Cynthia Roberts

Endothelial Nitric Oxide Synthase Regulates Brain-Derived Neurotrophic Factor Expression and Neurogenesis after Stroke in Mice

Here, we investigate the effects of endothelial nitric oxide synthase (eNOS) on angiogenesis, neurogenesis, neurotrophic factor expression, and neurological functional outcome after stroke. Wild-type and eNOS knock-out (eNOS-/-) mice were subjected to permanent occlusion of the right middle cerebral artery. eNOS-/- mice exhibited more severe neurological functional deficit after stroke than wild-type mice. Decreased subventricular zone (SVZ) progenitor cell proliferation and migration, measured using bromodeoxyuridine, Ki-67, nestin, and doublecortin immunostaining in the ischemic brain, and decreased angiogenesis, as demonstrated by reduced endothelial cell proliferation, vessel perimeter, and vascular density in the ischemic border, were evident in eNOS-/- mice compared with wild-type mice. eNOS-deficient mice also exhibited a reduced response to vascular endothelial growth factor (VEGF)-induced angiogenesis in a corneal assay. ELISAs showed that eNOS-/- mice have decreased brain-derived neurotrophic factor (BDNF) expression but not VEGF and basic fibroblast growth factor in the ischemic brain compared with wild-type mice. In addition, cultured SVZ neurosphere formation, proliferation, telomerase activity, and neurite outgrowth but not cell viability from eNOS-/- mice were significantly reduced compared with wild-type mice. BDNF treatment of SVZ cells derived from eNOS-/- mice restored the decreased neurosphere formation, proliferation, neurite outgrowth, and telomerase activity in cultured eNOS-/- SVZ neurospheres. SVZ explant cell migration also was significantly decreased in eNOS-/- mice compared with wild-type mice. These data indicate that eNOS is not only a downstream mediator for VEGF and angiogenesis but also regulates BDNF expression in the ischemic brain and influences progenitor cell proliferation, neuronal migration, and neurite outgrowth and affects functional recovery after stroke.

Angiopoietin1/Tie2 and VEGF/Flk1 induced by MSC treatment amplifies angiogenesis and vascular stabilization after stroke

Bone marrow stromal cells (MSCs) increase vascular endothelial growth factor (VEGF) expression and promote angiogenesis after stroke. Angiopoietin-1 (Ang1) and its receptor Tie2 mediate vascular integrity and angiogenesis as does VEGF and its receptors. In this study, we tested whether MSC treatment of stroke increases Ang1/Tie2 expression, and whether Ang1/Tie2 with VEGF / vascular endothelial growth factor receptor 2 (VEGFR2) (Flk1), in combination, induced by MSCs enhances angiogenesis and vascular integrity. Male Wistar rats were subjected to middle cerebral artery occlusion (MCAo) and treated with or without MSCs. Marrow stromal cell treatment significantly decreased blood—brain barrier (BBB) leakage and increased Ang1, Tie2, and occludin (a tight junction protein) expression in the ischemic border compared with MCAo control. To further test the mechanisms of MSC-induced angiogenesis and vascular stabilization, cocultures of MSCs with mouse brain endothelial cells (MBECs) or astrocytes were performed. Supernatant derived from MSCs cocultured with MBECs significantly increased MBEC expression of Ang1/Tie2 and Flk1 compared with MBEC alone. Marrow stromal cells cocultured with astrocytes also significantly increased astrocyte VEGF and Ang1/Tie2 expression compared with astrocyte culture alone. Conditioned media from MSCs alone, and media from cocultures of MSCs with astrocytes or MBECs, all significantly increased capillary tube-like formation of MBEC compared with control Dulbeccos modified Eagles medium media. Inhibition of Flk1 and/or Ang1 significantly decreased MSC-induced MBEC tube formation. Knockdown of Tie2 expression in MBECs significantly inhibited MSC-induced tube formation. Our data indicate MSC treatment of stroke promotes angiogenesis and vascular stabilization, which is at least partially mediated by VEGF/Flk1 and Ang1/Tie2.

Niaspan increases angiogenesis and improves functional recovery after stroke

High‐density lipoprotein (HDL) is implicated in the modulation of angiogenesis. In this study, we investigated whether the Niacin‐mediated increase of HDL regulates angiogenesis and thereby improves functional outcome after stroke.

Adverse Effects of Bone Marrow Stromal Cell Treatment of Stroke in Diabetic Rats

Background and Purpose— Cell therapy with bone marrow stromal cells (BMSCs) improves functional recovery after stroke in nondiabetic rats. However, its effect on diabetics with stroke is unknown. This study investigated the effect of BMSCs on stroke outcome in Type 1 diabetic (T1DM) rats. Methods— T1DM was induced in adult male Wistar rats by injecting streptozotocin. Nondiabetic and T1DM rats were subjected to 2 hours of middle cerebral artery occlusion (MCAO), treated with or without BMSCs (3×106) at 24 hours after MCAO, and monitored for 14 days. Results— Functional benefit was not detected in T1DM-MCAO treated with BMSC rats compared with corresponding T1DM-MCAO controls. BMSC treatment in T1DM-MCAO rats had increased mortality, blood–brain barrier leakage, brain hemorrhage, and angiogenesis. Internal carotid artery neointimal formation and cerebral arteriole narrowing/occlusion were also observed in T1DM-MCAO+BMSCs rats compared with T1DM-MCAO controls (P<0.05), but not in nondiabetic stroke rats. We further studied the underlying mechanisms responsible for BMSC-induced blood–brain barrier leakage and accelerated vascular damage in T1DM-MCAO rats. We found that the expression of angiogenin (an angiogenic factor) and ED1 (a marker for macrophages) was significantly increased in the T1DM-MCAO+BMSC rats in the ischemic brain and internal carotid artery compared with nontreated T1DM-MCAO rats, but not in nondiabetic stroke rats. Conclusions— BMSC therapy in T1DM-MCAO rats does not improve functional outcome. On the contrary, it increases blood–brain barrier leakage and cerebral artery neointimal formation, and arteriosclerosis, which possibly is due to increased expression of angiogenin. Thus, BMSC treatment starting 24 hours after MCAO may not be beneficial for diabetic subjects with stroke.

Axonal Outgrowth and Dendritic Plasticity in the Cortical Peri-Infarct Area After Experimental Stroke

Background and Purpose— Axonal remodeling is critical to brain repair after stroke. The present study investigated axonal outgrowth after stroke and the signaling pathways mediating axonal outgrowth in cortical neurons. Methods— Using a rodent model of middle cerebral artery occlusion, we examined high-molecular weight neurofilament (NFH) immunoreactive axons and myelin basic protein-positive oligodendrocytes in the peri-infarct area. In vitro, using cultured cortical neurons in a microfluidic chamber challenged by oxygen-glucose deprivation (OGD), we investigated mechanisms selectively regulating axonal outgrowth after OGD. Results— NFH+ axons and MBP+ oligodendrocytes substantially increased in the peri-infarct area during stroke recovery, concomitantly with an increase in dendrites and spines identified by Golgi-Cox staining. In vitro, cortical neurons subjected to OGD exhibited significant increases in axonal outgrowth and in phosphorylated NFH protein levels, concurrently with downregulation of phosphatase tensin homolog deleted on chromosome 10, activation of Akt, and inactivation of glycogen synthase kinase-3&bgr; in regenerated axons. Blockage of phosphoinositide 3-kinase with pharmacological inhibitors suppressed Akt activation and attenuated phosphorylation of glycogen synthase kinase-3&bgr;, which resulted in suppression of phosphorylated NFH and axonal outgrowth after OGD; whereas GSK-3 inhibitors augmented axonal regeneration and elevated phosphorylated NFH levels after OGD. Conclusions— Stroke induces axonal outgrowth and myelination in rodent ischemic brain during stroke recovery, and the phosphoinositide 3-kinase/Akt/glycogen synthase kinase-3&bgr; signaling pathway mediates axonal regeneration of cortical neurons after OGD.

Beneficial effects of gfap/vimentin reactive astrocytes for axonal remodeling and motor behavioral recovery in mice after stroke

The functional role of reactive astrocytes after stroke is controversial. To elucidate whether reactive astrocytes contribute to neurological recovery, we compared behavioral outcome, axonal remodeling of the corticospinal tract (CST), and the spatio‐temporal change of chondroitin sulfate proteoglycan (CSPG) expression between wild‐type (WT) and glial fibrillary acidic protein/vimentin double knockout (GFAP–/–Vim–/–) mice subjected to Rose Bengal induced cerebral cortical photothrombotic stroke in the right forelimb motor area. 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 CST axons. Compared with WT mice, the motor functional recovery and BDA‐positive CST axonal length in the denervated side of the cervical gray matter were significantly reduced in GFAP–/–Vim–/– mice (n = 10/group, P < 0.01). Immunohistological data showed that in GFAP–/–Vim–/– mice, in which astrocytic reactivity is attenuated, CSPG expression was significantly increased in the lesion remote areas in both hemispheres, but decreased in the ischemic lesion boundary zone, compared with WT mice (n = 12/group, P < 0.001). Our data suggest that attenuated astrocytic reactivity impairs or delays neurological recovery by reducing CST axonal remodeling in the denervated spinal cord. Thus, manipulation of astrocytic reactivity post stroke may represent a therapeutic target for neurorestorative strategies. GLIA 2014;62:2022–2033

White Matter Damage and the Effect of Matrix Metalloproteinases in Type 2 Diabetic Mice After Stroke

Background and Purpose— Diabetes mellitus leads to a higher risk of ischemic stroke and worse outcome compared to the general population. However, there have been few studies on white matter (WM) damage after stroke in diabetes mellitus. We therefore investigated WM damage after stroke in mice with diabetes mellitus. Methods— BKS.Cg-m+/+Leprdb/J (db/db) type 2 diabetes mellitus mice and db+ non-diabetes mellitus mice were subjected to middle cerebral artery occlusion. Functional outcome, immunostaining, zymography, Western blot, and polymerase chain reaction were used. Results— After stroke, mice with diabetes mellitus exhibited significantly increased lesion volume and brain hemorrhagic and neurological deficits compared to mice without diabetes mellitus. Bielshowsky silver, luxol fast blue, amyloid precursor protein, and NG2 expression were significantly decreased, indicating WM damage, and matrix metalloproteinase (MMP)-9 activity was significantly increased in the ischemic brain of mice with diabetes mellitus. Subanalysis of similar lesions in mice with and without diabetes mellitus demonstrated mice with diabetes mellitus had significantly increased WM damage than in mice without diabetes mellitus (P<0.05). To investigate the mechanism underlying diabetes mellitus-induced WM damage, oxygen–glucose deprivation-stressed premature oligodendrocyte and primary cortical neuron cultures were used. High glucose increased MMP-2, MMP-9, cleaved caspase-3 levels, and apoptosis, as well as decreased cell survival and dendrite outgrowth in cultured primary cortical neuron. High glucose increased MMP-9, cleaved caspase-3 level, and apoptosis, and decreased cell proliferation and cell survival in cultured oligodendrocytes. Inhibition of MMP by GM6001 treatment significantly decreased high glucose-induced cell death and apoptosis in cultured primary cortical neuron and oligodendrocytes but did not alter dendrite outgrowth in primary cortical neuron. Conclusions— Mice with diabetes mellitus have increased brain hemorrhage and show more severely injured WM than mice without diabetes mellitus after stroke. MMP-9 upregulated in mice with diabetes mellitus may exacerbate WM damage after stroke in mice with diabetes mellitus.

Nitric Oxide Donor Upregulation of Stromal Cell-Derived Factor-1/Chemokine (CXC Motif) Receptor 4 Enhances Bone Marrow Stromal Cell Migration into Ischemic Brain After Stroke

Stromal cell‐derived factor‐1 (SDF1) and its chemokine (CXC motif) receptor 4 (CXCR4), along with matrix metalloproteinases (MMPs), regulate bone marrow stromal cell (BMSC) migration. We tested the hypothesis that a nitric oxide donor, DETA‐NONOate, increases endogenous ischemic brain SDF1 and BMSC CXCR4 and MMP9 expression, which promotes BMSC migration into ischemic brain and thereby enhances functional outcome after stroke. C57BL/6J mice were subjected to middle cerebral artery occlusion (MCAo), and 24 hours later, the following were intravenously administered (n = 9 mice per group): (a) phosphate‐buffered saline; (b) BMSCs (5 × 105); (c) 0.4 mg/kg DETA‐NONOate; (d) combination of CXCR4‐inhibition BMSCs with DETA‐NONOate; and (e) combination of BMSCs with DETA‐NONOate. To elucidate the mechanisms underlying combination‐enhanced BMSC migration, transwell cocultures of BMSC with mouse brain endothelial cells (MBECs) or astrocytes were performed. Combination treatment significantly improved functional outcome after stroke compared with BMSC monotherapy and MCAo control, and it increased SDF1 expression in the ischemic brain compared with DETA‐NONOate monotherapy and MCAo control. The number of BMSCs in the ischemic brain was significantly increased after combination BMSC with DETA‐NONOate treatment compared with monotherapy with BMSCs. The number of engrafted BMSCs was significantly correlated with functional outcome after stroke. DETA‐NONOate significantly increased BMSC CXCR4 and MMP9 expression and promoted BMSC adhesion and migration to MBECs and astrocytes compared with nontreatment BMSCs. Inhibition of CXCR4 or MMPs in BMSCs significantly decreased DETA‐NONOate‐induced BMSC adhesion and migration. Our data demonstrate that DETA‐NONOate enhanced the therapeutic potency of BMSCs, possibly via upregulation of SDF1/CXCR4 and MMP pathways, and increased BMSC engraftment into the ischemic brain.

NEUROLOGICAL FUNCTIONAL RECOVERY AFTER THYMOSIN BETA4 TREATMENT IN MICE WITH EXPERIMENTAL AUTO ENCEPHALOMYELITIS

In the present study, we hypothesized that thymosin beta 4 (Tbeta4) is a potential therapy of multiple sclerosis (MS). To test this hypothesis, SJL/J mice (n=21) were subjected to experimental autoimmune encephalomyelitis (EAE), an animal model of MS. EAE mice were treated with saline or Tbeta4 (6 mg/kg, n=10) every 3 days starting on the day of myelin proteolipid protein (PLP) immunization for total five doses. Neurological function, inflammatory infiltration, oligodendrocyte progenitor cells (OPCs) and mature oligodendrocytes were measured in the brain of EAE mice. Double immunohistochemical staining was used to detect proliferation and differentiation of OPCs. Tbeta4 was used to treat N20.1 cells (premature oligodendrocyte cell line) in vitro, and proliferation of N20.1 cells was measured by bromodeoxyuridine (BrdU) immunostaining. Tbeta4 treatment improved functional recovery after EAE. Inflammatory infiltrates were significantly reduced in the Tbeta4 treatment group compared to the saline groups (3.6+/-0.3/slide vs 5+/-0.5/slide, P<0.05). NG2(+) OPCs (447.7+/-41.9 vs 195.2+/-31/mm(2) in subventricular zone (SVZ), 75.1+/-4.7 vs 41.7+/-3.2/mm(2) in white matter), CNPase(+) mature oligodendrocytes (267.5+/-10.3 vs 141.4+/-22.9/mm(2)), BrdU(+) with NG2(+) OPCs (32.9+/-3.7 vs 17.9+/-3.6/mm(2)), BrdU(+) with CNPase(+) mature oligodendrocytes (18.2+/-1.7 vs 10.7+/-2.2/mm(2)) were significantly increased in the Tbeta4 treated mice compared to those of saline controls (P<0.05). These data indicate that Tbeta4 treatment improved functional recovery after EAE, possibly, via reducing inflammatory infiltrates, and stimulating oligodendrogenesis.

Chemokine, vascular and therapeutic effects of combination Simvastatin and BMSC treatment of stroke

We investigated the additive therapeutic effect of the 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 (1x10(6)) or combination of Simvastatin and BMSCs starting at 24 h 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 was 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.