[show abstract][hide abstract] ABSTRACT: Stroke has a high incidence in the elderly. Stroke enters the chronic phase 3 months after initial stroke onset. Currently, there is no pharmaceutical treatment available for chronic stroke. We have demonstrated the therapeutic effects of the combination of stem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF) (SCF+G-CSF) on chronic stroke. However, it remains unclear how SCF+G-CSF repairs the brain in chronic stroke. In this study, we determined the effects of SCF+G-CSF on neuronal network remodeling in the aged brain of chronic stroke. Cortical brain ischemia was produced in 16-18 month-old transgenic mice expressing yellow fluorescent protein in layer V pyramidal neurons. SCF+G-CSF was subcutaneously injected for 7 days beginning at 3.5 months post-ischemia. Using both live brain imaging and immunohistochemistry, we observed that SCF+G-CSF increased the mushroom-type spines on the apical dendrites of layer V pyramidal neurons adjacent to the infarct cavities 2 and 6 weeks after treatment. SCF+G-CSF also augmented dendritic branches and post-synaptic density protein 95 puncta in the peri-infarct cortex 6 weeks after treatment. These data suggest that SCF+G-CSF treatment in chronic stroke remodels neural circuits in the aged brain. This study provides evidence to support the development of a new therapeutic strategy for chronic stroke.
PLoS ONE 01/2013; 8(6):e64684. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Gene regulation remains one of the major challenges for gene therapy in clinical trials. In the present study, we first generated a binary tetracycline-on (Tet-On) system based on two lentivirus vectors, one expressing both human glial cell line-derived neurotrophic factor (hGDNF) and humanized recombinant green fluorescent protein (hrGFP) genes under second-generation tetracycline response element (TRE), and the other expressing the advanced reverse tetracycline-controlled transactivator - rtTA2S-M2 under a human minimal cytomegalovirus immediate early (CMV-IE) promoter. This system allows simultaneous expression of hGDNF and hrGFP genes in the presence of doxycycline (Dox). Human bone marrow-derived mesenchymal stem cells (hMSCs) were transduced with the binary Tet-On lentivirus vectors and characterized in vitro in the presence (On) or absence (Off) of Dox. The expression of hGDNF and hrGFP transgenes in transduced hMSCs was tightly regulated as determined by flow cytometry (FCM), GDNF enzyme-linked immunosorbent assay (ELISA) and quantitative real time-polymerase chain reaction (qRT-PCR). There was a dose-dependent regulation for hrGFP transgene expression. The levels of hGDNF protein in culture medium were correlated with the mean fluorescence intensity (MFI) units of hrGFP. The levels of transgene background expression were very low in the absence of Dox. The treatment of the conditioned medium from cultures of transduced hMSCs in the presence of Dox protected SH-SY5Y cells against 6-hydroxydopamine (6-OHDA) toxicity as determined by cell viability using 3, [4,5-dimethylthiazol-2-yl]- diphenyltetrazolium bromide (MTT) assay. The treatment of the conditioned medium was also found to improve the survival of dopaminergic (DA) neurons of ventral mesencephalic (VM) tissue in serum-free culture conditions as assessed by cell body area, the number of neurites and dendrite branching points, and proportion of tyrosine hydroxylase (TH)-immunoreactive (IR) cells. Our inducible lentivirus-mediated hGDNF gene delivery system may provide useful tools for basic research on gene therapy for chronic neurological disorders such as Parkinson's disease (PD).
PLoS ONE 01/2013; 8(5):e64389. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have recently demonstrated that adeno-associated virus serotype 9 (AAV9)-mediated human erythropoietin (hEPO) gene delivery into the brain protects dopaminergic (DA) neurons in the substantia nigra in a rat model of Parkinson's disease. In the present study, we examined whether pre-exposure to AAV9-hEPO vectors with an intramuscular or intrastriatal injection would reduce AAV9-mediated hEPO transduction in rat brain. We first characterized transgene expression and immune responses against AAV9-hEPO vectors in rat striatum at 4 days, 3 weeks and 6 months, and with doses ranging from 10(11) to 10(13) viral genomes. To sensitize immune system, rats received an injection of AAV9-hEPO into either the muscle or the left striatum, and then sequentially an injection of AAV9-hEPO into the right striatum 3 weeks later. We observed that transgene expression exhibited in a time course and dose dependent manner, and inflammatory and immune responses displayed in a time course manner. Intramuscular, but not intrastriatal injections of AAV9-hEPO resulted in reduced levels of hEPO transduction and increased levels of the major histocompatibility complex (MHC) class I and class II antigen expression in the striatum following AAV9-hEPO re-administration. There were infiltration of the cluster of differentiation 4 (CD4)-and CD8-lymphacytes, and accumulation of activated microglial cells and astrocytes in the virally injected striatum. In addition, the sera from the rats with intramuscular injections of AAV9-hEPO contained greater levels of antibodies against both AAV9 capsid protein and hEPO protein than the other treatment groups. hEPO gene expression was negatively correlated with the levels of circulating antibodies against AAV9 capsid protein. Intramuscular and intrastriatal re-administration of AAV9-hEPO led to increased numbers of red blood cells in peripheral blood. Our results suggest that pre-immunization with an intramuscular injection can lead to the reduction of transgene expression in the striatal re-administration.
PLoS ONE 01/2013; 8(5):e63876. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Human albumin has recently been demonstrated to protect brain neurons from injury in rat ischemic brain. However, there is no information available about whether human albumin can prevent loss of tyrosine hydroxylase (TH) expression of dopaminergic (DA) neurons induced by 6-hydroxydopamine (6-OHDA) toxicity that is most commonly used to create a rat model of Parkinson's disease (PD). In the present study, two microliters of 1.25% human albumin were stereotaxically injected into the right striatum of rats one day before or 7 days after the 6-OHDA lesion in the same side. D-Amphetamine-induced rotational asymmetry was measured 7 days, 3 and 10 weeks after 6-OHDA lesion. We observed that intrastriatal administration of human albumin significantly reduced the degree of rotational asymmetry. The number of TH-immunoreactive neurons present in the substantia nigra was greater in 6-OHDA lesioned rats following human albumin-treatment than non-human albumin treatment. TH-immunoreactivity in the 6-OHDA-lesioned striatum was also significantly increased in the human albumin-treated rats. To examine the mechanisms underlying the effects of human albumin, we challenged PC12 cells with 6-OHDA as an in vitro model of PD. Incubation with human albumin prevented 6-OHDA-induced reduction of cell viability in PC12 cell cultures, as measured by MTT assay. Furthermore, human albumin reduced 6-OHDA-induced formation of reactive oxygen species (ROS) and apoptosis in cultured PC12 cells, as assessed by flow cytometry. Western blot analysis showed that human albumin inhibited 6-OHDA-induced activation of JNK, c-Jun, ERK, and p38 mitogen-activated protein kinases (MAPK) signaling in PC12 cultures challenged with 6-OHDA. Human albumin may protect against 6-OHDA toxicity by influencing MAPK pathway followed by anti-ROS formation and anti-apoptosis.
PLoS ONE 01/2012; 7(7):e41226. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Convincing evidence has shown that brain ischemia causes the proliferation of neural stem cells/neural progenitor cells (NSCs/NPCs) in both the subventricular zone (SVZ) and the subgranular zone (SGZ) of adult brain. The role of brain ischemia-induced NSC/NPC proliferation, however, has remained unclear. Here we have determined whether brain ischemia-induced amplification of the NSCs/NPCs in adult brain is required for brain self-protection. The approach of intracerebroventricular (ICV) infusion of cytosine arabinoside (Ara-C), an inhibitor for cell proliferation, for the first 7days after brain ischemia was used to block ischemia-induced NSC/NPC proliferation. We observed that ICV infusion of Ara-C caused a complete blockade of NSC/NPC proliferation in the SVZ and a dramatic reduction of NSC/NPC proliferation in the SGZ. Additionally, as a result of the inhibition of ischemia-induced NSC/NPC pool amplification, the number of neurons in the hippocampal CA1 and CA3 was significantly reduced, the infarction size was significantly enlarged, and neurological deficits were significantly worsened after focal brain ischemia. We also found that an NSC/NPC-conditioned medium showed neuroprotective effects in vitro and that adult NSC/NPC-released brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) are required for NSC/NPC-conditioned medium-induced neuroprotection. These data suggest that NSC/NPC-generated trophic factors are neuroprotective and that brain ischemia-triggered NSC/NPC proliferation is crucial for brain protection. This study provides insights into the contribution of endogenous NSCs/NPCs to brain self-protection in adult brain after ischemia injury.
Brain research 02/2010; 1327:91-102. · 2.46 Impact Factor
[show abstract][hide abstract] ABSTRACT: Thiopental is an anesthetic used for controlling high intracranial pressure (ICP) caused by brain surgery, brain trauma, and severe stroke. However, it remains controversial whether Thiopental is detrimental or beneficial in ischemic stroke. In this study, we used an animal model of ischemic stroke in spontaneously hypertensive rats to determine whether or not Thiopental is neuroprotective in the setting of brain ischemia. We observed that Thiopental caused a prolonged duration of unconsciousness with a high rate of mortality, that Thiopental created exaggerated neurological deficits that were revealed through limb placement tests at 4 days and 4 weeks after brain ischemia, and that infarct volume was increased in Thiopental-anesthetized rats. These data suggest that Thiopental is detrimental in ischemic stroke. Thus, our findings raise a caution about the use of Thiopental in the setting of ischemic stroke.
Brain research 08/2009; 1294:176-82. · 2.46 Impact Factor
[show abstract][hide abstract] ABSTRACT: Chronic stroke is a highly important but under-investigated scientific problem in neurologic research. We have reported earlier that stem cell factor (SCF) in combination with granulocyte-colony stimulating factor (G-CSF) treatment during chronic stroke improves functional outcomes. Here we have determined the contribution of bone marrow-derived cells in angiogenesis and neurogenesis, which are enhanced by SCF+G-CSF treatment during chronic stroke. Using bone marrow tracking, flow cytometry, 2-photon live brain imaging, and immunohistochemistry, we observed that the levels of circulating bone marrow stem cells (BMSCs) (CD34+/c-kit+) were significantly increased by SCF+G-CSF treatment. In addition, live brain imaging revealed that numerous bone marrow-derived cells migrate into the brain parenchyma in the treated mice. We also found that bone marrow-derived cells, bone marrow-derived endothelial cells, vascular density, and bone marrow-derived neurons were significantly augmented by SCF+G-CSF. It is interesting that, in addition to the increase in bone marrow-derived endothelial cells, the number of bone marrow-derived pericytes was reduced after SCF+G-CSF treatment during chronic stroke. These data suggest that SCF+G-CSF treatment can enhance repair of brain damage during chronic stroke by mobilizing BMSCs, and promoting the contribution of bone marrow-derived cells to angiogenesis and neurogenesis.
Journal of cerebral blood flow and metabolism: official journal of the International Society of Cerebral Blood Flow and Metabolism 02/2009; 29(4):759-70. · 5.46 Impact Factor
[show abstract][hide abstract] ABSTRACT: Stroke is the leading cause of adult disability worldwide. Currently, there is no effective treatment for stroke survivors. Stem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF) are the growth factors regulating hematopoiesis. We have previously observed that SCF and G-CSF have neuroprotective and functional effects on acute brain ischemia. In the present study, the beneficial effects of SCF and G-CSF on chronic brain ischemia were determined.
SCF, G-CSF, or SCF+G-CSF was administered subcutaneously to rats 3.5 months after induction of ischemic stroke by middle cerebral artery occlusion. Neurological deficits were evaluated by limb placement test and foot fault test over time. Field-evoked potential was performed 19 weeks after treatment. Infarct volume was histologically determined using serial coronal sections.
Significant functional improvement was seen in SCF+G-CSF-treated rats 1, 5, and 17 weeks after injections. SCF alone also improved functional outcome, but it did not show as stable improvement as SCF+G-CSF. No functional benefit was seen in G-CSF-treated rats. Field-evoked potential studies further confirmed the behavioral data that the normal pattern of neuronal activity was reestablished in the lesioned brain of the rats with good functional outcome. Interestingly, infarction volume was also significantly reduced in SCF+G-CSF-treated rats.
These data provide first evidence that functional restoration in chronic brain ischemia can be attained using hematopoietic growth factors.
[show abstract][hide abstract] ABSTRACT: Stem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF) are essential growth factors in hematopoiesis. We determined whether receptors for SCF and G-CSF exist in the brain and whether exogenous SCF and G-CSF are beneficial to brain repair after brain ischemia.
A well-established rat model of experimental stroke was used in this study. SCF, G-CSF, SCF+G-CSF, or saline was subcutaneously administered 3 hours to 7 days after brain ischemia. Bromodeoxyuridine was administered simultaneously. Sensorimotor function was evaluated with a limb placement test and foot fault test over time.
We observed that receptors for SCF and G-CSF were expressed in both neurogenic regions and neurons. SCF-treated rats showed the best functional restoration at 1 week that was maintained 4, 7, and 10 weeks after the final injection. G-CSF-induced functional recovery was limited and unstable. Interestingly, stable but delayed functional improvement was seen in SCF+G-CSF-treated rats. Infarction size was significantly reduced in all growth factor-treated rats. In addition, SCF and SCF+G-CSF enhanced neural progenitor cell proliferation in the subventricular zone bilaterally, whereas G-CSF and SCF+G-CSF treatment increased bromodeoxyuridine -positive cells in periinfarct areas.
SCF and G-CSF are neuroprotective and beneficial to functional restoration when administered during the acute phase after brain ischemia, indicating hematopoietic growth factors play a role in brain repair.