Exosomes are 30–150 nm vesicles secreted by a wide range of mammalian cells that can contain microRNA (miRNA). To test if marrow stromal cell (MSC) exosomes could be used as a vehicle for delivery of anti-tumor miRNAs, we transfected MSCs with a miR-146b expression plasmid, and harvested exosomes released by the MSCs. Intra-tumor injection of exosomes derived from miR-146-expressing MSCs significantly reduced glioma xenograft growth in a rat model of primary brain tumor.
Stroke remains the leading cause of adult disability. Thus, it is imperative to develop restorative therapies for ischemic stroke designed specifically to treat the intact brain tissue to stimulate functional benefit. Therapies targeting amplification of brain repair processes with nitric oxide (NO) donors and phosphodiesterase type 5 (PDE5) inhibitors in preclinical studies are emerging and showing improvement of functional recovery after stroke.
This review will mainly cover the effect of NO donors, which produce NO, and PDE5 inhibitors, which elevate cyclic guanosine 3′,5′-monophosphate (cGMP), on neural restorative events in ischemic brain and highlight mechanisms underlying their restorative therapeutic activity .
During stroke recovery, interwoven restorative events occur in ischemic brain, which include angiogenesis, neurogenesis, oligodendrogenesis, astrogliosis, and neurite outgrowth. Emerging preclinical data indicate that restorative therapies targeting multiple parenchymal cells including neural stem cells, cerebral endothelial cells, astrocytes, oligodendrocytes, neurons would be more effective than agents with a single cell target. Preclinical data suggest that elevated cGMP levels induced by NO donors and PDE5 inhibitors act on cerebral endothelial cells, neural stem cells, and oligodendrocyte progenitor cells to enhance stroke-induced angiogenesis, neurogenesis and oligodendrogenesis, respectively. These interacting remodeling events collectively improve neurological function after stroke.
Nitric oxide; cGMP; PDE5 inhibitor; stroke; angiogenesis; neurogenesis
Substantial effort is being expended on using micro-structural modeling of the white matter, with the goal of relating diffusion weighted magnetic resonance imaging (DWMRI) to the underlying structure of the tissue, such as axonal density. However, one of the important parameters affecting diffusion is the water exchange rate between the intra- and extra-axonal space, which has not been fully investigated and is a crucial marker of brain injury such as multiple sclerosis (MS), stroke, and traumatic brain injury (TBI). To our knowledge, there is no diffusion analytical model which includes the Water eXchange Rate (WXR) without the requirement of short gradient pulse (SGP) approximation. We therefore propose a new analytical model by deriving the diffusion signal for a permeable cylinder, assuming a clinically feasible pulse gradient spin echo (PGSE) sequence. Simulations based on Markov Random Walk confirm that the exchange parameter included in our model has a linear correlation (R2>0.88) with the actual WXR. Moreover, increasing WXR causes the estimated values of diameter and volume fraction of the cylinders to increase and decrease, respectively, which is consistent with our findings from histology measurements in tissues near TBI regions. This model was also applied to the diffusion signal acquired from ex vivo brains of 14 male (10 TBI and 4 normal) rats using hybrid diffusion imaging. The estimated values of axon diameter and axonal volume fraction are in agreement with their corresponding histological measurements in normal brains, with 0.96 intra-class correlation coefficient value resulting from consistency analysis. Moreover, a significant increase (p = 0.001) in WXR and diameter and decrease in axonal volume fraction in the TBI boundary were detected in the TBI rats compared with the normal rats.
Background and Purpose
We have previously demonstrated that 2-week treatment of experimental intracerebral hemorrhage (ICH) with a daily dose of 2 mg/kg statin starting 24 hours post-injury exerts a neuroprotective effect. The present study extends our previous investigation and tests the effect of acute high-dose (within 24 hours) statin therapy on experimental ICH.
Material and methods
Fifty-six male Wistar rats were subjected to ICH by stereotactic injection of 100 μl of autologous blood into the striatum. Rats were divided randomly into seven groups: saline control group (n = 8); 10, 20 and 40 mg/kg simvastatin-treated groups (n = 8); and 10, 20 and 40 mg/kg atorvastatin-treated groups (n = 8). Simvastatin or atorvastatin were administered orally at 3 and 24 hours after ICH. Neurological functional outcome was evaluated using behavioral tests (mNSS and corner turn test) at multiple time points after ICH. Animals were sacrificed at 28 days after treatment, and histological studies were completed.
Acute treatment with simvastatin or atorvastatin at doses of 10 and 20 mg/kg, but not at 40 mg/kg, significantly enhanced recovery of neurological function starting from 2 weeks post-ICH and persisting for up to 4 weeks post ICH. In addition, at doses of 10 mg/kg and 20 mg/kg, histological evaluations revealed that simvastatin or atorvastatin reduced tissue loss, increased cell proliferation in the subventricular zone and enhanced vascular density and synaptogenesis in the hematoma boundary zone when compared to saline-treated rats.
Treatment with simvastatin or atorvastatin at doses of 10 and 20 mg/kg significantly improves neurological recovery after administration during the first 24 hours after ICH. Decreased tissue loss, increased cell proliferation and vascularity likely contribute to improved functional recovery in rats treated with statins after ICH.
Simvastatin; atorvastatin; neurogenesis; synaptogenesis; vascular; intracerebral hemorrhage
Stroke is a major cause of mortality and disability in adults worldwide. Unfortunately, current therapy which targets vessel recanalization has a narrow treatment window, and at this time neuroprotective approaches are not effective for stroke treatment. However, after stroke the parenchymal and endothelial cells in the central nervous system (CNS) respond in concert to ischemic stressors and create a microenvironment in which successful recovery may ensue. Neurogenesis, synaptogenesis, axonal sprouting, glial cell activation, angiogenesis and vascular remodeling within the brain and the spinal cord are stimulated post stroke. Cell based-therapy amplifies these endogenous restorative effects within the CNS to promote functional outcome.
This article reviews current knowledge of cell-based therapy in the adult brain after stroke, including transplanted cell type, benefits and risks, with an emphasis on mechanisms of action.
Experimental studies and clinical trials with cell-based therapy in stroke appear promising. Cell-based therapy is not intended for the replacement of damaged cells, but for the remodeling of the CNS by promoting neuroplasticity, angiogenesis and immunomodulation. However, there are risks associated with the use of cell-based therapy, and adequate evaluation of these potential risks is a prerequisite before clinical application for stroke patients.
cell-based therapy; neurorestoration; stem cell; stroke
Tissue plasminogen activator (tPA) has been implicated in neurite outgrowth and neurological recovery post stroke. tPA converts the zymogen plasminogen (Plg) into plasmin. In this study, using plasminogen knockout (Plg-/-) mice and their Plg-native littermates (Plg+/+), we investigated the role of Plg in axonal remodeling and neurological recovery after stroke. Plg+/+ and Plg-/- mice (n = 10/group) were subjected to permanent intraluminal monofilament middle cerebral artery occlusion (MCAo). A foot-fault test and a single pellet reaching test were performed prior to and on day 3 after stroke, and weekly thereafter to monitor functional deficit and recovery. Biotinylated dextran amine (BDA) was injected into the left motor cortex to anterogradely label the corticospinal tract (CST). Animals were euthanized 4 weeks after stroke. Neurite outgrowth was also measured in primary cultured cortical neurons harvested from Plg+/+ and Plg-/- embryos. In Plg+/+ mice, the motor functional deficiency after stroke progressively recovered with time. In contrast, recovery in Plg-/- mice was significantly impaired compared to Plg+/+ mice (p<0.01). BDA-positive axonal density of the CST originating from the contralesional cortex in the denervated side of the cervical gray matter was significantly reduced in Plg-/- mice compared with Plg+/+ mice (p<0.05). The behavioral outcome was highly correlated with the midline-crossing CST axonal density (R2>0.82, p<0.01). Plg-/- neurons exhibited significantly reduced neurite outgrowth. Our data suggest that plasminogen-dependent proteolysis has a beneficial effect during neurological recovery after stroke, at least in part, by promoting axonal remodeling in the denervated spinal cord.
Background and Purpose
MRI was used to evaluate the effects of experimental intracerebral hemorrhage (ICH) on brain tissue injury and recovery.
Primary ICH was induced in rats (n=6) by direct infusion of autologous blood into the striatum. The evolution of ICH damage was assessed by MRI estimates of T2 and T1sat relaxation times, cerebral blood flow, vascular permeability, and susceptibility-weighted imaging before surgery (baseline) and at 2 hours and 1, 7, and 14 days post-ICH. Behavioral testing was done before and at 1, 7, and 14 days post-ICH. Animals were euthanized for histology at 14 days.
The MRI appearance of the hemorrhage and surrounding regions changed in a consistent manner over time. Two primary regions of interest were identified based on T2 values. These included a core, corresponding to the bulk of the hemorrhage, and an adjacent rim; both varied with time. The core was associated with significantly lower cerebral blood flow values at all post-ICH time points, whereas cerebral blood flow varied in the rim. Increases in vascular permeability were noted at 1, 7, and 14 days. Changes in T1sat were similar to those of T2. MRI and histological estimates of tissue loss were well correlated and showed approximately 9% hemispheric tissue loss.
Although the cerebral blood flow changes observed with this ICH model may not exactly mimic the clinical situation, our results suggest that the evolution of ICH injury can be accurately characterized with MRI. These methods may be useful to evaluate therapeutic interventions after experimental ICH and eventually in humans.
edema; intracerebral hemorrhage; MRI
This study was designed to investigate the beneficial effects of recombinant human erythropoietin (rhEPO) treatment of traumatic brain injury (TBI) in mice.
Adult male C57BL/6 mice were divided into 3 groups: 1) saline group (TBI + saline, n = 13); 2) EPO group (TBI + rhEPO, n = 12); and 3) sham group (sham + rhEPO, n = 8). TBI 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. rhEPO 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 sacrificed 35 days after injury and brain sections stained for immunohistochemistry.
TBI caused both tissue loss in the cortex and cell loss in the dentate gyrus (DG) and impaired sensorimotor function (footfaults) and spatial learning (Morris Water Maze). TBI alone stimulated cell proliferation and angiogenesis. As compared to saline treatment, rhEPO significantly reduced lesion volume in the cortex and cell loss in the DG after TBI and substantially improved sensorimotor function recovery and spatial learning performance. rhEPO enhanced neurogenesis in the injured cortex and the DG.
rhEPO initiated 6 hours post-TBI provides neuroprotection by decreasing lesion volume and cell loss as well as neurorestoration by enhancing neurogenesis, subsequently improving sensorimotor and spatial learning function. rhEPO is a promising neuroprotective and neurorestorative agent for TBI and warrants further investigation.
erythropoietin; mouse; sensorimotor; spatial learning; traumatic brain injury
Recombinant human erythropoietin (rhEPO) induces neurogenesis and angiogenesis. Using a coculture system of mouse brain endothelial cells (MBECs) and neural progenitor cells derived from the subventricular zone of adult mouse, we investigated the hypothesis that neural progenitor cells treated with rhEPO promote angiogenesis. Treatment of neural progenitor cells with rhEPO significantly increased their expression and secretion of vascular endothelial growth factor (VEGF) and activated phosphatidylinositol 3-kinase/Akt (PI3K/Akt) and extracellular signal-regulated kinase (ERK1/2). Selective inhibition of the Akt and ERK1/2 signaling pathways significantly attenuated the rhEPO-induced VEGF expression in neural progenitor cells. The supernatant harvested from neural progenitor cells treated with rhEPO significantly increased the capillary-like tube formation of MBECs. SU1498, a specific VEGF type-2 receptor (VEGFR2) antagonist, abolished the supernatant-enhanced angiogenesis. In addition, coculture of MBECs with neural progenitor cells treated with rhEPO substantially increased VEGFR2 mRNA and protein levels in MBECs. These in vitro results suggest that EPO enhances VEGF secretion in neural progenitor cells through activation of the PI3K/Akt and ERK1/2 signaling pathways and that neural progenitor cells treated with rhEPO upregulate VEGFR2 expression in cerebral endothelial cells, which along with VEGF secreted by neural progenitor cells promotes angiogenesis.
angiogenesis; mouse brain endothelial cell; neural progenitor cell; rhEPO
Biotinidase deficiency is an autosomal recessively inherited disorder characterized by neurological and cutaneous abnormalities. We have developed a transgenic knock-out mouse with biotinidase deficiency to better understand aspects of pathophysiology and natural history of the disorder in humans. Neurological deficits observed in symptomatic mice with biotinidase deficiency are similar to those seen in symptomatic children with the disorder. Using a battery of functional neurological assessment tests, the symptomatic mice performed poorly compared to wild-type mice. Demyelination, axonal degeneration, ventriculomegaly, and corpus callosum compression were found in the brains of untreated, symptomatic enzyme-deficient mice. With biotin treatment, the symptomatic mice improved neurologically and the white matter abnormalities resolved. These functional and anatomical findings and their reversal with biotin therapy are similar to those observed in untreated, symptomatic and treated individuals with biotinidase deficiency. The mouse with biotinidase deficiency appears to be an appropriate animal model in which to study the neurological abnormalities and the effects of treatment of the disorder.
Biotin; Biotin-responsive metabolic disorders; Biotinidase deficiency; Multiple carboxylase; Deficiency; Motor neuron impairment; Somatosensory impairment; Axonal damage; Transgenic knockout model
A consortium of translational stem cell and stroke experts from multiple academic institutes and biotechnology companies, under the guidance of the government (FDA/NIH), is missing. Here, we build a case for the establishment of this consortium if cell therapy for stroke is to advance from the laboratory to the clinic.
Stem cell transplantation; Tissue regeneration; Cellular therapy; Clinical translation
Recent laboratory findings suggest that it might be possible to promote cerebral plasticity and neurological recovery after stroke by use of exogenous pharmacological or cell-based treatments. Brain microvasculature and glial cells respond in concert to ischaemic stressors and treatment, creating an environment in which successful recovery can ensue. Neurons remote from and adjacent to the ischaemic lesion are enabled to sprout, and neural precursor cells that accumulate with cerebral microvessels in the perilesional tissue further stimulate brain plasticity and neurological recovery. These factors interact in a highly dynamic way, facilitating temporally and spatially orchestrated responses of brain networks. In view of the complexity of the systems involved, stroke treatments that stimulate and amplify these endogenous restorative mechanisms might also provoke unwanted side-effects. In experimental studies, adverse effects have been identified when neurorestorative treatments were administered to animals with severe associated illnesses, after thrombolysis with alteplase, and when therapies were initiated outside appropriate time windows. Balancing the opportunities and possible risks, we provide suggestions for the translation of restorative therapies from the laboratory to the clinic.
Pituitary adenylate cyclase activating peptide (PACAP), a potent neuropeptide which crosses the blood–brain barrier, is known to provide neuroprotection in rat stroke models of middle cerebral artery occlusion (MCAO) by mechanism(s) which deserve clarification. We confirmed that following i.v. injection of 30 ng/kg of PACAP38 in rats exposed to 2 h of MCAO focal cerebral ischemia and 48 h reoxygenation, 50 % neuroprotection was measured by reduced caspase-3 activity and volume of cerebral infarction. Similar neuroprotective effects were measured upon PACAP38 treatment of oxygen–glucose deprivation and reoxygenation of brain cortical neurons. The neuroprotection was temporally associated with increased expression of brain-derived neurotrophic factor, phosphorylation of its receptor—tropomyosin-related kinase receptor type B (trkB), activation of phosphoinositide 3-kinase and Akt, and reduction of extracellular signal-regulated kinases 1/2 phosphorylation. PACAP38 increased expression of neuronal markers beta-tubulin III, microtubule-associated protein-2, and growth-associated protein-43. PACAP38 induced stimulation of Rac and suppression of Rho GTPase activities. PACAP38 down-regulated the nerve growth factor receptor (p75NTR) and associated Nogo-(Neurite outgrowth-A) receptor. Collectively, these in vitro and in vivo results propose that PACAP exhibits neuroprotective effects in cerebral ischemia by three mechanisms: a direct one, mediated by PACAP receptors, and two indirect, induced by neurotrophin release, activation of the trkB receptors and attenuation of neuronal growth inhibitory signaling molecules p75NTR and Nogo receptor.
Stroke; Apoptosis; Neuroprotection; PACAP; BDNF; trkB; p75; NgR; Akt; Erk1/2
Traumatic brain injury (TBI) is a leading cause of mortality and morbidity in both civilian life and the battlefield worldwide. Survivors of TBI frequently experience long-term disabling changes in cognition, sensorimotor function and personality. Over the past three decades, animal models have been developed to replicate the various aspects of human TBI, to better understand the underlying pathophysiology and to explore potential treatments. Nevertheless, promising neuroprotective drugs, which were identified to be effective in animal TBI models, have all failed in phase II or phase III clinical trials. This failure in clinical translation of preclinical studies highlights a compelling need to revisit the current status of animal models of TBI and therapeutic strategies.
Astrocytes have not been a major therapeutic target for the treatment of stroke, with most research emphasis on the neuron. Given the essential role that astrocytes play in maintaining physiological function of the central nervous system and the very rapid and sensitive reaction astrocytes have in response to cerebral injury or ischemic insult, we propose to replace the neurocentric view for treatment with a more nuanced astrocytic centered approach. In addition, after decades of effort in attempting to develop neuroprotective therapies, which target reduction of the ischemic lesion, there are no effective clinical treatments for stroke, aside from thrombolysis with tissue plasminogen activator, which is used in a small minority of patients. A more promising therapeutic approach, which may affect nearly all stroke patients, may be in promoting endogenous restorative mechanisms, which enhance neurological recovery. A focus of efforts in stimulating recovery post stroke is the use of exogenously administered cells. The present review focuses on the role of the astrocyte in mediating the brain network, brain plasticity, and neurological recovery post stroke. As a model to describe the interaction of a restorative cell-based therapy with astrocytes, which drives recovery from stroke, we specifically highlight the subacute treatment of stroke with multipotent mesenchymal stromal cell therapy.
stroke; marrow stromal cells; microRNA; exosomes; Shh; tPA; restoration; plasticity
Invasion and angiogenesis are two major pathophysiological features of malignant gliomas. Anti-angiogenic treatment lead to enhanced tumor cell invasion and metastasis. In the current study, we tested invasion and angiogenesis related mRNA expression profiles of glioma cells via RT2Profiler PCR Array by employing an in vivo 9L homograft glioma tumor animal model and an in vitro hypoxic cell culture model. The miRNA profile was also obtained via miRNA array. Genes with mRNA expression that changed significantly in the mRNA array were selected to predict possible miRNAs that regulate mRNA expression using the TargetScan database, and were then matched with miRNA array results. Based on these criteria, NRP-2 with the matching miRNA miR-15b, and MMP-3 with the matching miRNA miR-152 were selected for further study, and to determine whether they regulate tumor microenviroment changes and affect glioma angiogenesis and invasion. The protein expression of NRP-2 and MMP-3 were verified in 9L glioma cells and were negatively correlated to miR-15b and miR-152 level, respectively. Rat astrocytes (primary and cell line), when co-cultured with 9L glioma cells, showed significantly elevated NRP-2, MMP-3 expression and reduced miR-15b, miR-152 expression compared to non co-cultured astrocytes. Luciferase activity assay confirmed that miR-15b and miR-152 attenuate expression of NRP-2 and MMP-3 protein by binding to NRP-2 and MMP-3 transcript, respectively. In vitro invasion assay data showed that miR-15b and miR-152 significantly decreased 9L cell invasiveness. Anti-miR-15b and anti-miR-152 inhibitors counteracted the inhibition of invasion caused by miR-15b and miR-152. In vitro tube formation assay data showed that miR-15b, but not miR-152, reduced tube formation in cultured endothelial cells, and anti-miR-15b inhibitor counteracted the inhibition of tube formation caused by miR-15b. A preliminary pathway study indicated that miR-15b and miR-152 deactivated the MEK-ERK pathway via NRP-2 and MMP-3 in 9L cells, respectively. In conclusion, our current study indicates that miR-15b reduces invasion of glioma cells and angiogenesis via NRP-2, and miR-152 reduces invasion of glioma cells through MMP-3.
Glioma; tumor model; angiogenesis; invasion; miR-15b; miR-152; NRP-2; MMP-3
The aim of this study was to investigate the effect of mild and severe TBI on young male Wistar rats' spatial learning.
Randomized repeated measure experimental design was used to examine spatial learning in three independent animal groups.
Methods and procedures
Twenty-four (severe n = 9, mild n = 8, sham n = 7) male rats were included in the study. Animals received controlled mild (1.5 mm), severe (2.5 mm) cortical impact injury or sham surgery. Spatial learning was assessed daily using a modified Morris water maze test, 20 days post-trauma, for 5 consecutive days. Percentage time travelled within each quadrant and escape latency were calculated. All animals' hippocampal brain regions were examined post-injury using neuron (MAP2) and pre-synaptic protein (Synaptophysin) biomarkers.
Main outcomes and results
It took the animals with mild injury until day 3 to reach the platform; and animals with mild and severe injury spent significantly less time in the target quadrant than the sham. The hippocampal neuron numbers differed proportionately between animals with severe and mild injury, but the percentage of synaptophysin density was significantly less in the dentate gyrus of both animals with mild and severe injury than sham group.
Persistent spatial learning deficits exist after mild TBI; these deficits appear equivalent to deficits exhibited after a more severe injury.
Hippocampus; synaptophysin; Morris water maze
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).
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.
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).
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.
Intracerebral hemorrhage; Atorvastatin; Occludin; Simvastatin; Blood brain barrier
Human Placenta-Derived Adherent Cells (PDAC®) are a novel mesenchymal-like cell population derived from normal human placental tissue. PDA-001 is a clinical formulation of PDAC® developed for intravenous administration. In this study, we investigated the efficacy of PDA-001 treatment in a rat model of transient middle cerebral artery occlusion (MCAo) in young adult (2–3 month old) and older rats (10–12 months old).
To evaluate efficacy and determine the optimal number of transplanted cells, young adult Wistar rats were subjected to MCAo and treated 1 day post MCAo with 1×106, 4×106 or 8×106 PDA-001 cells (i.v.), vehicle or cell control. 4×106 or 8×106 PDA-001 cells were also tested in older rats after MCAo. Treatment response was evaluated using a battery of functional outcome tests, consisting of adhesive-removal test, modified Neurological Severity Score (mNSS) and foot-fault test. Young adult rats were sacrificed 56 days after MCAo, older rats were sacrificed 29 days after MCAo, and lesion volumes were measured using H&E. Immunohistochemical stainings for bromodeoxyuridine (BrdU) and von Willebrand Factor (vWF), and synaptophysin were performed.
In young adult rats, treatment with 4×106 PDA-001 cells significantly improved functional outcome after stroke (p<0.05). In older rats, significant functional improvement was observed with PDA-001 cell therapy in both of the 4×106 and 8×106 treatment groups. Functional benefits in young adult and older rats were associated with significant increases in the number of BrdU immunoreactive endothelial cells, vascular density and perimeter in the ischemic brain, as well as significantly increased synaptophysin expression in the ischemic border zone (p<0.05).
PDA-001 treatment significantly improved functional outcome after stroke in both young adult and older rats. The neurorestorative effects induced by PDA-001 treatment may be related to increased vascular density and synaptic plasticity.
Background and Purpose
GW3965, a synthetic liver X receptor agonist, elevates high-density lipoprotein cholesterol (HDL-C) and has anti-atherosclerosis and anti-inflammation properties. We tested the hypothesis that GW3965 treatment of stroke increases vascular remodeling, promotes synapticprotein expression and axonal growth in the ischemic brain and improves functional outcome in mice.
Mice were subjected to transient middle cerebral artery occlusion (MCAo) and treated without or with different doses of GW3965 (5, 10 or 20 mg/kg) starting 24 hours after MCAo daily for 14 days. Neurological functional tests, blood HDL-C measurement, and immunostaining were performed. Mouse brain endothelial cells, primary cultured artery explants and primary cortical neurons cultures were also employed in vitro.
GW3965 treatment of stroke significantly increased blood HDL-C level, synaptic protein expression, axonal density, angiogenesis and arteriogenesis, and Angiopoietin1, Tie2 and occludin expression in the ischemic brain and improved functional outcome compared with MCAo control animals (P<0.05, n=10). In vitro, GW3965 and HDL also significantly increased capillary-like tube formation and artery explant cell migration as well as neurite outgrowth. Inhibition o f A n giopoietin1 attenuated GW3965-induced tube-formation, artery cell migration and neurite outgrowth (P<0.05, n=6/group).
These data indicate, for the first time, that GW3965 promotessynaptic protein expression, axonal growth, and increases vascular remodeling which may contribute to improvement of functional outcome after stroke. Increasing Angiopoietin1/Tie2 signaling activity may play an important role in GW3965-induced brain plasticity and neurological recovery from stroke.
LXR agonist; HDL-cholesterol; axonal plasticity; vascular remodeling; Angiopoietin1; stroke
Background and Purpose
We sought to demonstrate the contribution of axonal remodeling of the corticospinal tract (CST) in the spinal cord to functional outcome after stroke.
Bilateral pyramidotomy (BPT) or sham-BPT was performed in mice with transgenic yellow fluorescent protein labeling in the CST subjected to middle cerebral artery occlusion (MCAo). Foot-fault and single pellet reaching tests were performed 3 days after MCAo and weekly thereafter. Mice were euthanized at day 14 or 28 after stroke. Immunofluorescent staining for growth-associated protein-43 and Synaptophysin was performed on cervical sections.
Functional improvements were evident during the initial 14 days in both MCAo-sham-BPT and MCAo-BPT mice (P<0.01, versus day 3). Progressive recovery was present during the subsequent 14 days in MCAo-sham-BPT mice (P<0.001, versus day 14) but not in MCAo-BPT mice. In the stroke-affected cervical gray matter of MCAo-sham-BPT mice, growth-associated protein-43-Cy3 staining on CST axons were significantly increased at day 14 after stroke compared with normal mice (P<0.001), and CST axonal density and Synaptophysin-Cy3 staining of CST-yellow fluorescent protein axonal terminals were significantly increased at day 28 compared with day 14 after MCAo (P<0.001).
Our data demonstrate that voluntary motor recovery is associated with CST axonal outgrowth and synaptic formation in the denervated side of the spinal gray matter during the later phase after stroke, suggesting that the CST axonal plasticity in the spinal cord contributes to neurological recovery.
functional recovery; middle cerebral artery occlusion; neuronal plasticity; pyramidotomy
Bone marrow stem cells participate in tissue repair processes and may have a role in wound healing. Diabetes is characterised by delayed and poor wound healing. We investigated the potential of bone marrow-derived mesenchymal stromal cells (BMSCs) to promote healing of fascial wounds in diabetic rats. After manifestation of streptozotocin (STZ)-induced diabetic state for 5 weeks in male adult Sprague–Dawley rats, healing of fascial wounds was severely compromised. Compromised wound healing in diabetic rats was characterised by excessive polymorphonuclear cell infiltration, lack of granulation tissue formation, deficit of collagen and growth factor [transforming growth factor (TGF-β), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor PDGF-BB and keratinocyte growth factor (KGF)] expression in the wound tissue and significant decrease in biomechanical strength of wounds. Treatment with BMSC systemically or locally at the wound site improved the wound-breaking strength (WBS) of fascial wounds. The improvement in WBS was associated with an immediate and significant increase in collagen levels (types I–V) in the wound bed. In addition, treatment with BMSCs increased the expression of growth factors critical to proper repair and regeneration of the damaged tissue moderately (TGF-β, KGF) to markedly (EGF, VEGF, PDGF-BB). These data suggest that cell therapy with BMSCs has the potential to augment healing of the diabetic wounds.
Bone marrow stromal cells; Collagen; Diabetes; Growth factors; Wound healing
Serum response factor (SRF) is a transcription factor that transactivates actin associated genes, and has been implicated in oligodendrocyte (OL) differentiation. To date, it has not been investigated in cerebral ischemia. We investigated the dynamics of SRF expression after stroke in vivo and the role of SRF in oligodendrocyte differentiation in vitro. Using immunohistochemistry, we found that SRF was upregulated in OLs and OL precursor cells (OPCs) after stroke. Moreover, upregulation of SRF was concurrent with downregulation of the microRNAs (miRNAs) miR-9 and the miR-200 family in the ischemic white matter region, the corpus callosum. Inhibition of SRF activation by CCG-1423, a specific inhibitor of SRF function, blocked OPCs from differentiating into OLs. Over-expression of miR-9 and miR-200 in cultured OPCs suppressed SRF expression and inhibited OPC differentiation. Moreover, co-expression of miR-9 and miR-200 attenuated activity of a luciferase reporter assay containing the Srf 3′ untranslated region (UTR). Collectively, this study is the first to show that stroke upregulates SRF expression in OPCs and OLs, and that SRF levels are mediated by miRNAs and regulate OPC differentiation.
Thymosin beta 4 (Tβ4), a G-actin sequestering peptide, increases oligodendrogenesis and improves functional outcome in models of neurological injury. The molecular mechanisms of Tβ4 mediated oligodendrogenesis are unclear. The p38 mitogen-activated protein kinase (p38MAPK) regulates oligodendrocyte (OL) differentiation and myelin gene expression in other models. Therefore, we investigated p38MAPK signaling pathways. We used primary rat neural progenitor cells (NPCs) and a mouse oligodendrocyte progenitor cell (OPC) line (N20.1 cells) to investigate the molecular mechanisms of Tβ4-enhanced oligodendrogenesis. NPCs were isolated from rat subventricular zone (SVZ) of the lateral ventricles (n=12). Primary NPCs and N20.1 cells were grown in the presence of 0, 25 and 50 ng/ml of Tβ4 (RegeneRx Biopharmaceuticals Inc, Rockville, MD) for 14 days. Quantitative real-time PCR and Western blot data showed significant induction of both expression and phosphorylation of p38MAPK with simultaneous inhibition of phosphorylation of extracellular signal regulated kinase (ERK1), c-Jun N-terminal kinase 1 (JNK1), leading to reduction of phosphorylation of c-Jun, a potent negative regulator of transcription of myelin genes. These effects were reversed with transfection of Tβ4siRNA. Our data indicate that Tβ4 treatment induces OL differentiation by inducing p38MAPK with parallel inactivation of ERK1 and JNK1, thus preventing the accumulation of phosphorylated c-Jun.
N20.1 cells; subventricular zone; neruospheres; actin binding proteins
Peripheral neuropathy is one of the most common complications of diabetes mellitus. Using a mouse model of diabetic peripheral neuropathy, we tested the hypothesis that thymosin β 4 (Tβ4) ameliorates diabetes–induced neurovascular dysfunction in the sciatic nerve and promotes recovery of neurological function from diabetic peripheral neuropathy. Tβ4 treatment of diabetic mice increased functional vascular density and regional blood flow in the sciatic nerve, and improved nerve function. Tβ4 upregulated angiopoietin-1 (Ang1) expression, but suppressed Ang2 expression in endothelial and Schwann cells in the diabetic sciatic nerve. In vitro, incubation of Human Umbilical Vein Endothelial Cells (HUVECs) with Tβ4 under high glucose condition completely abolished high glucose-downregulated Ang1 expression and high glucose-reduced capillary-like tube formation. Moreover, incubation of HUVECs under high glucose with conditioned medium collected from Human Schwann cells (HSCs) treated with Tβ4 significantly reversed high glucose-decreased capillary-like tube formation. PI3K/Akt signaling pathway is involved in Tβ4-regulated Ang1 expression on endothelial and Schwann cells. These data indicate that Tβ4 likely acts on endothelial cells and Schwann cells to preserve and/or restore vascular function in the sciatic nerve which facilitates improvement of peripheral nerve function under diabetic neuropathy. Thus, Tβ4 has potential for the treatment of diabetic peripheral neuropathy.
Tβ4; peripheral neuropathy; diabetes; mice