Isolectin B4 Research Articles

A Novel Autoantibody against Plexin D1 in Patients with Neuropathic Pain.

To identify novel autoantibodies for neuropathic pain (NeP). We screened autoantibodies that selectively bind to mouse unmyelinated C-fiber type dorsal root ganglion (DRG) neurons using tissue-based indirect immunofluorescence assays (IFA) with sera from 110 NeP patients with various inflammatory and allergic neurologic diseases or other neuropathies, and 50 controls without NeP including 20 healthy subjects and 30 patients with neurodegenerative diseases or systemic inflammatory diseases. IgG purified from IFA-positive patients' sera was subjected to Western blotting (WB) and immunoprecipitation (IP) using mouse DRG lysates. Immunoprecipitates were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify target autoantigens. Antiunmyelinated C-fiber type DRG neuron antibodies were more frequent in patients with NeP than non-NeP subjects (10% vs 0%; p < 0.05). These autoantibodies were all from the IgG2 subclass and colocalized mostly with isolectin B4- and P2X3-positive pain-conducting small neurons but not with S100β-positive myelinated neurons. WB revealed a common immunoreactive band (approximately 220kDa). IP and LC-MS/MS studies identified plexin D1 as a target autoantigen. Immunoadsorption tests with recombinant human plexin D1 in IFA revealed that all 11 anti-small DRG neuron antibody-positive patients had anti-plexin D1 antibodies. Application of anti-plexin D1 antibody-positive patient sera to cultured DRG neurons increased membrane permeability, leading to cellular swelling. NeP patients with anti-plexin D1 antibodies commonly developed burning pain and current perception threshold abnormalities for C-fibers. Main comorbidities were atopy and collagen-vascular disease. Immunotherapies ameliorated NeP in 7 treated cases. Anti-plexin D1 antibodies are a novel biomarker for immunotherapy-responsive NeP. Ann Neurol 2018;84:208-224.

Inhibitory Effects On Retinal Neovascularization by Ranibizumab and sTie2-Fc in An Oxygen-Induced Retinopathy Mouse Model

ABSTRACTPurpose: To study retinal neovascularization (RNV) inhibition by intravitreal injections (IVs) of ranibizumab, sTie2 fusion protein (sTie2-Fc), and a combined therapy in an oxygen-induced retinopathy (OIR) model.Materials and methods: An OIR mouse model was used to simulate RNV in retinopathy of prematurity (ROP); and the effect of blocking the angiopoietin (Ang) and its receptor (Tie2) and the vascular endothelial growth factor (VEGF) and its receptor (VEGFR) signaling pathways was compared using an IV of sTie2-Fc (Ang inhibitor) and/or ranibizumab (aVEGF antagonist). The effects were assessed using fluorescein isothiocyanate (FITC)-dextran cardiac perfusion, isolectin B4 (IB4) staining with whole retinal mounting, and hematoxylin and eosin (HE) staining to count the endothelial cells (ECs) that broke through the internal limiting membrane (ILM). The mRNA and protein levels of VEGF-A, VEGFR-2, Ang1, Ang2, and Tie2 were also determined by reverse transcriptase polymerase chain reaction (RT-PCR) and western blot analysis.Results: Compared with the control group injected with phosphate-buffered saline (PBS), all three experimental groups, ranibizumab, sTie2-Fc, and ranibizumab + sTie2-Fc, had a significant decrease in micro-vessel densities and neovascular clusters, and fewer ECs broke through the ILM (all p < 0.05). The non-perfusion areas decreased in both mono-treated groups, although the combined therapy had larger non-perfusion areas. All three treatments decreased the mRNA and protein levels of VEGFA, Ang1, and Tie2.Conclusion: In this study, it was confirmed that blocking the Ang/Tie2 and/or VEGF/VEGFR pathways could inhibit RNV and decrease abnormal micro-vessel density; and the mono-blockage of Ang/Tie2 might cause a smaller non-perfusion area.

The Number of IgG-Positive Neurons in the Rat Hippocampus Increases after Dosed Traumatic Brain Injury

To evaluate the consequences of traumatic brain injury (TBI), we used a model of lateral fluid percussion brain injury in freely moving male Wistar rats. The immediate response to TBI included development of motor excitation and tonic–clonic seizures. Morphological analysis was performed 7 day after TBI. To localize IgG in the brain, rat brain slices were double stained with antibodies against IgG and NeuN (neuronal marker). To evaluate the state of microglia, we performed staining with Isolectin B4 (a microglial marker). The number of neurons was measured in sections stained using the Nissl method. The results show the IgG accumulation in neurons adjacent to cortical focus of trauma. In the hippocampus, IgG was accumulated in the neurons of the ipsilateral hippocampal CA1 and CA2 fields and the dentate gyrus, while in the contralateral hemisphere IgG was accumulated in the neurons of the CA1 field. These changes were accompanied by activation of microglia in the hippocampus, as well as by a decrease in neuronal density in the dentate gyrus of the ipsilateral hippocampus. The results show that TBI leads to bilateral damage to the hippocampus.

Sensorimotor Activity Partially Ameliorates Pain and Reduces Nociceptive Fiber Density in the Chronically Injured Spinal Cord.

A large proportion of patients suffering from spinal cord injury (SCI) develop chronic central neuropathic pain. Previously, we and others have shown that sensorimotor training early after SCI can prevent the development of mechanical allodynia. To determine whether training initiated in the subchronic/chronic phase remains effective, correlates of below-level neuropathic pain were analyzed in the hindpaws 5-10 weeks after a moderate T11 contusion SCI (50 kDyn) in adult female C57BL/6 mice. In a comparison of SCI and sham mice 5 weeks post-injury, about 80% of injured animals developed mechanical hypersensitivity to light mechanical stimuli, whereas testing of noxious stimuli revealed hypo-responsiveness. Thermal sensitivity testing showed a decreased response latency after injury. Without intervention, mechanical and thermal hyper-responsiveness were evident until the end of the experiment (10 weeks). In contrast, treadmill training (2 × 15 min/day; 5 × /week) initiated 6 weeks post-injury resulted in partial amelioration of pain behavior and this effect remained stable. Analysis of calcitonin gene-related peptide (CGRP)-labeled fibers in lamina III-IV of the lumbar dorsal horn revealed an increase in labeling density after SCI. This was not due to changes in the number or size distribution of CGRP-labeled lumbar dorsal root ganglion neurons. Treadmill training reduced the CGRP-labeling density in the spinal cord of injured mice, whereas the density of non-peptidergic isolectin-B4 (IB4)+ fibers showed no changes in lamina IIi and a slight reduction of sparse IB4 labeling in laminae III-IV. Thus, sensorimotor activity initiated in the subchronic/chronic phase of SCI remains effective in ameliorating pain behavior and influencing structural changes of the nociceptive system.

CD140b (PDGFRβ) signaling in adipose-derived stem cells mediates angiogenic behavior of retinal endothelial cells.

Adipose-derived stem cells (ASCs) are multipotent mesenchymal progenitor cells that have functional and phenotypic overlap with pericytes lining microvessels in adipose tissue. The role of CD140b [platelet-derived growth factor receptor- β (PDGFR-β)], a constitutive marker expressed by ASCs, in the angiogenic behavior of human retinal endothelial cells (HREs) is not known. CD140b was knocked down in ASCs using targeted siRNA and lipofectamine transfection protocol. Both CD140b+ and CD140b- ASCs were tested for their proliferation (WST-1 reagent), adhesion (laminin-1 coated plates), and migration (wound-scratch assay). Angiogenic effect of CD140b+ and CD140b- ASCs on HREs was examined by co-culturing ASCs:HREs in 12:1 ratio for 6 days followed by visualization of vascular network by Isolectin B4 staining. The RayBio® Membrane-Based Antibody Array was used to assess differences in human cytokines released by CD140b+ or CD140b- ASCs. Knockdown of CD140b in ASCs resulted in a significant 50% decrease in proliferation rate, 25% decrease in adhesion ability to Laminin-1, and 50% decrease in migration rate, as compared to CD140b+ ASCs. Direct contact of ASCs expressing CD140b+ with HREs resulted in robust vascular network formation that was significantly reduced with using CD140b- ASCs. Of the 80 proteins tested, 45 proteins remained unchanged (>0.5-<1.5 fold), 6 proteins including IL-10 downregulated (<0.5 fold) and 29 proteins including IL-16 & TNF-β were upregulated (>1.5 fold) in CD140b- ASCs compared to CD140b+ ASCs. Our data demonstrate a substantial role for CD140b in the intrinsic abilities of ASCs and their angiogenic influence on HREs. Future studies are needed to fully explore the signaling of CD140b in ASCs in vivo for retinal regeneration.

Differential aging‐related changes in neurophysiology and gene expression in IB4‐positive and IB4‐negative nociceptive neurons

SummaryDespite pain prevalence altering with age, the effects of aging on the properties of nociceptors are not well understood. Nociceptors, whose somas are located in dorsal root ganglia, are frequently divided into two groups based on their ability to bind isolectin B4 (IB4). Here, using cultured neurons from 1‐, 3‐, 5‐, 8‐, 12‐, and 18‐month‐old mice, we investigate age‐dependent changes in IB4‐positive and IB4‐negative neurons. Current‐clamp experiments at physiological temperature revealed nonlinear changes in firing frequency of IB4‐positive, but not IB4‐negative neurons, with a peak at 8 months. This was likely due to the presence of proexcitatory conductances activated at depolarized membrane potentials and significantly higher input resistances found in IB4‐positive neurons from 8‐month‐old mice. Repetitive firing in nociceptors is driven primarily by the TTX‐resistant sodium current, and indeed, IB4‐positive neurons from 8‐month‐old mice were found to receive larger contributions from the TTX‐resistant window current around the resting membrane potential. To further address the mechanisms behind these differences, we performed RNA‐seq experiments on IB4‐positive and IB4‐negative neurons from 1‐, 8‐, and 18‐month‐old mice. We found a larger number of genes significantly affected by age within the IB4‐positive than IB4‐negative neurons from 8‐month‐old mice, including known determinants of nociceptor excitability. The above pronounced age‐dependent changes at the cellular and molecular levels in IB4‐positive neurons point to potential mechanisms behind the reported increase in pain sensitivity in middle‐aged rodents and humans, and highlight the possibility of targeting a particular group of neurons in the development of age‐tailored pain treatments.

Connexin36 Expression in Primary Afferent Neurons in Relation to the Axon Reflex and Modality Coding of Somatic Sensation

Electrical coupling mediated by connexin36-containing gap junctions that form electrical synapses is known to be prevalent in the central nervous system, but such coupling was long ago reported also to occur between cutaneous sensory fibers. Here, we provide evidence supporting the capability of primary afferent fibers to engage in electrical coupling. In transgenic mice with enhanced green fluorescent protein (eGFP) serving as a reporter for connexin36 expression, immunofluorescence labeling of eGFP was found in subpopulations of neurons in lumbar dorsal root and trigeminal sensory ganglia, and in fibers within peripheral nerves and tissues. Immunolabeling of connexin36 was robust in the sciatic nerve, weaker in sensory ganglia than in peripheral nerve, and absent in these tissues from Cx36 null mice. Connexin36 mRNA was detected in ganglia from wild-type mice, but not in those from Cx36 null mice. Labeling of eGFP was localized within a subpopulation of ganglion cells containing substance P and calcitonin gene-releasing peptide, and in peripheral fibers containing these peptides. Expression of eGFP was also found in various proportions of sensory ganglion neurons containing transient receptor potential (TRP) channels, including TRPV1 and TRPM8. Ganglion cells labeled for isolectin B4 and tyrosine hydroxylase displayed very little co-localization with eGFP. Our results suggest that previously observed electrical coupling between peripheral sensory fibers occurs via electrical synapses formed by Cx36-containing gap junctions, and that some degree of selectivity in the extent of electrical coupling may occur between fibers belonging to subpopulations of sensory neurons identified according to their sensory modality responsiveness.

Activation of Mechanosensitive Transient Receptor Potential/Piezo Channels in Odontoblasts Generates Action Potentials in Cocultured Isolectin B4–negative Medium-sized Trigeminal Ganglion Neurons

IntroductionVarious stimuli to the dentin surface elicit dentinal pain by inducing dentinal fluid movement causing cellular deformation in odontoblasts. Although odontoblasts detect deformation by the activation of mechanosensitive ionic channels, it is still unclear whether odontoblasts are capable of establishing neurotransmission with myelinated A delta (Aδ) neurons. Additionally, it is still unclear whether these neurons evoke action potentials by neurotransmitters from odontoblasts to mediate sensory transduction in dentin. Thus, we investigated evoked inward currents and evoked action potentials form trigeminal ganglion (TG) neurons after odontoblast mechanical stimulation. MethodsWe used patch clamp recordings to identify electrophysiological properties and record evoked responses in TG neurons. ResultsWe classified TG cells into small-sized and medium-sized neurons. In both types of neurons, we observed voltage-dependent inward currents. The currents from medium-sized neurons showed fast inactivation kinetics. When mechanical stimuli were applied to odontoblasts, evoked inward currents were recorded from medium-sized neurons. Antagonists for the ionotropic adenosine triphosphate receptor (P2X3), transient receptor potential channel subfamilies, and Piezo1 channel significantly inhibited these inward currents. Mechanical stimulation to odontoblasts also generated action potentials in the isolectin B4–negative medium-sized neurons. Action potentials in these isolectin B4–negative medium-sized neurons showed a short duration. Overall, electrophysiological properties of neurons indicate that the TG neurons with recorded evoked responses after odontoblast mechanical stimulation were myelinated Aδ neurons. ConclusionsOdontoblasts established neurotransmission with myelinated Aδ neurons via P2X3 receptor activation. The results also indicated that mechanosensitive TRP/Piezo1 channels were functionally expressed in odontoblasts. The activation of P2X3 receptors induced an action potential in the Aδ neurons, underlying a sensory generation mechanism of dentinal pain.

Interaction of Phospho‐Moesin and CD44 in Pericytes Attenuated the Maturation of Neovessles in AGE‐induced Angiogenesis

BackgroundWe have previously demonstrated that advanced glycation endproducts (AGEs) promoted angiogenesis by inducing ERM phosphorylation, especially, moesin phosphorylation in human umbilical vein endothelial cells (HUVECs). This study was to investigate the effect of moesin phosphorylation on angiogenesis and neovessel maturation in aortic ring and retina in AGE‐treated mice. This study also focus on the impact of interaction between phospho‐moesin and CD44 on AGE‐induced retinal microvascular pericyte (RMP) migration and neovessel maturation.MethodsAGE‐treated mouse model was developed in WT mice, in receptor for AGEs (RAGE)‐knockout (RAGE(−/−)) mice and in WT postnatal mice by intraperitoneal injection of AGEs. retinal vessels were observed by whole mount immunofluorescence staining for isolectin B4 (IB4). Mouse aorta rings were embedded in 3D collagen and the sprouted vascular endothelial cells was identified by immunofluorescent staining IB4. Pericytes were identified with NG2 or α‐SMA in retina and aortic ring. Primary RMPs were isolated, purified from weanling rats, and identified by cellular markers α‐SMA, PDGFR‐β, NG2 and desmin using immunofluorescence microscopy. Migration of RMPs was induced in different condition and detected with transwell. The phosphorylation of moesin was measured using western blotting. Colocalization and interaction between CD44, phospho‐moesin and F‐actin was detected by confocal laser scanning microscopy and coimmunoprecipitation.ResultsThe number of neovessels in retina and the phosphorylation of moesin were increased in WT mice after AGE treatment for 1, 2, and 6 months, and the increase of retinal neovessels was more obvious with time extension. There were no significant alteration in RAGE(−/−) mice after AGE treatment for six months. The phosphorylation of moesin and the number of neovessels in retina were increased in WT postnatal mice after AGE treatment for 7 d. Both in vivo and in vitro AGE treatment could enhanced the phosphorylation of moesin and promoted neovessel sprouting in mouse aortic ring, while there were not significant changes in RAGE(−/−) mouse aortic ring. The pericyte cover rate was reduced in neovessels in AGE‐treated mouse aortic ring, as well as in retinal microvessels in AGE‐treated mice. AGE‐BSA increased moesin phosphorylation in RMPs, which was in consistent with RMP migration in a dose‐ and time‐dependent manner. AGE‐BSA promoted CD44 and phospho‐moesin interaction, as well as the F‐actin stress fiber reassembly. The inhibition of ROCK by specific inhibitor Y27632 decreased AGE‐induced moesin phosphorylation and subsequently attenuated pericyte migration, suppressed the formation of CD44‐phospho‐ERM complex and redistribution of F‐actin. The inhibiting mutant of phosphorylation of Thr 558 in moesin suppressed AGE‐induced migration and F‐actin rearrangement, while the activating mutant of moesin phosphorylation of Thr 558 promoted cell migration and F‐actin alteration.ConclusionsAGEs promoted angiogenesis in mice by inducing moesin phosphorylation. The interaction of phospho‐moesin and CD44 participates in the pathogenesis of AGE‐induced RMP migration through ROCK activation and might hamper the maturation of neovessels.Support or Funding InformationSponsored by General Program from Natural Science Foundation of China (81370226, 81170297, and 80971201)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Repurposing Griseofulvin as a Non‐Toxic Angiogenesis Inhibitor

BackgroundOcular neovascularization causes vision loss in major blinding diseases such as proliferative diabetic retinopathy, retinopathy of prematurity, and neovascular age‐related macular degeneration. Over 30% of patients are refractive to current therapies, hence developing small molecules targeting ocular neovascularization is critical. We previously identified the heme biosynthesis enzyme ferrochelatase (FECH) as an angiogenesis mediator. FECH is upregulated in human and mouse eyes undergoing neovascularization, and knockdown or mutation abolished angiogenesis in vitro and in vivo. FECH inhibition is therefore a promising therapeutic approach for blocking neovascularization. The FDA‐approved anti‐fungal drug, griseofulvin, inhibits FECH as an off‐target effect and blocks choroidal neovascularization ex‐vivo and in mice. Here, we investigated the anti‐angiogenic effect of griseofulvin in the mouse oxygen‐induced retinopathy (OIR) model of retinal neovascularization, and explored its ocular toxicity.MethodsIn the OIR model, newborn mice are exposed to 75% oxygen at postnatal day 7 (P7) for 5 days, causing retinal vascular regression. At P12, upon return to normoxia, the retina becomes ischemic, forming pre‐retinal neovascularization. Temporal expression of FECH was analyzed at various ages in mouse OIR retinas. Griseofulvin was intravitreally injected in OIR mice at P12 and retinal neovascularization assessed at P17 using Isolectin B4 staining. Tip cell filopodia and retinal sprouting were assessed by murine retinal ex‐vivo assays. For toxicity studies, adult mice were intravitreally injected with griseofulvin and retinal damage analyzed after 7 and 14 days. Structural and morphological analyses were performed using optical coherence tomography (OCT) and histology. Vascular leakage was assessed by fluorescein angiography (FA). Functional analysis of the retina was performed using electroretinogram (ERG). Apoptosis and immune cell activation were assessed by immunohistochemistry.ResultsTemporal FECH upregulation was observed in mouse OIR retinas. Griseofulvin inhibited retinal neovascularization in OIR and also decreased the avascular area in the OIR retina. Further, griseofulvin inhibited retinal tip cell filopodia and retinal sprouting ex‐vivo. OCT and FA revealed that retinal structure was maintained and no vascular leakage was found in griseofulvin injected eyes. ERG showed that retinal function was normal in griseofulvin treated mice, and histology showed normal retinal stratification in griseofulvin treated eyes. Immunostaining revealed no aberrant cell death, microglia activation or gliosis in griseofulvin treated eyes, suggesting no cellular toxicity or stress.ConclusionsGriseofulvin inhibits retinal neovascularization and also promotes revascularization, suggesting a dual effect on vascular repair. Griseofulvin lacks ocular toxicity. Together, these findings suggest that griseofulvin could be repurposed to treat neovascularization in ocular diseases.Support or Funding InformationNIH/NEI R01EY025641, Research to Prevent Blindness, Inc.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Novel Role for Lymphotactin (XCL1) Signaling in the Nervous System: XCL1 Acts via its Receptor XCR1 to Increase Trigeminal Neuronal Excitability

Chemokines are known to have a role in the nervous system, influencing a range of processes including the development of chronic pain. To date there are very few studies describing the functions of the chemokine lymphotactin (XCL1) or its receptor (XCR1) in the nervous system. We investigated the role of the XCL1-XCR1 axis in nociceptive processing, using a combination of immunohistochemical, pharmacological and electrophysiological techniques. Expression of XCR1 in the rat mental nerve was elevated 3 days following chronic constriction injury (CCI), compared with 11 days post-CCI and sham controls. XCR1 co-existed with neuronal marker PGP9.5, leukocyte common antigen CD45 and Schwann cell marker S-100. In the trigeminal root and white matter of the brainstem, XCR1-positive cells co-expressed the oligodendrocyte marker Olig2. In trigeminal subnucleus caudalis (Vc), XCR1 immunoreactivity was present in the outer laminae and was colocalized with vesicular glutamate transporter 2 (VGlut2), but not calcitonin gene-related peptide (CGRP) or isolectin B4 (IB4). Incubation of brainstem slices with XCL1 induced activation of c-Fos, ERK and p38 in the superficial layers of Vc, and enhanced levels of intrinsic excitability. These effects were blocked by the XCR1 antagonist viral CC chemokine macrophage inhibitory protein-II (vMIP-II). This study has identified for the first time a role for XCL1-XCR1 in nociceptive processing, demonstrating upregulation of XCR1 at nerve injury sites and identifying XCL1 as a modulator of central excitability and signaling via XCR1 in Vc, a key area for modulation of orofacial pain, thus indicating XCR1 as a potential target for novel analgesics.

Spatial and temporal recruitment of the neurovascular unit during development of the mouse blood-retinal barrier.

The inner blood-retinal barrier (BRB) is made up by the neurovascular unit, consisting of endothelial cells, pericytes and glial cells. The BRB maintains homeostasis of the neural retina, but in pathological eye conditions the neurovascular unit is often disrupted, causing BRB loss. Here, we investigated in detail temporal and spatial recruitment of the neurovascular unit in the neonatal mouse retina from postnatal day (P)3 to P25 employing immunohistochemical staining of vascular endothelium (isolectin B4), pericytes (α-SMA and NG2) and astrocytes (GFAP). In addition, we investigated gene expression of polarized astrocytic end-feet markers aquaporin-4 and laminin α2 chain with qPCR. We observed GFAP-positive cells migrating ahead of the retinal vasculature during the first postnatal week, suggesting that the retinal vasculature follows an astrocytic meshwork. From P9 onwards, astrocytes acquired a mature phenotype, with a more stellate shape and increased expression of aquaporin-4. NG2-positive cells and tip cells co-localized at P5 and invaded the retina together as a vascular sprouting front. In summary, these data suggest that recruitment of the cell types of the neurovascular unit is a prerequisite for proper retinal vascularization and BRB formation.

ROR2 in post-natal vessel formation

Vessel formation requires a precise orchestration of morphometric and molecular events controlled by a multitude of angiogenic factors and morphogens. Growing evidence has shown that Wnt/PCP pathway is important for vessel formation. We previously demonstrated that ubiquitin ligase E3 PDZRN3 drives non-canonical Wnt/PCP signaling to control post-natal angiogenesis in the retina. Endothelial cell polarity is essential to control vascular properties and endothelial cell polarization. Orphan tyrosine receptor kinase ROR2 has been described to regulate Wnt/PCP signaling. The goal of this study was to investigate the role of ROR2 during vascular formation and its role in EC polarity. For this purpose we first analyzed the expression of ROR2 during the kinetic of vascular formation in the retina. After in toto immunofluorescence staining, ROR2 is observed in different cells in the retina and in CD31+ cells. By Western blot and qRT-PCR, we showed that ROR2 is strongly expressed at P4-P7 and decreased after P8. To analyze the role of ROR2 in the endothelium, transgenic mice deleted for ROR2 specifically in the endothelium (CDH5-iCre;ROR2 F/F and Pdgfb-iCre; ROR2 F/F mice) were generated. The retinas of pups sacrificed 5, 7 and 11 days after birth were immunostained (CD31, isolectin B4, NG2, SMA, ColIV, KI67) to study the kinetics of vascular plexus formation. Deletion of ROR2 gene induced a significant decrease in vessel plexus formation at P5, P7 and P11 in Pdgfb-ROR2 iECKO and CDH5-ROR2 iECKO mice compared to littermates. This study shows that ROR2 is required for the formation of primary but also ternary vascular network. Investigation of the ROR2 signaling and molecular mechanisms are under investigation to understand whether and how ROR2/PCP pathway could regulate EC properties.

Neuronal Expression of Junctional Adhesion Molecule-C is Essential for Retinal Thickness and Photoreceptor Survival.

Photoreceptor cell death is a key pathology of retinal degeneration diseases. To date, the molecular mechanisms for this pathological process remain largely unclear. Junctional adhesion molecule-c (Jam-c) has been shown to play important roles in different biological events. However, its effect on retinal neuronal cells is unknown. To determine the effect of Jam-c on adult mouse eyes, particularly, on retinal structure, vasculature and photoreceptor cells, in order to explore potential important target molecules for ocular diseases. Jam-c global knockout mice, endothelial-specific and neuronal-specific Jam-c conditional knockout mice using Tie2-Cre and Nestin-Cre mice respectively were used in this study. Mouse eyes were harvested from the different groups and eye size examined. Cryosections of the eyes were made and stained with Hematoxylin and Eosin (H&E) and the thicknesses of retinal layers measured. Retinal blood vessels and cone and rod photoreceptors were analyzed using isolectin B4, peanut agglutinin and rhodopsin as markers respectively. In vivo Jam-c knockdown in mouse eyes was performed by intravitreal injection of Jam-c shRNA. Jam-c expression in the retinae was quantified by real-time PCR. Global Jam-c gene deletion in mice resulted in smaller eyes and decreased the diameters of lens and iris. Jam-c-/- mice display marked thinning of the outer nuclear layer (ONL), less numbers of photoreceptor cells, and abnormal retinal vasculature. Importantly, neuronal-specific Jam-c deletion led to similar phenotype, whereas no obvious defect was observed in endothelial-specific Jam-c knockout mice. Moreover, Jam-c knockdown by shRNA also decreased ONL thickness and photoreceptor numbers. We found that Jam-c is critically required for the normal size and retinal structure. Particularly, Jam-c plays important roles in maintaining the normal retinal thickness, vasculature and photoreceptor numbers. Jam-c thus may therefore have important roles in various ocular diseases.

Nitrosative damage during retrovirus infection-induced neuropathic pain

BackgroundPeripheral neuropathy is currently the most common neurological complication in HIV-infected individuals, occurring in 35–50% of patients undergoing combination anti-retroviral therapy. Data have shown that distal symmetric polyneuropathy develops in mice by 6 weeks following infection with the LP-BM5 retrovirus mixture. Previous work from our laboratory has demonstrated that glial cells modulate antiviral T-cell effector responses through the programmed death (PD)-1: PD-L1 pathway, thereby limiting the deleterious consequences of unrestrained neuroinflammation.MethodsUsing the MouseMet electronic von Frey system, we assessed hind-paw mechanical hypersensitivity in LP-BM5-infected wild-type (WT) and PD-1 KO animals. Using multi-color flow cytometry, we quantitatively assessed cellular infiltration and microglial activation. Using real-time RT-PCR, we assessed viral load, expression of IFN-γ, iNOS, and MHC class II. Using western blotting, we measured protein nitrosylation within the lumbar spinal cord (LSC) and dorsal root ganglion (DRG). Histochemical staining was performed to analyze the presence of CD3, ionized calcium binding adaptor molecule (Iba)-1, MHCII, nitrotyrosine, isolectin B4 (IB4) binding, and neurofilament 200 (NF200). Statistical analyses were carried out using graphpad prism.ResultsHind-paw mechanical hypersensitivity observed in LP-BM5-infected animals was associated with significantly increased lymphocyte infiltration into the spinal cord and DRG. We also observed elevated expression of IFN-γ (in LSC and DRG) and MHC II (on resident microglia in LSC). We detected elevated levels of 3-nitrotyrosine within the LSC and DRG of LP-BM5-infected animals, an indicator of nitric oxide (NO)-induced protein damage. Moreover, we observed 3-nitrotyrosine in both small (IB4+) and large (NF200+) DRG sensory neurons. Additionally, infected PD-1 KO animals displayed significantly greater mechanical hypersensitivity than WT or uninfected mice at 4 weeks post-infection (p.i.). Accelerated onset of hind-paw hypersensitivity in PD-1 KO animals was associated with significantly increased infiltration of CD4+ and CD8+ T lymphocytes, macrophages, and microglial activation at early time points. Importantly, we also observed elevated levels of 3-nitrotyrosine and iNOS in infected PD-1 KO animals when compared with WT animals.ConclusionsResults reported here connect peripheral immune cell infiltration and reactive gliosis with nitrosative damage. These data may help elucidate how retroviral infection-induced neuroinflammatory networks contribute to nerve damage and neuropathic pain.

(138) - Mechanical sensitization indicates neuropathic allodynia in a rat model of fabry disease

Fabry disease is an X-linked lysosomal disease, and recent newborn screening reveals a US incidence that approaches 1:3000. Beginning in childhood, patients experience significant neuropathic pain, which includes acroparesthesia (i.e., extremity tingling/burning) and episodic crises (i.e., agonizing pain lasting minutes to days). The molecular defect of Fabry disease is deficiency of the lysosomal enzyme, α-galactosidase A. When this enzyme is deficient, glycosphingolipid substrates, such as globotriaosylceramide (Gb3), accumulate in lysosomes of several cell types, including dorsal root ganglia (DRG) neurons. The neuronal glycosphingolipid accumulation likely initiates pathogenesis, but the cellular mechanisms that drive or maintain the pain development in Fabry disease are unknown. We generated a knockout (KO) rat model using CRISPR/Cas9 technology. Behavioral assays were used to longitudinally assess mechanosensation, and KO males and females were found to have increased sensitivity to noxious sharp force and decreased mechanical thresholds compared to wild type (WT) rats. In contrast, no differences in cold or heat sensitivity were detected. DRG sections show numerous inclusions and dramatically greater isolectin B4 staining in KO compared to WT neurons. These inclusions are observed primarily in smaller diameter neurons, while large diameter neurons are spared. Ultrastructural examination via electron microscopy demonstrates that the inclusions resemble “zebra-bodies,” which are characteristic in Fabry disease. Mass spectrometry reveals that compared to WT controls, Gb3 is elevated 127-fold in KO DRG. During whole cell patch clamping with focal mechanical stimulation, KO sensory neurons exhibit larger mechanically gated currents than WT. The behavioral, cellular, and molecular findings indicate that Fabry rats recapitulate pain phenotypes observed in patients, and thus this is an excellent model to study pain mechanisms and test therapies. Supported by NIH grants F30DK113641, P41GM103490, R21AI129873, R21NS095627, R24HL114474, T32GM080202.

The 50/10 Oxygen-Induced Retinopathy Model Serves as a Hyperoxia and Hypoxia Model of Bronchopulmonary Dysplasia

BackgroundAnimal models of bronchopulmonary dysplasia (BPD) are mainly created by hyperoxia exposure. However, these models do not fully recapitulate BPD pathophysiology as observed in clinical practice. To find a better BPD model, we established a rat 50/10 oxygen-induced retinopathy (OIR) model and analyzed the pathologic features of the lungs. MethodsThe rat OIR model was established by exposing newborn rats (P0) to 50% and 10% oxygen (hyperoxia and hypoxia) on alternating days for 14 days. Lungs were harvested immediately on postnatal day 14 (P14) and on P18 after 4 days of normoxia exposure for hematoxylin and eosin staining, antialpha smooth muscle actin (α-SMA) immunohistochemistry and Picrosirius red staining of collagen. Retinas were obtained to confirm successful model establishment by isolectin B4 staining of retinal vasculature. ResultsOIR rats presented with fewer and enlarged alveoli, and the septal walls were thicker than those in age-matched controls. α-SMA immunohistochemistry indicated increased abundance of myofibroblasts in OIR rats. At P18, α-SMA-positive myofibroblasts were present at extremely low levels from the alveolar walls of control rats, while OIR rats showed myofibroblast persistence. The amount of collagen in OIR rats was also higher than that in control rats at both P14 and P18 as evidenced by Picrosirius red staining. ConclusionsAlveolar changes observed by hematoxylin and eosin staining, prolonged and stronger α-SMA expression and augmented collagen accumulation resemble the histopathology of BPD, suggesting that the rat 50/10 OIR model is suitable for use in BPD research.

Abstract 73: Triggering Receptor Expressed on Myeloid Cells-2 Expression derived From Microglia Contributes to Neurological Recovery in Experimental Ischemic Stroke

Background and Purpose: We previously reported that triggering receptor expressed on myeloid cells-2 (TREM2) expression promotes post-ischemic phagocytosis and leads to neurological recovery. However, the precise underlying mechanism is not fully understood yet. In present study, we aimed to clarify which pool of myeloid cells (brain resident microglia versus macrophage derived from circulation) are contributing to its beneficial role in post-ischemic recovery, with the usage of bone marrow (BM) chimeric mice. Methods: BM chimeric mice were created by BM transplanting BM from TREM2 knockout (KO) and wild type (Wt) mice into KO and Wt recipient mice. Thus, 4 groups (Wt (donor) to Wt (recipient), KO to Wt, Wt to KO, KO to KO; n=6/each) of BM chimeric mice were produced, then were subjected to dMCAO surgery. Neurological function was observed until 14 days after surgery. Infarct volume and immunohistological analysis (myeloid cell activation and infiltration: isolectin B4 (IB4) and CD11b; phagocytosis: oil red o and CD68; TREM2 expression) was also undergone. Results: Compared to mice lacking brain TREM2 (WT to KO and KO to KO), mice with intact TREM2 in brain microglia (KO to WT and WT to WT) showed better neurological recovery (p&lt;0.05), and smaller infarct volume (p&lt;0.01). Myeloid cell activation and infiltration (IB4) was significantly decreased in order of WT to WT, KO to Wt, Wt to KO, KO to KO (p&lt;0.05). Likewise, number of TREM2 positive myeloid cells (CD11b) showed similar decreases. Oil red o staining demonstrated that mice with intact TREM2 in microglia (KO to WT and WT to WT) showed more accumulation of foamy macrophages, compared to mice lacking TREM2 in brain (Wt to KO and KO to KO). Furthermore, both the number and proportion of TREM2 positive phagocytes (CD68) was significantly higher in mice with intact TREM2 in the brain, compared to mice lacking brain TREM2. Conclusions: Although our results suggest TREM2 expression in both microglia and circulating macrophage were crucial for post-ischemic recovery, its beneficial effect was greater when TREM2 was present in brain resident microglia.

Abstract TP255: Calcium Release-activated Calcium Channel Inhibition Protects Against Inflammation in Ischemic Stroke

Purpose: Inflammatory responses after ischemic stroke contribute to worsening of ischemic brain injury. Calcium release-activated calcium (CRAC) channels are known to be involved in the activation of immune responses in microglia, and have been studied indirectly through non-specific calcineurin inhibitors such as cyclosporine and FK506. Since these compounds have many off-target effects and unacceptable side effects, we studied a novel CRAC channel inhibitor which is specific to the CRAC channel and appears safe in preclinical studies in anticipation of human clinical trials for other indications. We explored whether this inhibitor might improve outcome in experimental stroke by inhibiting microglial activation. Subjects and Methods: 2-3 month old C57/BL6 male mice, were exposed to transient middle cerebral artery occlusion (tMCAO, filament model) for 2h followed by reperfusion. Some were treated with the CRAC channel inhibitor (CM-EX-137, 5mg/kg IP; tMCAO-CM) or vehicle control (tMCAO) daily beginning immediately after occlusion and the following two days. Brains were collected 3d post-stroke. Neurological function was evaluated using a Bederson score, elevated body swing test, adhesive removal test, and corner test at baseline, at 3, 24 and 72 h. Relative cerebral blood flow (rCBF) was studied in some mice using laser Doppler flowmetry. Ischemic lesion size was evaluated by 2,3,5-triphenyl tetrazolium chloride (TTC) staining. Microglial/monocyte activation was assessed by isolectin B4 histochemistry. Results: A total of 18 mice were studied (tMCAO-CM: 8; tMCAO: 8, sham:2). rCBF was no different between experimental groups, and is consistent with the notion that the compound should not alter blood flow. Neurological function at 1 &amp; 3d after ischemia was significantly improved in tMCAO-CM group compared to tMCAO (p&lt;0.001). The CM compound also significantly reduced infarct volume by ~60% (p&lt;0.05). Isolectin B4 staining was markedly reduced in the tMCAO-CM group compared to tMCAO 3d post ischemia (p&lt;0.01). Conclusion: CRAC channel inhibition protects against experimental stroke by decreasing lesion size, improving neurological outcomes and decreasing microglial activation.

The promoting effects of SNAI1 activating matrix metalloproteinase on choroidal neovascularization under hypoxia

Objective To investigate whether vascular endothelial cells in choroidal neovascularization whether hypoxia condition can up-regulate SNAI1 and activate matrix metalloproteinase (MMP)2 and MMP9 therefore to participate in choroidal neovascularization(CNV). Methods Sixteen SPF male C57 mice aged 6-8 weeks were divided into control group and model group.CNV models were induced by retinal laser photocoagulation, and flat-mount and frozen sections of retinal pigment epithelium (RPE)-choroid-sclera compound were prepared at 7 days after modeling.The CNV in flat-mount was examined by Isolectin B4 staining, and the location of SNAI1, MMP2 and MMP9 in frozen sections was determined by immunofluorescence technology.The expression of SNAI1, MMP2 and MMP9 at mRNA level in CNV was detected by real-time fluorescence quantitative PCR (real-time PCR). The use and care of experimental animals complied with Statement for the Use of Animals in Ophthalmic and Visual Research.The RF/6A cells were divided into normoxia group and hypoxia group and cultured for 24 hours in 5%CO2 condition and mix condition of 94%N2, 5%CO2 and 1%O2, respectively.The expression of SNAI1, MMP2 and MMP9 in the cells at mRNA and protein levels was detected by real-time PCR and Western blot assay, respectively.Small interfering RNA of SNAI1 (siSNAI1) was transfected into the cells, and then the expression of MMP2 in the cells at mRNA and protein levels was detected by real-time PCR and Western blot assay, respectively, and the migrating number of the cells was assayed by Transwell chamber assay. Results CD31 and SNAI1 positive-response cells were seen in RPE-choroid-sclera flat-mounts under the laser scanning confocal microscope.The relative expression levels of SNAI1 mRNA and MMP2 mRNA in RPE-choroid-sclera tissues were higher in the model group than those in the control group (SNAI1 mRNA: 1.291±0.060 vs.0.759±0.074, P=0.001; MMP2 mRNA: 1.610±0.424 vs.0.772±0.080, P=0.044). The expression of MMP9 mRNA was not significantly elevated between model group and control group (P>0.05). The relative expression level of MMP2 mRNA was higher in comparison with MMP9 mRNA in the model group (P 0.05). The relative expressions of MMP2 mRNA in the cells were 0.217±0.036 and 0.818±0.105, and those of MMP2 protein in the cells were 0.236±0.009 and 1.043±0.120 in the hypoxia+ siSNAI1 group and only hypoxia group, respectively, with significant differences between them (P=0.002, 0.003). The migrating number of the cells was (254.60±71.31)/field in the hypoxia+ siSNAI1 group, which was significantly less than (534.10±96.21) /field in the control group (P=0.029). Conclusions The hypoxic environment at CNV can activate MMP2 by up-regulating the expression of SNAI1, which promotes the migration of vascular endothelial cells and therefore participates in CNV formation, and the intervention of SNAI1 activation under the hypoxic condition can inhibit this process. Key words: Choroidal neovascularization; Matrix metalloproteinase; Small interfering RNA; Transcriptional factor SNAI1; Vascular endothelial cells; Disease model, C57 mice; Hypoxia