Ruizhuo Ning

Treatment of traumatic brain injury in mice with bone marrow stromal cell–impregnated collagen scaffolds

OBJECT This study was designed to investigate new ways of delivering human marrow stromal cells (hMSCs) to the injured brain by impregnating them into collagen scaffolds in a mouse model of traumatic brain injury (TBI). METHODS Eight C57BL/6 J mice were injured with controlled cortical impact and received transplantation into the lesion cavity of 0.3 x 10(6) hMSCs impregnated into 3D porous collagen scaffolds. Additional experimental groups of 8 mice each received scaffolds implanted alone into the lesion cavity, hMSCs administered alone intracerebrally or intravenously, or saline injected into the lesion core. All treatments were performed 7 days after TBI. Spatial learning was measured using a modified Morris water maze test, and brain tissue samples were processed for histopathological analysis. RESULTS The results showed that hMSC-impregnated scaffolds were more effective than hMSCs administered alone (either intravenously or intracerebrally) in improving spatial learning, reducing lesion volume, and increasing vascular density after TBI. CONCLUSIONS Collagen scaffolds populated with hMSCs may be a new way to reconstruct injured brain tissue and improve neurological function after TBI.

Erythropoietin promotes neurovascular remodeling and long-term functional recovery in rats following traumatic brain injury☆

Erythropoietin (EPO) improves functional recovery after traumatic brain injury (TBI). This study was designed to investigate long-term (3 months) effects of EPO on brain remodeling and functional recovery in rats after TBI. Young male Wistar rats were subjected to unilateral controlled cortical impact injury. TBI rats were divided into the following groups: (1) saline group (n=7); (2) EPO-6h group (n=8); and (3) EPO-24h group (n=8). EPO (5000 U/kg in saline) was administered intraperitoneally at 6h, and 1 and 2 days (EPO-6h group) or at 1, 2, and 3 days (EPO-24h group) postinjury. Neurological function was assessed using a modified neurological severity score, footfault and Morris water maze tests. Animals were sacrificed at 3 months after injury and brain sections were stained for immunohistochemical analyses. Compared to the saline, EPO-6h treatment significantly reduced cortical lesion volume, while EPO-24h therapy did not affect the lesion volume (P<0.05). Both the EPO-6h and EPO-24h treatments significantly reduced hippocampal cell loss (P<0.05), promoted angiogenesis (P<0.05) and increased endogenous cellular proliferation (BrdU-positive cells) in the injury boundary zone and hippocampus (P<0.05) compared to saline controls. Significantly enhanced neurogenesis (BrdU/NeuN-positive cells) was seen in the dentate gyrus of both EPO groups compared to the saline group. Both EPO treatments significantly improved long-term sensorimotor and cognitive functional recovery after TBI. In conclusion, the beneficial effects of posttraumatic EPO treatment on injured brain persisted for at least 3 months. The long-term improvement in functional outcome may in part be related to the neurovascular remodeling induced by EPO.

Neurorestorative Therapy of Stroke in Type 2 Diabetes Mellitus Rats Treated With Human Umbilical Cord Blood Cells

Background and Purpose— Diabetes mellitus is a high-risk factor for ischemic stroke. Diabetic stroke patients suffer worse outcomes, poor long-term recovery, risk of recurrent strokes, and extensive vascular damage. We investigated the neurorestorative effects and the underlying mechanisms of stroke treatment with human umbilical cord blood cells (HUCBCs) in type 2 diabetes mellitus (T2DM) rats. Methods— Adult male T2DM rats were subjected to 2 hours of middle cerebral artery occlusion (MCAo). Three days after MCAo, rats were treated via tail-vein injection with (1) PBS and (2) HUCBCs (5×106), n=10 per group. Results— HUCBC stroke treatment initiated 3 days after MCAo in T2DM rats did not significantly decrease blood–brain barrier leakage (P=0.1) and lesion volume (P=0.078), but significantly improved long-term functional outcome and decreased brain hemorrhage (P<0.05) when compared with the PBS-treated T2DM MCAo control group. HUCBC treatment significantly promoted white matter remodeling as indicated by increased expression of Bielschowsky silver (axons marker), Luxol fast blue (myelin marker), SMI-31 (neurofilament), and Synaptophysin in the ischemic border zone. HUCBC promoted vascular remodeling and significantly increased arterial and vascular density. HUCBC treatment of stroke in T2DM rats significantly increased M2 macrophage polarization (increased M2 macrophage, CD163and CD 206; decreased M1 macrophage, ED1 and inducible nitric oxide synthase expression) in the ischemic brain compared with PBS-treated T2DM MCAo controls (P<0.05). HUCBC also significantly decreased proinflammatory factors, that is, matrix metalloproteinase 9, receptor for advanced glycation end products and toll-like receptor 4 expression in the ischemic brain. Conclusions— HUCBC treatment initiated 3 days after stroke significantly increased white matter and vascular remodeling in the ischemic brain as well as decreased neuroinflammatory factor expression in the ischemic brain in T2DM rats and promoted M2 macrophage polarization. HUCBC reduction of neuroinflammation and increased vascular and white matter axonal remodeling may contribute to the HUCBC-induced beneficial effects in T2DM stroke rats.

Persistent Cerebrovascular Damage After Stroke in Type Two Diabetic Rats Measured by Magnetic Resonance Imaging

Background and Purpose— Diabetes mellitus is a disease with vascular components. Consequently, the blood–brain barrier disruption after stroke may differ between diabetic and nondiabetic animals. However, few studies have documented the longitudinal blood–brain barrier disruption afte stroke in diabetic animals. In this study, using MRI, we noninvasively evaluated the blood–brain barrier damage after middle cerebral artery occlusion in diabetic and nondiabetic rats. Methods— Type 2 diabetes mellitus (T2DM) was induced in adult male Wistar rats by administration of a high-fat diet in combination with a single intraperitoneal injection (35 mg/kg) of streptozotocin. T2DM rats (n=9) and nondiabetic wild-type (WT) rats (n=9) were subjected to middle cerebral artery occlusion for 2 hours using the filament model. MRI was performed 1 day and then weekly for 5 weeks after middle cerebral artery occlusion for all rats. Results— The ischemic lesion volumes after stroke as measured using T2 maps were not significantly different between the T2DM and WT rats. Compared with the WT rats, the volumes of blood–brain barrier disruption evaluated using contrast-enhanced T1-weighted imaging with gadolinium-diethylenetriamine penta-acetic acid and the cerebral hemorrhagic volumes measured with susceptibility-weighted imaging were significantly (P<0.05) larger in the T2DM rats from 1 to 5 weeks after stroke; values of diffusion fractional anisotropy were significantly lower in T2DM rats (P<0.03) than in WT rats after stroke. These MRI measurements were consistent with histological data. Conclusions— Using MRI, T2-weighted imaging did not detect significant differences of the ischemic lesion volumes between T2DM and WT rats. In contrast to the WT rats, however, contrast-enhanced T1-weighted imaging and susceptibility-weighted imaging identified much more severe ischemic vascular damage, whereas fractional anisotropy demonstrated lower axonal density in the T2DM rats after stroke.

Combination treatment of stroke with sub-therapeutic doses of Simvastatin and human umbilical cord blood cells enhances vascular remodeling and improves functional outcome

Human umbilical cord blood cells (HUCBCs) have been employed as a restorative treatment for experimental stroke. In this study, we investigated whether transplantation of sub-therapeutic doses of HUCBCs and Simvastatin enhances cerebral vascular remodeling after stroke. Adult male Wistar rats (n=34) were subjected to transient middle cerebral artery occlusion (MCAo) and treated with: phosphate-buffered solution (PBS, gavaged daily for 7 days); Simvastatin (0.5mg/kg, gavaged daily for 7 days); HUCBCs (1×10(6), injected once via tail vein); and combination Simvasatin with HUCBCs, starting at 24h after MCAo. There was no significant difference between Simvastatin- or HUCBC-monotherapy and MCAo-alone group. Combination treatment 24h post-stroke significantly increased the perimeter of von Willebrand factor (vWF)-positive vessels, the diameter and density of alpha smooth muscle actin (αSMA)-positive arteries, and the percentage of 5-bromodeoxyuridine (BrdU)-positive endothelial cells (ECs) in the ischemic boundary zone (IBZ) compared with MCAo-alone or HUCBC-monotherapy 14 days after MCAo (p<0.05, n=8/group); Combination treatment significantly increased the densities of vWF-vessels and αSMA-arteries as well as the densities of BrdU-ECs and BrdU-positive smooth muscle cells (SMCs) in vascular walls in the IBZ compared with Simvastatin-monotherapy. Moreover, the increased BrdU-ECs and BrdU-SMCs were significantly correlated with neurological functional outcome 14 days after MCAo. Combination treatment also significantly increased the expression of Angiopoietin-1 (Ang1), Tie2 and Occludin in the IBZ (p<0.05, n=8/group). The in vitro experiments showed that combination treatment and Ang1 significantly increased capillary-like tube formation and arterial cell migration; anti-Ang1 significantly reduced combination treatment-induced tube-formation and artery cell migration (p<0.05, n=6/group). These findings indicated that a combination of sub-therapeutic doses of Simvastatin and HUCBCs treatment of stroke increases Ang1/Tie2 and Occludin expression in the ischemic brain, amplifies endogenous angiogenesis and arteriogenesis, and enhances vascular remodeling which in concert may contribute to functional outcome after stroke.

Combination BMSC and Niaspan Treatment of Stroke Enhances White Matter Remodeling and Synaptic Protein Expression in Diabetic Rats

Objective White matter remodeling plays an important role in neurological recovery after stroke. Bone marrow stromal cells (BMSCs) and Niaspan, an agent which increases high density lipoprotein (HDL), each induces neurorestorative effects and promotes white matter remodeling after stroke in non-diabetic rats. In this study, we test whether combination of BMSCs with Niaspan induces an enhanced white matter remodeling in the ischemic brain of diabetic rats. Research design and methods Type-1 diabetes (T1DM) rats were subjected to transient middle cerebral artery occlusion (MCAo) and treated with or without BMSCs; Niaspan; and the combination of BMSCs + Niaspan daily for 14 days after MCAo. Immunostaining for white matter remodeling and synaptic protein expression including NG2; CNPase; BS (Bielschowsky silver); LFB (luxol fast blue); Synaptophysin and SMI-31 immunostaining were performed. Results BMSC monotherapy did not regulate NG2 and CNPase expression compared to T1DM control rats. Both, combination of BMSCs + Niaspan treatment, and Niaspan monotherapy significantly increase NG2 and CNPase expression compared to T1DM control. While combination BMSC+Niaspan, BMSC monotherapy and Niaspan monotherapy groups all increase BS, LFB, synaptophysin, and SMI-31 expression in the ischemic brain compared to T1DM-MCAo control. In addition, the combination treatment significantly enhances LFB, SMI-31, and Synaptophysin expression compared to BMSC monotherapy. Conclusions Combination treatment of stroke with BMSCs and Niaspan in T1DM rats increases white matter remodeling and additively increases BMSC monotherapy induced myelination and synaptic plasticity after stroke in T1DM rats.

Neurorestorative Responses to Delayed Human Mesenchymal Stromal Cells Treatment of Stroke in Type 2 Diabetic Rats

Background and Purpose— Comorbidity of diabetes mellitus and stroke results in worse functional outcome, poor long-term recovery, and extensive vascular damage. We investigated the neurorestorative effects and mechanisms of stroke treatment with human bone marrow–derived mesenchymal stromal cells (hMSCs) in type 2 diabetes mellitus (T2DM) rats. Methods— Adult male Wistar rats were induced with T2DM, subjected to 2 hours of middle cerebral artery occlusion (MCAo) and treated via tail-vein injection with (1) PBS (n=8) and (2) hMSCs (n=10; 5×106) at 3 days after MCAo. Results— In T2DM rats, hMSCs administered at 3 days after MCAo significantly improves neurological function without affecting blood glucose, infarct volume, and incidence of brain hemorrhage in comparison to T2DM-MCAo PBS-treated rats. Delayed hMSC treatment of T2DM stroke significantly improves blood–brain barrier integrity, increases vascular and arterial density and cerebral vascular perfusion, and promotes neuroblast cell migration and white matter remodeling as indicated by increased doublecortin, axon, myelin, and neurofilament density, respectively. Delayed hMSC treatment significantly increases platelet-derived growth factor expression in the ischemic brain, decreases proinflammatory M1 macrophage and increases anti-inflammatory M2 macrophage compared to PBS-treated T2DM-MCAo rats. In vitro data show that hMSCs increase subventricular zone explant cell migration and primary cortical neuron neurite outgrowth, whereas inhibition of platelet-derived growth factor decreases hMSC-induced subventricular zone cell migration and axonal outgrowth. Conclusions— In T2DM stroke rats, delayed hMSC treatment significantly improves neurological functional outcome and increases neurorestorative effects and M2 macrophage polarization. Increasing brain platelet-derived growth factor expression may contribute to hMSC-induced neurorestoration.

Deficiency of brain ATP-binding cassette transporter A-1 exacerbates blood-brain barrier and white matter damage after stroke.

Background and Purpose— The ATP-binding cassette transporter A-1 (ABCA1) gene is a key target of the transcription factors liver X receptors. Liver X receptor activation has anti-inflammatory and neuroprotective effects in animal ischemic stroke models. Here, we tested the hypothesis that brain ABCA1 reduces blood–brain barrier (BBB) and white matter (WM) impairment in the ischemic brain after stroke. Methods— Adult brain-specific ABCA1–deficient (ABCA1−B/−B) and floxed-control (ABCA1fl/fl) mice were subjected to permanent distal middle cerebral artery occlusion and were euthanized 7 days after distal middle cerebral artery occlusion. Functional outcome, infarct volume, BBB leakage, and WM damage were analyzed. Results— Compared with ABCA1fl/fl mice, ABCA1−B/−B mice showed marginally (P=0.052) increased lesion volume but significantly increased BBB leakage and WM damage in the ischemic brain and more severe neurological deficits. Brain ABCA1–deficient mice exhibited increased the level of matrix metalloproteinase-9 and reduced the level of insulin-like growth factor 1 in the ischemic brain. BBB leakage was inversely correlated (r=−0.073; P<0.05) with aquaporin-4 expression. Reduction of insulin-like growth factor 1 and aquaporin-4, but upregulation of matrix metalloproteinase-9 expression were also found in the primary astrocyte cultures derived from ABCA1−B/−B mice. Cultured primary cortical neurons derived from C57BL/6 wild-type mice with ABCA1−B/−B astrocyte–conditioned medium exhibited decreased neurite outgrowth compared with culture with ABCA1fl/fl astrocyte–conditioned medium. ABCA1−B/−B primary cortical neurons show significantly decreased neurite outgrowth, which was attenuated by insulin-like growth factor 1 treatment. Conclusions— We demonstrate that brain ABCA1 deficiency increases BBB leakage, WM/axonal damage, and functional deficits after stroke. Concomitant reduction of insulin-like growth factor 1 and upregulation of matrix metalloproteinase-9 may contribute to brain ABCA1 deficiency–induced BBB and WM/axonal damage in the ischemic brain.

The treatment of traumatic brain injury with velcade.

Traumatic brain injury (TBI) elicits a strong inflammatory response that contributes to the acute pathological processes seen following TBI, including cerebral edema and disruption of the blood-brain barrier (BBB), in addition to longer-term neurological damage and cognitive impairment. Proteasome inhibitors reduce vascular thrombotic and inflammatory events and consequently protect vascular function. In the present study we evaluated the neuroprotective effect of Velcade (bortezomib), a potent and selective inhibitor of proteasomes, which is in clinical use for the treatment of multiple myeloma. When administered within 2 h after TBI onset, Velcade reduced inflammatory responses, lesion volume, and neurological functional deficits, and enhanced neuronal survival. Western blot and ELISA showed that Velcade decreased the expression of NF-κB. These results suggest that in the experimental setting, Velcade is an effective neuroprotective agent for the treatment of TBI.

D-4F Decreases White Matter Damage After Stroke in Mice

Background and Purpose— Stroke-induced neuroinflammation and white matter damage are associated with neurological deficits. Whether D-4F, an apolipoprotein A-I mimetic peptide, treatment of stroke decreases neuroinflammation and white matter damage and improves functional outcome has not been investigated. Methods— Adult male C57BL/6 mice were subjected to permanent middle cerebral artery occlusion (MCAo) and were orally administered saline as a vehicle control and different doses of D-4F (2, 4, 8, 16, or 32 mg/kg) starting at 2 h after MCAo and daily until euthanized at 7 days after MCAo. D-4F treatment did not alter the blood levels of high-density lipoprotein, total cholesterol, triglyceride, blood–brain barrier leakage, and infarction volume compared with control group. Results— D-4F (16 mg/kg) treatment of stroke significantly improved functional outcome, increased the white matter density and the number of oligodendrocyte progenitor cells in the ischemic boundary zone of the ipsilateral striatum, and increased myelin basic protein, insulin-like growth factor-1 (IGF1), but decreased inflammatory factor Toll-like receptor-4 and tumor necrosis factor-&agr; expression in the ischemic brain 7 days after MCAo (P<0.05, n=11/group). The neurite/axonal outgrowth in primary cultured neurons was significantly increased when treated with D-4F (100 ng/mL) and IGF1 (100 ng/mL) compared with the nontreatment control. Inhibition of IGF1 significantly attenuated D-4F or IGF1 treatment–induced axonal outgrowth. D-4F-treatment did not increase oligodendrocyte–progenitor cell proliferation but decreased oligodendrocyte–progenitor cell death. Conclusions— D-4F treatment initiated 2 h after MCAo decreases neuroinflammation and white matter damage and improves functional outcome after stroke. D-4F-induced increase in IGF1 may contribute to D-4F–induced neurite/axonal outgrowth after stroke.