Isolectin B4 Research Articles

Cutaneous TRPM8-expressing sensory afferents are a small population of neurons with unique firing properties.

It has been well documented that the transient receptor potential melastatin 8 (TRPM8) receptor is involved in environmental cold detection. The role that this receptor plays in nociception however, has been somewhat controversial since conflicting reports have shown different neurochemical identities and responsiveness of TRPM8 neurons. In order to functionally characterize cutaneous TRMP8 fibers, we used two ex vivo somatosensory recording preparations to functionally characterize TRPM8 neurons that innervate the hairy skin in mice genetically engineered to express GFP from the TRPM8 locus. We found several types of cold‐sensitive neurons that innervate the hairy skin of the mouse but the TRPM8‐expressing neurons were found to be of two specific populations that responded with rapid firing to cool temperatures. The first group was mechanically insensitive but the other did respond to high threshold mechanical deformation of the skin. None of these fibers were found to contain calcitonin gene‐related peptide, transient receptor potential vanilloid type 1 or bind isolectin B4. These results taken together with other reports suggest that TRPM8 containing sensory neurons are environmental cooling detectors that may be nociceptive or non‐nociceptive depending on the sensitivity of individual fibers to different combinations of stimulus modalities.

Peripheral and central anatomical organization of cutaneous afferent subtypes in a rat nociceptive intersegmental spinal reflex.

Stimulation of rat segmental dorsal cutaneous nerves (DCNs) evokes the nociceptive intersegmental cutaneus trunci muscle (CTM) reflex. The reflex consists of early and late responses, mediated by Aδ and C fibers, respectively, based on required stimulation strengths, and shows segmental differences in terms of amplitude and duration. We have now investigated whether the peripheral or central anatomy of nociceptive afferent subtypes in different DCNs also vary in a segmental manner. The numbers of different axon subtypes, determined by axon diameter, were analyzed across peripheral DCNs from T6 to L1. The central projections of T7 and T13 DCN afferents were traced using DCN injections of cholera toxin subunit B (CTB) for myelinated A fibers and isolectin B4 (IB4) for unmyelinated C fibers and both labels were quantified in the dorsal horns. Peripheral axon subtype numbers did not differ significantly across DCNs. Centrally, IB4+ , but not CTB+ , projection areas were different between T7 and T13, consistent with different segmental CTM neurogram responses. At both levels, A fibers projected to deeper layers of the dorsal horn than did C fibers. These termination sites are consistent with both mono- and polysynaptic connections between these afferents and the ascending propriospinal interneurons of the reflex. Also analyzed were the spatial distribution, the synaptic termination, and the glutamatergic transporter profiles of DCN A and C fibers and their relationship to calcitonin gene-related peptide (CGRP) staining in the dorsal horn.

Disabled-1 dorsal horn spinal cord neurons co-express Lmx1b and function in nociceptive circuits.

The Reelin-signaling pathway is essential for correct neuronal positioning within the central nervous system. Mutant mice with a deletion of Reelin, its lipoprotein receptors, or its intracellular adaptor protein Disabled-1 (Dab1), exhibit nociceptive abnormalities: thermal (heat) hyperalgesia and reduced mechanical sensitivity. To determine dorsal horn alterations associated with these nociceptive abnormalities, we first characterized the correctly positioned Dab1 neurons in wild-type and mispositioned neurons in Reelin-signaling pathway mutant lumbar spinal cord. Using immunofluorescence, we found that 70% of the numerous Dab1 neurons in Reln+/+ laminae I-II and 67% of those in the lateral reticulated area and lateral spinal nucleus (LSN) co-express the LIM-homeobox transcription factor 1 beta (Lmx1b), an excitatory glutamatergic neuron marker. Evidence of Dab1- and Dab1-Lmx1b neuronal positioning errors was found within the isolectin B4 terminal region of Reln-/- lamina IIinner and in the lateral reticulated area and LSN, where about 50% of the Dab1-Lmx1b neurons are missing. Importantly, Dab1-Lmx1b neurons in laminae I-II and the lateral reticulated area express Fos after noxious thermal or mechanical stimulation and thus participate in these circuits. In another pain relevant locus - the lateral cervical nucleus (LCN), we also found about a 50% loss of Dab1-Lmx1b neurons in Reln-/- mice. We suggest that extensively mispositioned Dab1 projection neurons in the lateral reticulated area, LSN, and LCN and the more subtle positioning errors of Dab1 interneurons in laminae I-II contribute to the abnormalities in pain responses found in Reelin-signaling pathway mutants.

Corneal Nerve Fiber Structure, Its Role in Corneal Function, and Its Changes in Corneal Diseases.

Recently, in vivo confocal microscopy is used to examine the human corneal nerve fibers morphology. Corneal nerve fiber architecture and its role are studied in healthy and pathological conditions. Corneal nerves of rats were studied by nonspecific acetylcholinesterase (NsAchE) staining. NsAchE-positive subepithelial (stromal) nerve fiber has been found to be insensitive to capsaicin. Besides, NsAchE-negative but capsaicin-sensitive subbasal nerve (leash) fibers formed thick mesh-like structure showing close interconnections and exhibit both isolectin B4- and transient receptor potential vanilloid channel 1- (TRPV1-) positive. TRPV1, TRPV3, TRPA (ankyrin) 1, and TRPM (melastatin) 8 are expressed in corneal nerve fibers. Besides the corneal nerve fibers, the expressions of TRPV (1, 3, and 4), TRPC (canonical) 4, and TRPM8 are demonstrated in the corneal epithelial cell membrane. The realization of the importance of TRP channels acting as polymodal sensors of environmental stresses has identified potential drug targets for corneal disease. The pathophysiological conditions of corneal diseases are associated with disruption of normal tissue innervation, especially capsaicin-sensitive small sensory nerve fibers. The relationships between subbasal corneal nerve fiber morphology and neurotrophic keratopathy in corneal diseases are well studied. The recommended treatment for neurotrophic keratopathy is administration of preservative free eye drops.

Continuous Intrathecal Infusion of Cannabinoid Receptor Agonists Attenuates Nerve Ligation-Induced Pain in Rats.

Cannabinoid receptors (CB1R/CB2R) are known to play important roles in pain transmission. In this study, we investigated the effects of continuous intrathecal infusion of CB1/2R agonists in the L5/6 spinal nerve ligation pain model. Under isoflurane anesthesia, rats received nerve ligation and intrathecal catheter connected to an infusion pump. After surgery, saline (1 μL/h), CB1/2R agonist WIN55,212-2, CB1R agonist ACEA, or CB2R agonist AM1241 (1 μmol/h) was given intrathecally for 7 days. The mechanical and thermal sensitivities of rat hindpaw were determined by von Frey hair and radiant heat tests. The expression of CB1/2R and protein levels of CB1/2R, Iba1, glial fibrillary acidic protein, and tumor necrosis factor α were examined by immunofluorescence study and Western blotting. On postligation day 7, rats that received WIN55,212-2, ACEA or AM1241 had significantly higher mean withdrawal thresholds (6.8, 8.4, and 10.2 g) and latencies (6.3, 7.3, and 9.1 seconds) than did saline-treated rats (1.7 g, 2.2 seconds). Cannabinoid receptors were expressed not only in IB4 (isolectin B4) and CGRP (calcitonin gene-related peptide) dorsal root ganglion neurons, their central terminals, and peripheral axons, but also in neurons, microglia, and astrocytes in spinal cord. Cannabinoid receptor agonists enhanced nerve ligation-induced up-regulation of cannabinoid receptor in spinal cord and dorsal root ganglion. Treatment with WIN55,212-2 or AM1241, but not ACEA, markedly reduced nerve ligation-induced up-regulation of Iba1, glial fibrillary acidic protein, and tumor necrosis factor α in spinal cord. Continuous intrathecal infusion of CB1/2R agonists elicits antinociception in the pain model. The mechanisms might involve their actions on neurons and glial cells. CB2R, but not CB1R, seems to play an important role in the regulation of nerve injury-induced neuroinflammation.

Ryk receptors on unmyelinated nerve fibers mediate excitatory synaptic transmission and CCL2 release during neuropathic pain induced by peripheral nerve injury.

BackgroundNeuropathic pain is a major pathology of the central nervous system associated with neuroinflammation. Ryk (receptor-like tyrosine kinase) receptors act as repulsive axon-guidance molecules during development of central nervous system and neural injury. Increasing evidence suggests the potential involvement of Wnt/Ryk (wingless and Int) signaling in the pathogenesis of neuropathic pain. However, its underlying mechanism remains unknown.ResultsThe expression and location of Ryk receptor as well as its ligand Wnt1 were detected by qPCR, Western blot, and immunohistochemistry. We found that Ryk, a specific Wnt receptor, was expressed in IB4+ (Isolectin B4) and CGRP+ (calcitonin gene-related peptide) dorsal root ganglia neurons and their ascending unmyelinated fibers in the dorsal horn of the spinal cord. Ryk was upregulated after spinal nerve ligation surgery. Wnt1 was also increased in activated astrocytes in the dorsal horn after spinal nerve ligation. The presynaptic mechanism of Ryk in regulation of neuropathic pain was determined by electrophysiology in spinal slice. Spinal nerve ligation model was established, and the therapeutic potential of inhibiting Ryk receptor was determined. Spine-specific blocking of the Wnt/Ryk receptor signaling attenuated the spinal nerve ligation-induced mechanical allodynia but not thermal hyperalgesia. Further, it also blocked Ca2+-dependent signals including CaMKII and PKCγ, subsequent release of CCL2 (CCR-like protein) in the dorsal horn. An in vitro study showed that inactivating Ryk receptors with anti-Ryk antibodies or lentiviral Ryk shRNA led to the inactivation of Wnt1 for excitatory synaptic transmission in spinal slices and subsequent decrease in CCL2 expression in the dorsal root ganglia neurons.ConclusionThese studies demonstrate the existence of critical crosstalk between astrocytes and unmyelinated fibers, which indicate the presynaptic mechanism of Ryk in cytokine transmission of neuropathic pain and the therapeutic potential for Wnt/Ryk signaling pathway in the treatment of neuropathic pain.

Dedifferentiated Adipocytes Improve Heart Function Post-Myocardial Infarction

Background:The development of pluripotent stem cell-based strategies for regenerative medicine offers hope of one day having a limitless source of therapeutic cells for the repairing of damaged cardiac tissue resulting from myocardial infarction (MI). Dedifferentiated adipose cells (DFAT) have recently gained attention in this regard for their in vitro and in vivo ability to express vascular and cardiac-specific markers. To date, there is limited knowledge with respect to their capacity to improve cardiac function and contractility when introduced into the myocardium after the onset of MI. To that end, this study investigated the in vivo potential of intramyocardially injected mouse DFAT (mDFAT) cells to differentiate towards cardiovascular lineages and enhance cardiac function in a murine model of MI. Methods: Mature adipocytes were harvested from GFP-transgenic mice and dedifferentiated in vitro to produce pluripotent mDFAT cells. Mice that underwent LAD ligation surgery were injected intramyocardially with a dose of therapeutic mDFAT cells (n=6) shortly after the confirmation of successful MI. Results: Consistent with previous findings by the authors, we observed an immunofluorescent stain pattern in the infarct area of mDFAT cells adjacent to or co-localized with Troponin T, Cx-43, CD31, isolectin B4 and α-SMA up to eight weeks after cell transplantation. Furthermore, echocardiography and hemodynamic catheterization measurements indicated an overall improvement in global cardiac function, including ejection fraction, fractional shortening and contractility. Conclusions: This study is the first to report that the transplantation of mDFAT cells into a murine model of MI can preserve cardiac function up to eight weeks after the onset of infarction, suggesting a potential role for DFAT cells as a therapeutic pluripotent cell source in cardiovascular tissue repair.

The Antiangiogenic Effects of Gold Nanoparticles on Experimental Choroidal Neovascularization in Mice.

The purpose of this study was to evaluate the antiangiogenic effect of gold nanoparticles (AuNPs) on experimental choroidal neovascularization (CNV) in mice. Choroidal neovascularization was induced by rupturing the Bruch's membrane using laser photocoagulation in C57BL/6 mice. The following day, intravitreal injections of AuNPs were administered. The control group received PBS injection of the same volume. Two weeks after laser injury, CNV lesions were evaluated by examination of choroidal flat-mounts using fluorescein-labeled dextran and immunofluorescence staining with isolectin B4. The effects of AuNPs on endothelial cell tube formation, proliferation, and cytotoxicity were evaluated using human umbilical vein endothelial cells (HUVECs) or human RPE cells. The activity of extracellular signal-regulated kinase (ERK)1/2, protein kinase B (Akt), and focal adhesion kinase (FAK) signaling pathways was also analyzed. The AuNPs reduced the extent of CNV. Mice treated with intravitreal AuNPs injections exhibited a 67.9% reduction in the extent of CNV lesions compared with the control group (P < 0.001). The size of the isolectin B4-labeled area was also significantly smaller in AuNP-treated groups compared with the control group (P < 0.001). Gold nanoparticles decreased vascular endothelial growth factor-induced HUVEC tube formation and proliferation but showed no RPE cell toxicity with the treatment doses administered. The phosphorylation of ERK1/2, Akt, and FAK in HUVECs was suppressed by AuNPs. Gold nanoparticles can inhibit laser-induced CNV in mice and may have an indication for the treatment of CNV.

Early Postnatal Hyperoxia in Mice Leads to Severe Persistent Vitreoretinopathy.

To describe a mouse model of hyperoxia-induced vitreoretinopathy that replicated some of the clinical and pathologic features encountered in infants with severe retinopathy of prematurity and congenital ocular conditions such as persistent hyperplastic primary vitreous. Experimental mice (C57BL/6J) were exposed to 65% oxygen between postnatal days (P)0 to P7 and studied at P10, P14, and 3, 5, 8, 20, and 40 weeks. Controls were exposed to normoxic conditions. Fundus imaging and fluorescein angiography were performed at all time points, and spectral-domain optical coherence tomography (SD-OCT) and electroretinography were performed at 8- and 20-week time points. Eyes were processed for resin histology, frozen sections, and retinal whole mounts. Immunostaining was performed to visualize vasculature isolectin B4 (Ib4), collagen type IV, glial fibrillary acidic protein, and α-smooth muscle actin. Early exposure to hyperoxia resulted in bilateral vitreous hemorrhages at 3 weeks. From 5 weeks onward there were extensive zones of retinal degeneration, scarring or gliosis, retinal folding, and detachments caused by traction of α-smooth muscle actin-positive vitreous membranes. Tortuous retinal vessels, together with hyperplastic and persistence of hyaloid vessels are evident into adulthood. In the early stages (P10-3 weeks), branches from the tunica vasculosa lentis (TVL) supplied the marginal retina until retinal vessels were established. The peripheral retina remained poorly vascularized into adulthood. Electroretinography revealed 50% to 60% diminution in retinal function in adult mice that strongly correlated with vitreal changes identified using SD-OCT. This animal model displays a mixture of vitreoretinal pathologic changes that persist into adulthood. The model may prove valuable in experimental investigations of therapeutic approaches to blinding conditions caused by vitreous and retinal abnormalities.

Dorsal root ganglion neurons become hyperexcitable and increase expression of voltage-gated T-type calcium channels (Cav3.2) in paclitaxel-induced peripheral neuropathy.

Here, it is shown that paclitaxel-induced neuropathy is associated with the development of spontaneous activity (SA) and hyperexcitability in dorsal root ganglion (DRG) neurons that is paralleled by increased expression of low-voltage-activated calcium channels (T-type; Cav3.2). The percentage of DRG neurons showing SA and the overall mean rate of SA were significantly higher at day 7 in rats receiving paclitaxel treatment than in rats receiving vehicle. Cav3.2 expression was increased in L4-L6 DRG and spinal cord segments in paclitaxel-treated rats, localized to small calcitonin gene-related peptide and isolectin B4 expressing DRG neurons and to glial fibrillary acidic protein-positive spinal cord cells. Cav3.2 expression was also co-localized with toll-like receptor 4 (TLR4) in both the DRG and the dorsal horn. T-type current amplitudes and density were increased at day 7 after paclitaxel treatment. Perfusion of the TLR4 agonist lipopolysaccharide directly activated DRG neurons, whereas this was prevented by pretreatment with the specific T-type calcium channel inhibitor ML218 hydrochloride. Paclitaxel-induced behavioral hypersensitivity to mechanical stimuli in rats was prevented but not reversed by spinal administration of ML218 hydrochloride or intravenous injection of the TLR4 antagonist TAK242. Paclitaxel induced inward current and action potential discharges in cultured human DRG neurons, and this was blocked by ML218 hydrochloride pretreatment. Furthermore, ML218 hydrochloride decreased firing frequency in human DRG, where spontaneous action potentials were present. In summary, Cav3.2 in concert with TLR4 in DRG neurons appears to contribute to paclitaxel-induced neuropathy.

S100β-Positive Cells of Mesenchymal Origin Reside in the Anterior Lobe of the Embryonic Pituitary Gland.

The anterior and intermediate lobes of the pituitary gland develop through invagination of the oral ectoderm and as they are endocrine tissues, they participate in the maintenance of vital functions via the synthesis and secretion of numerous hormones. We recently observed that several extrapituitary cells invade the anterior lobe of the developing pituitary gland. This raised the question of the origin(s) of these S100β-positive cells, which are not classic endocrine cells but instead comprise a heterogeneous cell population with plural roles, especially as stem/progenitor cells. To better understand the roles of these S100β-positive cells, we performed immunohistochemical analysis using several markers in S100β/GFP-TG rats, which express GFP in S100β-expressing cells under control of the S100β promoter. GFP-positive cells were present as mesenchymal cells surrounding the developing pituitary gland and at Atwell's recess but were not present in the anterior lobe on embryonic day 15.5. These cells were negative for SOX2, a pituitary stem/progenitor marker, and PRRX1, a mesenchyme and pituitary stem/progenitor marker. However, three days later, GFP-positive and PRRX1-positive (but SOX2-negative) cells were observed in the parenchyma of the anterior lobe. Furthermore, some GFP-positive cells were positive for vimentin, p75, isolectin B4, DESMIN, and Ki67. These data suggest that S100β-positive cells of extrapituitary origin invade the anterior lobe, undergoing proliferation and diverse transformation during pituitary organogenesis.

Chemokine receptor CXCR2 in dorsal root ganglion contributes to the maintenance of inflammatory pain.

Chemokines and their receptors have been demonstrated to be important contributors to the development and maintenance of chronic pain. Recent studies showed that chemokine (C-X-C motif) ligand 1 (CXCL1) and its major receptor CXCR2 are respectively expressed in astrocytes and neurons in the spinal cord and are involved in the maintenance of neuropathic pain and inflammatory pain via astrocytic-neuronal interaction. Here we investigated how CXCL1 and CXCR2 are regulated in the dorsal root ganglion (DRG) after peripheral injection of complete Freund's adjuvant (CFA) and its implication in inflammatory pain. CFA induced rapid increase of CXCL1 mRNA and protein in the DRG. Double immunostaining showed that CXCL1 was colocalized with calcitonin gene-related peptide (CGRP), isolectin B4 (IB4), and neurofilament 200 (NF200). Furthermore, CXCR2 mRNA was increased 3h after CFA injection and maintained for more than 3 days. CXCR2 protein was also increased and colocalized with CGRP, IB4, and NF200. Finally, perisciatic nerve injection of CXCR2 siRNA to specifically knock down CXCR2 in the DRG effectively attenuated CFA-induced mechanical allodynia and heat hyperalgesia, and the effect maintained for more than 5 days. Taken together, our results demonstrated that CXCL1 and CXCR2 may regulate the maintenance of inflammatory pain via an autocrine/paracrine way in DRG neurons.

The Anti‐angiogenic Effects of Gold Nanoparticles on Experimental Choroidal Neovascularization in Mice

PurposeTo evaluate the anti‐angiogenic effect of gold nanoparticles (AuNPs) on experimental choroidal neovascularization (CNV) in mice.MethodsCNV was induced by rupturing Bruch's membrane using laser photocoagulation in C57BL/6 mice. The following day, intravitreal AuNPs injections were administered. Two weeks after laser injury, CNV lesions were evaluated by examination of choroidal flat‐mounts using fluorescein‐labeled dextran and immunofluorescence staining with isolectin B4. The effects of AuNPs on endothelial cell tube formation, proliferation, and cytotoxicity were evaluated using human umbilical vein endothelial cells (HUVECs) or human retinal pigment epithelial cells (PRE). The activity of ERK1/2, Akt, and FAK signaling pathways were also analyzed.ResultsAuNPs reduced the extent of CNV. Mice treated with intravitreal AuNPs injections exhibited a 67.9% reduction in the extent of CNV lesions compared to the control group (p &lt; 0.001). The size of the isolectin B4‐labeled area was also significantly smaller in AuNP‐treated groups (p &lt; 0.001). AuNPs decreased vascular endothelial growth factor‐induced HUVEC tube formation and proliferation in a dose‐dependent manner, but showed no cytotoxicity with the treatment doses administered. The phosphorylation of ERK1/2, Akt, and FAK were also suppressed by AuNPs.ConclusionsAuNPs can inhibit laser‐induced CNV in mice and may have therapeutic potential for the treatment of CNV development secondary to neovascular AMD.

SMA+perivascular cells evaluation in VEGF induced blood‐retinal barrier breakdown in rabbit model

PurposeDiabetic Retinopathy (DR) is one of the hallmark microvascular diseases secondary to diabetes. Endothelial cells and perivascular cells are key players in the pathogenesis. It has been shown that VEGF coordinates interactions between endothelial and perivascular cells. In rodent studies, VEGF injected into the eyes results in vascular retinal permeability with pericyte loss. Immunochemistry used for quantification of perivascular cells is well described for murine species but not for rabbit. The aim of the present study was to evaluate perivascular cell pattern in rabbit induced retinal permeability model.MethodsTen pigmented rabbits were induced by intravitreal injection of 500 ng rhVEGF165 into the right eye. At day 2, quantification of vascular leakage was evaluated by fluorometry in both eyes. At day 3, both eye were sampled and fixed. Anti‐ SMA and Isolectin B4 staining was performed on flat‐mounted retina. The evaluation of SMA+cells was performed along the microvasculature.ResultsArea of SMA+cells decreased with distal progression along the arterioles and venules: from 78% to 47% of coverage for arteriolar tree and 69% to 47% for venular tree, less coverage for capillary (30%). Flat‐mounted retina immunostaining analysis revealed that VEGF‐induced treated eyes displayed a remodeling and a loss of the SMA+cells coverage in comparison with left non‐induced eyes: Radial arteriole coverage decreased by 27% (A2) and 23% (A3) in VEGF‐induced eye. Radial venular coverage decreased by 22% (V2) in VEGF‐induced eye. No difference was observed on capillary vessel type with SMA immunolabelling.ConclusionsThis study showed the SMA+cells coverage pattern of retinal microvasculature in rabbit eyes and indicates that VEGF decreased SMA+cells coverage of both arteriolar and venular tree in an induced blood‐retinal barrier breakdown rabbit model.

Itraconazole inhibits laser‐induced choroidal neovascularization in rats by suppressing VEGFR2 expression

PurposeTo demonstrate the anti‐angiogenic effect of itraconazole on laser‐induced choroidal neovascularization (CNV) in rats.MethodsSix laser burns were induced in the peripapillary area of each eye of male Brown Norway rats (200–250 g) to cause CNV. Right eyes were administered intravitreal injections of 1 μg/10 μl itraconazole; left eyes received 10 μl balanced salt solution (BSS) as controls. On day 14 after laser induction, fluorescein angiography (FA) was used to assess abnormal vascular leakage. Flattened retinal pigment epithelium (RPE)‐choroid tissue complex was stained with Alexa Fluor 488‐conjugated isolectin B4 to measure the CNV area with an image analysis program. Vascular endothelial growth factor receptor 2 (VEGFR2) mRNA and protein expression were determined by quantitative RT‐PCR or Western blot.ResultsIntravitreal itraconazole significantly reduced leakage from CNV as assessed by FA and CNV area on flat mounts of the RPE‐choroid complex when compared with intravitreal BSS on day 14 after laser induction (p = 0.002 for CNV leakage, p &lt; 0.001 for CNV area). Quantitative RT‐PCR showed significantly lower expression of VEGFR2 mRNA in RPE‐choroid complexes from itraconazole‐injected eyes than from BSS‐injected eyes (p &lt; 0.001). Western blots indicated that VEGFR2 was downregulated after 14 days of itraconazole treatment.ConclusionsOur study demonstrated that intravitreal itraconazole significantly inhibited the development of laser‐induced CNV in rats. Itraconazole had anti‐angiogenic activity by reducing VEGFR2 expression. Itraconazole may prove beneficial for treating CNV as an alternative or adjunct to other therapies.

Highly efficient generation of GGTA1 knockout pigs using a combination of TALEN mRNA and magnetic beads with somatic cell nuclear transfer

The transcription activator-like effector nuclease (TALEN) technique combined with the somatic cell nuclear transfer (SCNT) method has been successfully applied for creating genetically modified pigs. However, methods for isolating cells with biallelic indels requires further improvement because of the relatively low enrichment efficiency of mutated somatic cells. Moreover, little is known regarding the off-target effects of the TALEN system and the heredity of TALEN-modified pigs. In this study, an efficient method to increase the enrichment efficiency of TALEN-mediated biallelic knockout (KO) cells was established, and corresponding genetically modified pigs with the expected genotype were generated whose off-target effect, fertility and heredity characteristics were aslo evaluated. Two TALEN pairs were constructed to target the porcine α-1,3-galactosyltransferase (GGTA1) gene locus. TALEN mRNA was transfected into the ear fibroblasts followed by the enrichment of α-Gal null cells of minipigs using isolectin B4 (IB4) lectin and magnetic beads. A total of 115 cell colonies were formed and validated to be GGTA1 KO cells by sequencing and 10 biallelic KO cell colonies were used as nuclear donors for SCNT. Thirty GGTA1 biallelic KO piglets were successfully delivered and grew normally. Seventeen potential off-target sites were investigated, and no off-target events were detected in the live piglets. To determine the fertility and heredity characteristics of TALEN-modified pigs, 10 mature founders were mated with each other and the mutations were determined to be transmitted to the F1 piglets. We established a robust and safe technology for developing genetically modified pig lines with expected genotypes for agricultural breeding and biomedical application.

Tissue and cellular characterisation of nucleolin in a murine model of corneal angiogenesis.

Corneal neovascularisation (CNV), with consequent loss of transparency, is due to an imbalance of proangiogenic factors. Cell-surface nucleolin (NCL) has been associated with neo-angiogenesis. There are studies identifying NCL translocation from nucleus to the cell surface, which is essential for endothelial cell proliferation. To find the possible role of NCL in the generation of corneal neovessels, the aim of this study is to characterise the NCL presence and cell-localisation in non-injured corneas, as well as to describe the changes in NCL cell and tissue localisation in CNV, and to analyse the effect of bevacizumab on NCL cellular and tissular distribution. Suture-induced CNV was performed in mice. The corneal tissues were obtained and the histological and co-immunofluorescence assays were performed using different proteins, such as CD31, cadherin and isolectin B4. To determine the possible role of VEGF in NCL presence and localisation in our CNV model, bevacizumab was concomitantly used. Nucleolin was principally observed in the nucleus of the basal epithelial cells of normal corneas. Interestingly, angiogenesis-induced changes were observed in the localisation of NCL, not only in tissue but also at the cellular level where NCL was extranuclear in epithelial cells, stromal cells and neovessels. In contrast, these changes were reverted when bevacizumab was used. Besides, NCL was able to stain only aberrant corneal neovessels in comparison with retinal vessels. NCL mobilisation outside the nucleus during angiogenesis could have a possible role as a proangiogenic molecule in the corneal tissue.

Blockage of PI3K/mTOR Pathways Inhibits Laser-Induced Choroidal Neovascularization and Improves Outcomes Relative to VEGF-A Suppression Alone.

Choroidal neovascularization (CNV) is a major cause of visual loss with age-related macular degeneration (AMD). We evaluated whether blockade of phosphatidyl-inositol-3-kinase (PI3K) and the mammalian target of rapamycin (mTOR), by impairing VEGF-A and other growth factor receptors like platelet-derived growth factor (PDGF), would reduce laser-induced CNV in mice. Choroidal neovascularization lesions were induced in C57BL/6 mice. Two groups of mice received oral GSK2126458 (3 mg/kg) or vehicle for 14 days following laser, whereas three groups were treated with GSK2126458 (6 μg/eye), aflibercept (2 μL/eye), or vehicle intravitreally on days 0 and 7 after laser. Vascular leakage was measured by fluorescein angiography (FA) on day 14. Choroidal neovascularization membranes were evaluated on choroidal flat mounts following FITC-dextran perfusion, as well as ED1 and isolectin B4 (IB4) immunohistochemistry. Oral and intravitreal (IVT) GSK2126458 reduced leakage and area of CNV lesions. Greater probability of leaking lesions (∼60%; P < 0.05) was observed in both vehicle groups. Fluorescein isothiocyanate-dextran-labeled total CNV burden area (total lesion area/eye) was reduced ∼67% (P < 0.05) and 35% (P = 0.0528) after oral and IVT GSK2126458 administration. GSK2126458 treatment reduced lesion size by ∼80% (P < 0.05) and 50% (P < 0.05) for oral and IVT control groups. Aflibercept did not alter lesion size (∼27% reduction). Phosphatidyl-inositol-3-kinase/mTOR is involved in laser-induced CNV angiogenic processes. GSK2126458 effectively reduces CNV size and leakage. Choroidal neovascularization size following IVT GSK2126458 was smaller than after oral administration. Therefore, inhibition of PI3K/mTOR pathways may be more effective due to blockade of action of multiple growth factors.

Cutaneous tissue damage induces long-lasting nociceptive sensitization and regulation of cellular stress- and nerve injury-associated genes in sensory neurons

Tissue damage is one of the major etiological factors in the emergence of chronic/persistent pain, although mechanisms remain enigmatic. Using incision of the back skin of adult rats as a model for tissue damage, we observed sensitization in a nociceptive reflex enduring to 28days post-incision (DPI). To determine if the enduring behavioral changes corresponded with a long-term impact of tissue damage on sensory neurons, we examined the temporal expression profile of injury-regulated genes and the electrophysiological properties of traced dorsal root ganglion (DRG) sensory neurons. The mRNA for the injury/stress-hub gene Activating Transcription Factor 3 (ATF3) was upregulated and peaked within 4 DPI, after which levels declined but remained significantly elevated out to 28 DPI, a time when the initial incision appears healed and tissue-inflammation largely resolved. Accordingly, stereological image analysis indicated that some neurons expressed ATF3 only transiently (mostly medium-large neurons), while in others it was sustained (mostly small neurons), suggesting cell-type-specific responses. In retrogradely-traced ATF3-expressing neurons, Calcium/calmodulin-dependent protein kinase type IV (CAMK4) protein levels and isolectin-B4 (IB4)-binding were suppressed whereas Growth Associated Protein-43 (GAP-43) and Neuropeptide Y (NPY) protein levels were enhanced. Electrophysiological recordings from DiI-traced sensory neurons 28 DPI showed a significant sensitization limited to ATF3-expressing neurons. Thus, ATF3 expression is revealed as a strong predictor of single cells displaying enduring pain-related electrophysiological properties. The cellular injury/stress response induced in sensory neurons by tissue damage and indicated by ATF3 expression is positioned to contribute to pain which can occur after tissue damage.

Enhanced Expression of TREK-1 Is Related with Chronic Constriction Injury of Neuropathic Pain Mouse Model in Dorsal Root Ganglion

Neuropathic pain is a complex state showing increased pain response with dysfunctional inhibitory neurotransmission. The TREK family, one of the two pore domain K+ (K2P) channel subgroups were focused among various mechanisms of neuropathic pain. These channels influence neuronal excitability and are thought to be related in mechano/thermosensation. However, only a little is known about the expression and role of TREK-1 and TREK-2, in neuropathic pain. It is performed to know whether TREK-1 and/or 2 are positively related in dorsal root ganglion (DRG) of a mouse neuropathic pain model, the chronic constriction injury (CCI) model. Following this purpose, Reverse Transcription Polymerase Chain Reaction (RT-PCR) and western blot analyses were performed using mouse DRG of CCI model and compared to the sham surgery group. Immunofluorescence staining of isolectin-B4 (IB4) and TREK were performed. Electrophysiological recordings of single channel currents were analyzed to obtain the information about the channel. Interactions with known TREK activators were tested to confirm the expression. While both TREK-1 and TREK-2 mRNA were significantly overexpressed in DRG of CCI mice, only TREK-1 showed significant increase (∼9 fold) in western blot analysis. The TREK-1-like channel recorded in DRG neurons of the CCI mouse showed similar current-voltage relationship and conductance to TREK-1. It was easily activated by low pH solution (pH 6.3), negative pressure, and riluzole. Immunofluorescence images showed the expression of TREK-1 was stronger compared to TREK-2 on IB4 positive neurons. These results suggest that modulation of the TREK-1 channel may have beneficial analgesic effects in neuropathic pain patients.