CX3CR1-CCR2-dependent monocyte-microglial signaling modulates neurovascular leakage and acute injury in a mouse model of childhood stroke.

Stroke is among the top 10 causes of death in children. The developmental stage of the brain is central to stroke pathophysiology. The incidence of childhood arterial ischemic stroke (CAIS) is lower than of perinatal arterial ischemic stroke but the rate of recurrence is strikingly high. Vascular inflammation is seen as major contributor to CAIS but the mechanisms that govern structural-functional basis of vascular abnormalities remain poorly understood. To identify the contribution of immune-neurovascular interactions to CAIS, we established stroke model in postnatal day 21 (P21) mice. We demonstrate acute functional deficits and histological injury and chronic MRI-identifiable injury, brain atrophy and marked derangements in the vascular network. In contrast to negligible albumin leakage and neutrophil infiltration following acute perinatal stroke, CAIS leads to significantly increased albumin leakage and neutrophil infiltration in injured regions of wild type mice and mice with functional CX3CR1-CCR2 receptors. In mice with dysfunctional CX3CR1-CCR2 signaling, extravascular albumin leakage is significantly attenuated, infiltration of injurious Ccr2+-monocytes essentially aborted, accumulation of Ly6G+ neutrophils reduced and acute injury attenuated. Unique identifiers of microglia and monocytes revealed phenotypic changes in each cell subtype of the monocyte lineage after CAIS. Taken together, CX3CR1-CCR2-dependent microglia-monocyte signaling contributes to cerebrovascular leakage, inflammation and CAIS injury.

Mesenchymal stem cells attenuate MRI-identifiable injury, protect white matter, and improve long-term functional outcomes after neonatal focal stroke in rats.

Cell therapy has emerged as a potential treatment for many neurodegenerative diseases including stroke and neonatal ischemic brain injury. Delayed intranasal administration of mesenchymal stem cells (MSCs) after experimental hypoxia-ischemia and after a transient middle cerebral artery occlusion (tMCAO) in neonatal rats has shown improvement in long-term functional outcomes, but the effects of MSCs on white matter injury (WMI) are insufficiently understood. In this study we used longitudinal T2-weighted (T2W) and diffusion tensor magnetic resonance imaging (MRI) to characterize chronic injury after tMCAO induced in postnatal day 10 (P10) rats and examined the effects of delayed MSC administration on WMI, axonal coverage, and long-term somatosensory function. We show unilateral injury- and region-dependent changes in diffusion fraction anisotropy 1 and 2 weeks after tMCAO that correspond to accumulation of degraded myelin basic protein, astrocytosis, and decreased axonal coverage. With the use of stringent T2W-based injury criteria at 72 hr after tMCAO to randomize neonatal rats to receive intranasal MSCs or vehicle, we show that a single MSC administration attenuates WMI and enhances somatosensory function 28 days after stroke. A positive correlation was found between MSC-enhanced white matter integrity and functional performance in injured neonatal rats. Collectively, these data indicate that the damage induced by tMCAO progresses over time and is halted by administration of MSCs. © 2016 Wiley Periodicals, Inc.

Genetic deletion of galectin-3 enhances neuroinflammation, affects microglial activation and contributes to sub-chronic injury in experimental neonatal focal stroke.

The pathophysiology of neonatal stroke and adult stroke are distinct in many aspects, including the inflammatory response. We previously showed endogenously protective functions of microglial cells in acute neonatal stroke. We asked if galectin-3 (Gal3), a pleotropic molecule that mediates interactions between microglia/macrophages and the extracellular matrix (ECM), plays a role in early injury after transient middle cerebral occlusion (tMCAO) in postnatal day 9-10 mice. Compared to wild type (WT) pups, in Gal3 knockout pups injury was worse and cytokine/chemokine production altered, including further increase of MIP1α and MIP1ß levels and reduced IL6 levels 72h after tMCAO. Lack of Gal3 did not affect morphological transformation or proliferation of microglia but markedly attenuated accumulation of CD11b+/CD45med-high cells after injury, as determined by multi-color flow cytometry. tMCAO increased expression of αV and ß3 integrin subunits in CD11b+/CD45low microglial cells and cells of non-monocyte lineage (CD11b-/CD45-), but not in CD11b+/CD45med-high cells within injured regions of WT mice or Gal3-/- mice. αV upregulated in areas occupied and not occupied by CD68+ cells, most prominently in the ECM, lining blood vessels, with expanded αV coverage in Gal3-/- mice. Cumulatively, these data show that lack of Gal3 worsens subchronic injury after neonatal focal stroke, likely by altering the neuroinflammatory milieu, including an imbalance between pro- and anti-inflammatory molecules, effects on microglial activation, and deregulation of the composition of the ECM.

Microglial Cells Prevent Hemorrhage in Neonatal Focal Arterial Stroke.

Perinatal stroke leads to significant morbidity and long-term neurological and cognitive deficits. The pathophysiological mechanisms of brain damage depend on brain maturation at the time of stroke. To understand whether microglial cells limit injury after neonatal stroke by preserving neurovascular integrity, we subjected postnatal day 7 (P7) rats depleted of microglial cells, rats with inhibited microglial TGFbr2/ALK5 signaling, and corresponding controls, to transient middle cerebral artery occlusion (tMCAO). Microglial depletion by intracerebral injection of liposome-encapsulated clodronate at P5 significantly reduced vessel coverage and triggered hemorrhages in injured regions 24 h after tMCAO. Lack of microglia did not alter expression or intracellular redistribution of several tight junction proteins, did not affect degradation of collagen IV induced by the tMCAO, but altered cell types producing TGFß1 and the phosphorylation and intracellular distribution of SMAD2/3. Selective inhibition of TGFbr2/ALK5 signaling in microglia via intracerebral liposome-encapsulated SB-431542 delivery triggered hemorrhages after tMCAO, demonstrating that TGFß1/TGFbr2/ALK5 signaling in microglia protects from hemorrhages. Consistent with observations in neonatal rats, depletion of microglia before tMCAO in P9 Cx3cr1(GFP/+)/Ccr2(RFP/+) mice exacerbated injury and induced hemorrhages at 24 h. The effects were independent of infiltration of Ccr2(RFP/+) monocytes into injured regions. Cumulatively, in two species, we show that microglial cells protect neonatal brain from hemorrhage after acute ischemic stroke.

Brain tissue oxygen tension and its response to physiological manipulations: influence of distance from injury site in a swine model of traumatic brain injury.

OBJECTIVE The optimal site for placement of tissue oxygen probes following traumatic brain injury (TBI) remains unresolved. The authors used a previously described swine model of focal TBI and studied brain tissue oxygen tension (PbtO2) at the sites of contusion, proximal and distal to contusion, and in the contralateral hemisphere to determine the effect of probe location on PbtO2 and to assess the effects of physiological interventions on PbtO2 at these different sites. METHODS A controlled cortical impact device was used to generate a focal lesion in the right frontal lobe in 12 anesthetized swine. PbtO2 was measured using Licox brain tissue oxygen probes placed at the site of contusion, in pericontusional tissue (proximal probe), in the right parietal region (distal probe), and in the contralateral hemisphere. PbtO2 was measured during normoxia, hyperoxia, hypoventilation, and hyperventilation. RESULTS Physiological interventions led to expected changes, including a large increase in partial pressure of oxygen in arterial blood with hyperoxia, increased intracranial pressure (ICP) with hypoventilation, and decreased ICP with hyperventilation. Importantly, PbtO2 decreased substantially with proximity to the focal injury (contusion and proximal probes), and this difference was maintained at different levels of fraction of inspired oxygen and partial pressure of carbon dioxide in arterial blood. In the distal and contralateral probes, hypoventilation and hyperventilation were associated with expected increased and decreased PbtO2 values, respectively. However, in the contusion and proximal probes, these effects were diminished, consistent with loss of cerebrovascular CO2 reactivity at and near the injury site. Similarly, hyperoxia led to the expected rise in PbtO2 only in the distal and contralateral probes, with little or no effect in the proximal and contusion probes, respectively. CONCLUSIONS PbtO2 measurements are strongly influenced by the distance from the site of focal injury. Physiological alterations, including hyperoxia, hyperventilation, and hypoventilation substantially affect PbtO2 values distal to the site of injury but have little effect in and around the site of contusion. Clinical interpretations of brain tissue oxygen measurements should take into account the spatial relation of probe position to the site of injury. The decision of where to place a brain tissue oxygen probe in TBI patients should also take these factors into consideration.

Lack of the scavenger receptor CD36 alters microglial phenotypes after neonatal stroke.

The stage of brain development at the time of stroke has a major impact on the pathophysiological mechanisms of ischemic damage, including the neuroinflammatory response. Microglial cells have been shown to contribute to acute and subchronic injury in adult stroke models, whereas in neonatal rodents we showed that microglial cells serve as endogenous neuroprotectants early following transient middle cerebral artery occlusion, limiting neuroinflammation and injury. In the neonate, microglial depletion or lack of the scavenger receptor CD36 exacerbates injury. In this study we asked if lack of CD36 affects microglial phenotypes after neonatal stroke. Using RT-PCR we characterized the patterns of gene expression in microglia isolated from injured regions following acute transient middle cerebral artery occlusion in postnatal day 10 mice and showed that expression of several pro-inflammatory genes, including Toll-like receptors, remains largely unaffected in activated microglia in injured regions. Using multiple biochemical assays we demonstrated that lack of CD36 alters several functions of microglia in acutely injured neonatal brain: it further enhances accumulation of the chemokine MCP-1, affects the number of CD11b(+) /CD45(+) cells, along with protein expression of its co-receptor, Toll-like receptor 2, but does not affect accumulation of superoxide in microglia or the cytokines TNFα and IL-1ß in injured regions.

LSR/angulin-1 is a tricellular tight junction protein involved in blood-brain barrier formation.

The blood-brain barrier (BBB) is a term used to describe the unique properties of central nervous system (CNS) blood vessels. One important BBB property is the formation of a paracellular barrier made by tight junctions (TJs) between CNS endothelial cells (ECs). Here, we show that Lipolysis-stimulated lipoprotein receptor (LSR), a component of paracellular junctions at points in which three cell membranes meet, is greatly enriched in CNS ECs compared with ECs in other nonneural tissues. We demonstrate that LSR is specifically expressed at tricellular junctions and that its expression correlates with the onset of BBB formation during embryogenesis. We further demonstrate that the BBB does not seal during embryogenesis in Lsr knockout mice with a leakage to small molecules. Finally, in mouse models in which BBB was disrupted, including an experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis and a middle cerebral artery occlusion (MCAO) model of stroke, LSR was down-regulated, linking loss of LSR and pathological BBB leakage.

Improved approach for chondrogenic differentiation of human induced pluripotent stem cells.

Human induced pluripotent stem cells (hiPSCs) have demonstrated great potential for hyaline cartilage regeneration. However, current approaches for chondrogenic differentiation of hiPSCs are complicated and inefficient primarily due to intermediate embryoid body formation, which is required to generate endodermal, ectodermal, and mesodermal cell lineages. We report a new, straightforward and highly efficient approach for chondrogenic differentiation of hiPSCs, which avoids embryoid body formation. We differentiated hiPSCs directly into mesenchymal stem /stromal cells (MSC) and chondrocytes. hiPSC-MSC-derived chondrocytes showed significantly increased Col2A1, GAG, and SOX9 gene expression compared to hiPSC-MSCs. Following transplantation of hiPSC-MSC and hiPSC-MSC-derived chondrocytes into osteochondral defects of arthritic joints of athymic rats, magnetic resonance imaging studies showed gradual engraftment, and histological correlations demonstrated hyaline cartilage matrix production. Results present an efficient and clinically translatable approach for cartilage tissue regeneration via patient-derived hiPSCs, which could improve cartilage regeneration outcomes in arthritic joints.

Magnetic resonance imaging of stem cell apoptosis in arthritic joints with a caspase activatable contrast agent.

About 43 million individuals in the U.S. encounter cartilage injuries due to trauma or osteoarthritis, leading to joint pain and functional disability. Matrix-associated stem cell implants (MASI) represent a promising approach for repair of cartilage defects. However, limited survival of MASI creates a significant bottleneck for successful cartilage regeneration outcomes and functional reconstitution. We report an approach for noninvasive detection of stem cell apoptosis with magnetic resonance imaging (MRI), based on a caspase-3-sensitive nanoaggregation MRI probe (C-SNAM). C-SNAM self-assembles into nanoparticles after hydrolysis by caspase-3, leading to 90% amplification of (1)H MR signal and prolonged in vivo retention. Following intra-articular injection, C-SNAM causes significant MR signal enhancement in apoptotic MASI compared to viable MASI. Our results indicate that C-SNAM functions as an imaging probe for stem cell apoptosis in MASI. This concept could be applied to a broad range of cell transplants and target sites.

Reduced brain edema and infarct volume in aquaporin-4 deficient mice after transient focal cerebral ischemia.

Aquaporin-4 (AQP4) is a water channel expressed in astrocyte end-feet lining the blood-brain barrier. AQP4 deletion in mice is associated with improved outcomes in global cerebral ischemia produced by transient carotid artery occlusion, and focal cerebral ischemia produced by permanent middle cerebral artery occlusion (MCAO). Here, we investigated the consequences of 1-h transient MCAO produced by intraluminal suture blockade followed by 23 h of reperfusion. In nine AQP4(+/+) and nine AQP4(-/-) mice, infarct volume was significantly reduced by an average of 39 ± 4% at 24h in AQP4(-/-) mice, cerebral hemispheric edema was reduced by 23 ± 3%, and Evans Blue extravasation was reduced by 31 ± 2% (mean ± SEM). Diffusion-weighted magnetic resonance imaging showed greatest reduction in apparent diffusion coefficient around the occlusion site after reperfusion, with remarkably lesser reduction in AQP4(-/-) mice. The reduced infarct volume in AQP4(-/-) mice following transient MCAO supports the potential utility of therapeutic AQP4 inhibition in stroke.

Iron administration before stem cell harvest enables MR imaging tracking after transplantation.

PURPOSE: To determine whether intravenous ferumoxytol can be used to effectively label mesenchymal stem cells (MSCs) in vivo and can be used for tracking of stem cell transplants. MATERIALS AND METHODS: This study was approved by the institutional animal care and use committee. Sprague-Dawley rats (6-8 weeks old) were injected with ferumoxytol 48 hours prior to extraction of MSCs from bone marrow. Ferumoxytol uptake by these MSCs was evaluated with fluorescence, confocal, and electron microscopy and compared with results of traditional ex vivo-labeling procedures. The in vivo-labeled cells were subsequently transplanted in osteochondral defects of 14 knees of seven athymic rats and were evaluated with magnetic resonance (MR) imaging up to 4 weeks after transplantation. T2 relaxation times of in vivo-labeled MSC transplants and unlabeled control transplants were compared by using t tests. MR data were correlated with histopathologic results. RESULTS: In vivo-labeled MSCs demonstrated significantly higher ferumoxytol uptake compared with ex vivo-labeled cells. With electron microscopy, iron oxide nanoparticles were localized in secondary lysosomes. In vivo-labeled cells demonstrated significant T2 shortening effects in vitro and in vivo when they were compared with unlabeled control cells (T2 in vivo, 15.4 vs 24.4 msec; P < .05) and could be tracked in osteochondral defects for 4 weeks. Histologic examination confirmed the presence of iron in labeled transplants and defect remodeling. CONCLUSION: Intravenous ferumoxytol can be used to effectively label MSCs in vivo and can be used for tracking of stem cell transplants with MR imaging. This method eliminates risks of contamination and biologic alteration of MSCs associated with ex vivo-labeling procedures.

Acute and chronic vascular responses to experimental focal arterial stroke in the neonate rat.

The presence of active developmental angiogenesis and vascular outgrowth in the postnatal brain may differentially affect vascular responses to stroke in newborns and adults, but very little is known about the dynamics of vascular injury and re-growth after stroke during the neonatal period. In this study we used a clinically relevant animal model of ischemic arterial stroke in neonate rats, a transient middle cerebral artery occlusion (MCAO) in postnatal day 7 (P7), to characterize the effects of injury on vascular density and angiogenesis from acute through the chronic phase. A marked vessel degeneration and suppressed endothelial cell proliferation occur in the ischemic regions early after neonatal stroke. In contrast to what has been described in adult animals, endothelial cell proliferation and vascular density are not increased in the peri-ischemic regions during the first week after MCAO in neonates. By two weeks after injury, endothelial cell proliferation is increased in the cortical peri-ischemic region but these changes are not accompanied by an increased vascular density. Suppressed angiogenesis in injured postnatal brain that we report may limit recovery after neonatal stroke. Thus, enhancement of angiogenesis after neonatal stroke may be a promising strategy for the long-term recovery of the affected newborns.

Ferumoxytol: a new, clinically applicable label for stem-cell tracking in arthritic joints with MRI.

AIM: To develop a clinically applicable MRI technique for tracking stem cells in matrix-associated stem-cell implants, using the US FDA-approved iron supplement ferumoxytol. MATERIALS & METHODS: Ferumoxytol-labeling of adipose-derived stem cells (ADSCs) was optimized in vitro. A total of 11 rats with osteochondral defects of both femurs were implanted with ferumoxytol- or ferumoxides-labeled or unlabeled ADSCs, and underwent MRI up to 4 weeks post matrix-associated stem-cell implant. The signal-to-noise ratio of different matrix-associated stem-cell implant was compared with t-tests and correlated with histopathology. RESULTS: An incubation concentration of 500 µg iron/ml ferumoxytol and 10 µg/ml protamine sulfate led to significant cellular iron uptake, T2 signal effects and unimpaired ADSC viability. In vivo, ferumoxytol- and ferumoxides-labeled ADSCs demonstrated significantly lower signal-to-noise ratio values compared with unlabeled controls (p < 0.01). Histopathology confirmed engraftment of labeled ADSCs, with slow dilution of the iron label over time. CONCLUSION: Ferumoxytol can be used for in vivo tracking of stem cells with MRI.

Mesenchymal stem cell transplantation attenuates brain injury after neonatal stroke.

BACKGROUND AND PURPOSE: Brain injury caused by stroke is a frequent cause of perinatal morbidity and mortality with limited therapeutic options. Mesenchymal stem cells (MSC) have been shown to improve outcome after neonatal hypoxic-ischemic brain injury mainly by secretion of growth factors stimulating repair processes. We investigated whether MSC treatment improves recovery after neonatal stroke and whether MSC overexpressing brain-derived neurotrophic factor (MSC-BDNF) further enhances recovery. METHODS: We performed 1.5-hour transient middle cerebral artery occlusion in 10-day-old rats. Three days after reperfusion, pups with evidence of injury by diffusion-weighted MRI were treated intranasally with MSC, MSC-BDNF, or vehicle. To determine the effect of MSC treatment, brain damage, sensorimotor function, and cerebral cell proliferation were analyzed. RESULTS: Intranasal delivery of MSC- and MSC-BDNF significantly reduced infarct size and gray matter loss in comparison with vehicle-treated rats without any significant difference between MSC- and MSC-BDNF-treatment. Treatment with MSC-BDNF significantly reduced white matter loss with no significant difference between MSC- and MSC-BDNF-treatment. Motor deficits were also improved by MSC treatment when compared with vehicle-treated rats. MSC-BDNF-treatment resulted in an additional significant improvement of motor deficits 14 days after middle cerebral artery occlusion, but there was no significant difference between MSC or MSC-BDNF 28 days after middle cerebral artery occlusion. Furthermore, treatment with either MSC or MSC-BDNF induced long-lasting cell proliferation in the ischemic hemisphere. CONCLUSIONS: Intranasal administration of MSC after neonatal stroke is a promising therapy for treatment of neonatal stroke. In this experimental paradigm, MSC- and BNDF-hypersecreting MSC are equally effective in reducing ischemic brain damage.

Erythropoietin increases neurogenesis and oligodendrogliosis of subventricular zone precursor cells after neonatal stroke.

BACKGROUND AND PURPOSE: Stroke is a common cause of neonatal brain injury. The subventricular zone is a lifelong source of newly generated cells in rodents, and erythropoietin (EPO) treatment has shown benefit in different animal models of brain injury. The purpose of this study is to investigate the specific role of exogenous EPO on subventricular zone progenitor cell populations in response to neonatal stroke. METHODS: Intraventricular injections of green fluorescent protein (GFP)-expressing lentivirus to label subventricular zone precursor cells were made in postnatal day 1 (P1) Long-Evans rats, which then underwent transient middle cerebral artery occlusion on P7. Middle cerebral artery occlusion and sham rats were treated with either vehicle or EPO (1000 U/kg) at reperfusion, 24 hours, and 7 days later. The density of double-labeled DCx+/GFP+, NeuN+/GFP+, O4+/GFP+, GFAP+/GFP+, as well as single-labeled GFP+ and Ki67+ cells, was calculated to determine cell fate outcome in the striatum at 72 hours and 2 weeks after stroke. RESULTS: There was a significant increase in DCx+/GFP+ and NeuN+/GFP+ neurons and O4+/GFP+ oligodendrocyte precursors, with decreased GFAP+/GFP+ astrocytes at both time points in EPO-middle cerebral artery occlusion animals. There was also a significant increase in GFP+ cells and Ki67+ proliferating cells in EPO compared with vehicle-middle cerebral artery occlusion animals. CONCLUSIONS: These data suggest that subventricular zone neural progenitor cells proliferate and migrate to the site of injury after neonatal stroke and multiple doses of EPO, with a shift in cell fate toward neurogenesis and oligodendrogliosis at both early and late time points. The contribution of local cell proliferation and neurogenesis remains to be determined.

Intravenous ferumoxytol allows noninvasive MR imaging monitoring of macrophage migration into stem cell transplants.

PURPOSE: To develop a clinically applicable imaging technique for monitoring differential migration of macrophages into viable and apoptotic matrix-associated stem cell implants (MASIs) in arthritic knee joints. MATERIALS AND METHODS: With institutional animal care and use committee approval, six athymic rats were injected with intravenous ferumoxytol (0.5 mmol iron per kilogram of body weight) to preload macrophages of the reticuloendothelial system with iron oxide nanoparticles. Forty-eight hours later, all animals received MASIs of viable adipose-derived stem cells (ADSCs) in an osteochondral defect of the right femur and mitomycin-pretreated apoptotic ADSCs in an osteochondral defect of the left femur. One additional control animal each received intravenous ferumoxytol and bilateral scaffold-only implants (without cells) or bilateral MASIs without prior ferumoxytol injection. All knees were imaged with a 7.0-T magnetic resonance (MR) imaging unit with T2-weighted fast spin-echo sequences immediately after, as well as 2 and 4 weeks after, matrix-associated stem cell implantation. Signal-to-noise ratios (SNRs) of viable and apoptotic MASIs were compared by using a linear mixed-effects model. MR imaging data were correlated with histopathologic findings. RESULTS: All ADSC implants showed a slowly decreasing T2 signal over 4 weeks after matrix-associated stem cell implantation. SNRs decreased significantly over time for the apoptotic implants (SNRs on the day of matrix-associated stem cell implantation, 2 weeks after the procedure, and 4 weeks after the procedure were 16.9, 10.9, and 6.7, respectively; P = .0004) but not for the viable implants (SNRs on the day of matrix-associated stem cell implantation, 2 weeks after the procedure, and 4 weeks after the procedure were 17.7, 16.2, and 15.7, respectively; P = .2218). At 4 weeks after matrix-associated stem cell implantation, SNRs of apoptotic ADSCs were significantly lower than those of viable ADSCs (mean, 6.7 vs 15.7; P = .0013). This corresponded to differential migration of iron-loaded macrophages into MASIs. CONCLUSION: Iron oxide loading of macrophages in the reticuloendothelial system by means of intravenous ferumoxytol injection can be utilized to monitor differential migration of bone marrow macrophages into viable and apoptotic MASIs in a rat model.

Blood-brain barrier permeability is increased after acute adult stroke but not neonatal stroke in the rat.

The immaturity of the CNS at birth greatly affects injury after stroke but the contribution of the blood-brain barrier (BBB) to the differential response to stroke in adults and neonates is poorly understood. We asked whether the structure and function of the BBB is disrupted differently in neonatal and adult rats by transient middle cerebral artery occlusion. In adult rats, albumin leakage into injured regions was markedly increased during 2-24 h reperfusion but leakage remained low in the neonates. Functional assays employing intravascular tracers in the neonates showed that BBB permeability to both large (70 kDa dextran) and small (3 kDa dextran), gadolinium (III)-diethyltriaminepentaacetic acid tracers remained largely undisturbed 24 h after reperfusion. The profoundly different functional integrity of the BBB was associated with the largely nonoverlapping patterns of regulated genes in endothelial cells purified from injured and uninjured adult and neonatal brain at 24 h (endothelial transcriptome, 31,042 total probe sets). Within significantly regulated 1266 probe sets in injured adults and 361 probe sets in neonates, changes in the gene expression of the basal lamina components, adhesion molecules, the tight junction protein occludin, and matrix metalloproteinase-9 were among the key differences. The protein expression of collagen-IV, laminin, claudin-5, occludin, and zonula occludens protein 1 was also better preserved in neonatal rats. Neutrophil infiltration remained low in acutely injured neonates but neutralization of cytokine-induced neutrophil chemoattractant-1 in the systemic circulation enhanced neutrophil infiltration, BBB permeability, and injury. The markedly more integrant BBB in neonatal brain than in adult brain after acute stroke may have major implications for the treatment of neonatal stroke.

Magnetic resonance imaging of ferumoxide-labeled mesenchymal stem cells in cartilage defects: in vitro and in vivo investigations.

The purpose of this study was to (1) compare three different techniques for ferumoxide labeling of mesenchymal stem cells (MSCs), (2) evaluate if ferumoxide labeling allows in vivo tracking of matrix-associated stem cell implants (MASIs) in an animal model, and (3) compare the magnetic resonance imaging (MRI) characteristics of ferumoxide-labeled viable and apoptotic MSCs. MSCs labeled with ferumoxide by simple incubation, protamine transfection, or Lipofectin transfection were evaluated with MRI and histopathology. Ferumoxide-labeled and unlabeled viable and apoptotic MSCs in osteochondral defects of rat knee joints were evaluated over 12 weeks with MRI. Signal to noise ratios (SNRs) of viable and apoptotic labeled MASIs were tested for significant differences using t-tests. A simple incubation labeling protocol demonstrated the best compromise between significant magnetic resonance signal effects and preserved cell viability and potential for immediate clinical translation. Labeled viable and apoptotic MASIs did not show significant differences in SNR. Labeled viable but not apoptotic MSCs demonstrated an increasing area of T2 signal loss over time, which correlated to stem cell proliferation at the transplantation site. Histopathology confirmed successful engraftment of viable MSCs. The engraftment of iron oxide-labeled MASIs by simple incubation can be monitored over several weeks with MRI. Viable and apoptotic MASIs can be distinguished via imaging signs of cell proliferation at the transplantation site.

Heparin-modified small-diameter nanofibrous vascular grafts.

Due to high incidence of vascular bypass procedures, an unmet need for suitable vessel replacements exists, especially for small-diameter vascular grafts. Here we produced 1-mm diameter vascular grafts with nanofibrous structure via electrospinning, and successfully modified the nanofibers by the conjugation of heparin using di-amino-poly(ethylene glycol) (PEG) as a linker. Antithrombogenic activity of these heparin-modified scaffolds was confirmed in vitro. After 1 month implantation using a rat common carotid artery bypass model, heparin-modified grafts exhibited 85.7% patency, versus 57.1% patency of PEGylated grafts and 42.9% patency of untreated grafts. Post-explant analysis of patent grafts showed complete endothelialization of the lumen and neovascularization around the graft. Smooth muscle cells were found in the surrounding neo-tissue. In addition, greater cell infiltration was observed in heparin-modified grafts. These findings suggest heparin modification may play multiple roles in the function and remodeling of nanofibrous vascular grafts, by preventing thrombosis and maintaining patency, and by promoting cell infiltration into the three-dimensional nanofibrous structure for remodeling.

Genetic deletion of CD36 enhances injury after acute neonatal stroke.

OBJECTIVE: The scavenger receptor CD36 is injurious in acute experimental focal stroke and neurodegenerative diseases in the adult. We investigated the effects of genetic deletion of CD36 (CD36ko) on acute injury, and oxidative and inflammatory signaling after neonatal stroke. METHODS: Postnatal day 9 CD36ko and wild-type (WT) mice were subjected to a transient middle cerebral artery occlusion (MCAO). Injury, phagocytosis of dying cells, and CD36 inflammatory signaling were determined. RESULTS: While the volume of tissue at risk by diffusion-weighted magnetic resonance imaging during MCAO was similar in neonatal CD36ko and WT mice, by 24 hours after reperfusion, injury was more severe in CD36ko and was associated with increased caspase-3 cleavage and reduced engulfment of neurons expressing cleaved caspase-3 by activated microglia. No significant superoxide generation was observed in activated microglia in injured WT, whereas increased superoxide production in vessels and nuclear factor (NF)-κB activation induced by MCAO were unaffected by lack of CD36. Lyn expression was higher in injured CD36ko, and cell type-specific patterns of Lyn expression were altered; Lyn was expressed in endothelial cells and microglia in WT but predominantly in dying neurons in CD36ko. INTERPRETATION: Lack of CD36 results in poorer short-term outcome from neonatal focal stroke due to lack of attenuation of NF-κB-mediated inflammation and diminished removal of apoptotic neuronal debris. Although inhibition of CD36 does not seem to be a good therapeutic target for protection after acute neonatal stroke, as it is after adult stroke, seeking better understanding of CD36 signaling in particular cell populations may reveal important therapeutic targets for neonatal stroke.