesis in the ICH border zone. This brain region also shows correlative evidence of neuronal recovery with increased vascularity.

Improvement in recovery after experimental intracerebral hemorrhage using a selective cathepsin B and L inhibitor

Spontaneous intracerebral hemorrhage (ICH) represents 10–15% of all strokes, and it exerts a much worse prognosis than injury caused by ischemic stroke.9 The size of the hematoma formed in the brain parenchyma from the rupture of a cerebral vessel is a significant predictor of poor outcome.40 Inflammation and toxicity triggered by extravascular blood products increase the cerebral damage observed in the early phases after hematoma formation and also lead to worsening edema around the clot.41 Effective surgical and drug approaches for clinical practice have been elusive.9,10,14 Cathepsins B and L are intracellular/lysosomal and secreted cysteine proteases that have been implicated in the process of neuronal cell death after experimental global and focal cerebral ischemia and after experimental spinal cord injury.1,6,,11,16,17,34,38 The levels of these cathepsins are significantly increased and relocate from the lysosomes to the cytoplasm following global cerebral ischemia.. Cathepsin B is one of the causative factors of microglia-induced neuronal apoptosis.32,34 In our previous study, CP-1, a non-toxic cysteine protease inhibitor which is selective for cathepsins B and L, but not the calpains or caspases, was shown to be effective at reducing infarct volume and improving functional scores when administered intravenously to rats after 2 hours of middle cerebral artery occlusion (MCAO) and reperfusion.35 The role of cathepsins in ICH has not been studied. However, results from models of cerebral ischemia suggest that inhibition of cathepsin B and L may be efficacious in preventing neuronal cell death. In this study, we investigated the ability of CP-1 to promote functional and histological recovery after ICH.OBJECT This study investigates a potential novel application of a selective cathepsin B and L inhibitor in experimental intracerebral hemorrhage (ICH) in rats. METHODS Forty adult male Wistar rats received an ICH by stereotactic injection of 100 μl of autologous blood or sham via needle insertion into the right striatum. The rats were treated with a selective cathepsin B and L inhibitor (CP-1) or 1% dimethyl sulfoxide sterile saline intravenously at 2 and 4 hours after injury. Modified neurological severity scores were obtained and corner turn tests were performed at 1, 4, 7, and 14 days after ICH. The rats were sacrificed at 3 and 14 days after ICH for immunohistological analysis of tissue loss, neurogenesis, angiogenesis, and apoptosis. RESULTS The animals treated with CP-1 demonstrated significantly reduced apoptosis as well as tissue loss compared with controls (p < 0.05 for each). Neurological function as assessed by modified neurological severity score and corner turn tests showed improvement after CP-1 treatment at 7 and 14 days (p < 0.05). Angiogenesis and neurogenesis parameters demonstrated improvement after CP-1 treatment compared with controls (p < 0.05) at 14 days. CONCLUSIONS This study is the first report of treatment of ICH with a selective cathepsin B and L inhibitor. Cathepsin B and L inhibition has been shown to be beneficial after cerebral ischemia, likely because of its upstream regulation of the other prominent cysteine proteases, calpains, and caspases. While ICH may not induce a major component of ischemia, the cellular stress in the border zone may activate these proteolytic pathways. The observation that cathepsin B and L blockade is efficacious in this model is provocative for further investigation.

Localization of bone marrow stromal cells to the injury site after intracerebral hemorrhage in rats: Laboratory investigation

OBJECT Previous studies demonstrated that intravascular injection of bone marrow stromal cells (BMSCs) significantly improved neurological functional recovery in a rat model of intracerebral hemorrhage (ICH). To further investigate the fate of transplanted cells, we examined the effect of male rat BMSCs administered to female rats after ICH. METHODS Twenty-seven female Wistar rats were subjected to ICH surgery. At 24 hours after ICH, these rats were randomly divided into 3 groups and injected intravenously with 1 ml phosphate-buffered saline or 0.5 million or 1 million male rat BMSCs in phosphate-buffered saline. To evaluate the neurological functional outcome, each rat was subjected to a series of behavioral tests (modified neurological severity score and corner turn test) at 1, 7, and 14 days after ICH. The rats were anesthetized intraperitoneally and killed, and the brain tissues were processed at Day 14 after ICH. Immunohistochemistry and in situ hybridization were used to identify cell-specific markers. RESULTS The male rat BMSCs significantly improved the neurological functional outcome and also significantly diminished tissue loss when intravenously transplanted into the rats after ICH. Immunoassay for bromodeoxyuridine (BrdU) and neuronal markers demonstrated a significant increase in the number of BrdU-positive cells, which indicated endogenous neurogenesis, and a significant increase in the number of cells positive for immature neuronal markers. In situ hybridization showed that more BMSCs resided around the hematoma of the rats treated with the 1-million-cell dose compared with the 0.5-million-cell-dose group. In addition, a subfraction of Y chromosome-positive cells were co-immunostained with the neuronal marker microtubule-associated protein-2 or the astrocytic marker glial fibrillary acidic protein. CONCLUSIONS Male rat BMSCs improve neurological outcome and increase histochemical parameters of neurogenesis when administered to female rats after ICH. This study has shown that the intravenously administered male rat BMSCs enter the brain, migrate to the perihematomal area, and express parenchymal markers.

Vascular recovery promoted by atorvastatin and simvastatin after experimental intracerebral hemorrhage: Magnetic resonance imaging and histological study: Laboratory investigation

Statins, 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitors, provide neuroprotection with beneficial effects on the nervous and vascular systems by improving or restoring endothelial function, decreasing oxidative stress and vascular inflammation, and enhancing angiogenesis and neurogenesis.10,23 In previous studies we found that atorvastatin and simvastatin enhanced functional outcome, and that atorvastatin enhanced angiogenesis and synaptogenesis after ischemic stroke.3 Similar beneficial effects of simvastatin and atorvastatin have been suggested in relation to hemorrhagic stroke since ischemic stroke and hemorrhagic stroke have a number of common injury-mechanisms.19,26 Contrast mechanisms in magnetic resonance imaging (MRI) are based on a wide variety of physical parameters, and MRI has been successfully applied to experimental ischemic stroke.27 MRI has a high sensitivity for assessing the temporal evolution of ICH and may be useful in evaluating the impact of therapeutic interventions after experimental ICH in animal models16,20, and eventually in humans. To date, little is known about the longitudinal multiparametric MRI assessment of therapeutic intervention on the experimental model of ICH. In the present study, we used longitudinal multiparametric MRI and immunohistochemistry to assess the therapeutic effects after ICH in rats. We hypothesized that 1) statins reduce edema and vascular injury while promoting angiogenesis and synaptogenesis, and 2) multiparametric MRI can assess the temporal evolution of brain tissue injury and repair and the effects of therapeutic intervention after experimental ICH.OBJECT Longitudinal multiparametric MR imaging and histological studies were performed on simvastatin- or atorvastatin-treated rats to evaluate vascular repair mechanisms after experimental intracerebral hemorrhage (ICH). METHODS Primary ICH was induced in adult Wistar rats by direct infusion of 100 μl of autologous blood into the striatal region adjacent to the subventricular zone. Atorvastatin (2 mg/kg), simvastatin (2 mg/kg), or phosphate-buffered saline was given orally at 24 hours post-ICH and continued daily for 7 days. The temporal evolution of ICH in each group was assessed by MR imaging measurements of T2, T1(sat), and cerebral blood flow in brain areas corresponding to the bulk of the hemorrhage (core) and edematous border (rim). Rats were killed after the final MR imaging examination at 28 days, and histological studies were performed. A small group of sham-operated animals was also studied. Neurobehavioral testing was performed in all animals. Analysis of variance methods were used to compare results from the treatment and control groups, with significance inferred at p ≤ 0.05. RESULTS Using histological indices, animals treated with simvastatin and atorvastatin had significantly increased angiogenesis and synaptogenesis in the hematoma rim compared with the control group (p ≤ 0.05). The statin-treated animals exhibited significantly increased cerebral blood flow in the hematoma rim at 4 weeks, while blood-brain barrier permeability (T1(sat)) and edema (T2) in the corresponding regions were reduced. Both statin-treated groups showed significant neurological improvement from 2 weeks post-ICH onward. CONCLUSIONS The results of the present study demonstrate that simvastatin and atorvastatin significantly improve the recovery of rats from ICH, possibly via angiogenesis and synaptic plasticity. In addition, in vivo multiparametric MR imaging measurements over time can be effectively applied to the experimental ICH model for longitudinal assessment of the therapeutic intervention.

Atorvastatin and Simvastatin Promote Vascular Recovery after Experimental Intracerebral Hemorrhage: MRI and Histological Study

Statins, 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitors, provide neuroprotection with beneficial effects on the nervous and vascular systems by improving or restoring endothelial function, decreasing oxidative stress and vascular inflammation, and enhancing angiogenesis and neurogenesis.10,23 In previous studies we found that atorvastatin and simvastatin enhanced functional outcome, and that atorvastatin enhanced angiogenesis and synaptogenesis after ischemic stroke.3 Similar beneficial effects of simvastatin and atorvastatin have been suggested in relation to hemorrhagic stroke since ischemic stroke and hemorrhagic stroke have a number of common injury-mechanisms.19,26 Contrast mechanisms in magnetic resonance imaging (MRI) are based on a wide variety of physical parameters, and MRI has been successfully applied to experimental ischemic stroke.27 MRI has a high sensitivity for assessing the temporal evolution of ICH and may be useful in evaluating the impact of therapeutic interventions after experimental ICH in animal models16,20, and eventually in humans. To date, little is known about the longitudinal multiparametric MRI assessment of therapeutic intervention on the experimental model of ICH. In the present study, we used longitudinal multiparametric MRI and immunohistochemistry to assess the therapeutic effects after ICH in rats. We hypothesized that 1) statins reduce edema and vascular injury while promoting angiogenesis and synaptogenesis, and 2) multiparametric MRI can assess the temporal evolution of brain tissue injury and repair and the effects of therapeutic intervention after experimental ICH.OBJECT Longitudinal multiparametric MR imaging and histological studies were performed on simvastatin- or atorvastatin-treated rats to evaluate vascular repair mechanisms after experimental intracerebral hemorrhage (ICH). METHODS Primary ICH was induced in adult Wistar rats by direct infusion of 100 μl of autologous blood into the striatal region adjacent to the subventricular zone. Atorvastatin (2 mg/kg), simvastatin (2 mg/kg), or phosphate-buffered saline was given orally at 24 hours post-ICH and continued daily for 7 days. The temporal evolution of ICH in each group was assessed by MR imaging measurements of T2, T1(sat), and cerebral blood flow in brain areas corresponding to the bulk of the hemorrhage (core) and edematous border (rim). Rats were killed after the final MR imaging examination at 28 days, and histological studies were performed. A small group of sham-operated animals was also studied. Neurobehavioral testing was performed in all animals. Analysis of variance methods were used to compare results from the treatment and control groups, with significance inferred at p ≤ 0.05. RESULTS Using histological indices, animals treated with simvastatin and atorvastatin had significantly increased angiogenesis and synaptogenesis in the hematoma rim compared with the control group (p ≤ 0.05). The statin-treated animals exhibited significantly increased cerebral blood flow in the hematoma rim at 4 weeks, while blood-brain barrier permeability (T1(sat)) and edema (T2) in the corresponding regions were reduced. Both statin-treated groups showed significant neurological improvement from 2 weeks post-ICH onward. CONCLUSIONS The results of the present study demonstrate that simvastatin and atorvastatin significantly improve the recovery of rats from ICH, possibly via angiogenesis and synaptic plasticity. In addition, in vivo multiparametric MR imaging measurements over time can be effectively applied to the experimental ICH model for longitudinal assessment of the therapeutic intervention.