Fluorescein Dextran Research Articles

Corrigendum for Roche et al., volume 298, 2010, p. C298–C312

CorrigendumCorrigendum for Roche et al., volume 298, 2010, p. C298–C312Published Online:09 Dec 2022https://doi.org/10.1152/ajpcell.00122.2009_COROriginal articleMoreSectionsPDF (1 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations Roche JA, Lovering RM, Roche R, Ru LW, Reed PW, Bloch RJ. Extensive mononuclear infiltration and myogenesis characterize recovery of dysferlin-null skeletal muscle from contraction-induced injuries. Am J Physiol Cell Physiol 298: C298–C312, 2010. First published November 18, 2009. doi: 10.1152/ajpcell.00122.2009.The original version of Fig. 2 mistakenly duplicated the panel for day 7 after 15 R injury of A/J fibers by large-strain eccentric injury in the panel for day 14 postinjury. A corrected version of this figure is presented below. The original figure legend remains the same. The authors apologize for this error and state that the correction does not change the scientific conclusions of the article.Figure 2.Membrane damage and resealing after injury. Representative images are shown of tibialis anterior (TA) muscle cross sections obtained from muscles exposed to fluorescein dextran (FDx) before injury and collected at different times thereafter. After 15 R injury, the same percentage of myofibers in TA muscles of A/WySnJ and A/J mice took up FDx. These FDx-positive (FDx+) fibers were retained through D14 in A/WySnJ mice but were lost in A/J mice. After 150 R injury, the same percentage of myofibers in TA muscles of A/WySnJ and A/J mice took up the dye. By D14, however, these fibers were lost in both mouse strains and the values for FDx-labeled myofibers returned to preinjury levels.Download figureDownload PowerPointThis article has no references to display. Download PDF Previous Back to Top FiguresReferencesRelatedInformation Related ArticlesExtensive mononuclear infiltration and myogenesis characterize recovery of dysferlin-null skeletal muscle from contraction-induced injuries 01 Feb 2010American Journal of Physiology-Cell Physiology More from this issue > Volume 323Issue 6December 2022Pages C1872-C1872 Crossmark Copyright & PermissionsCopyright © 2022 the American Physiological Society.https://doi.org/10.1152/ajpcell.00122.2009_CORPubMed36484642History Published online 9 December 2022 Published in print 1 December 2022 Metrics

TLR2 signaling contributes to the angiogenesis of oxygen-induced retinopathy

PurposeTo evaluate the role of Toll-like receptor 2 (TLR2) signaling in retinal neovascularization in a mouse model of oxygen-induced retinopathy (OIR). Materials and methodsThe OIR model was established in C57BL/6J wild type (WT) mice and TLR2−/− mice. Retinal neovascularization in the OIR model was measured by counting new vascular cell nuclei above the internal limiting membrane and analyzing flat-mounted retinas perfused with fluorescein dextran and immunostained with Griffonia Simplicifolia (GS) isolectin. The expression of TLR2 and VEGF in the retina was detected by immunofluorescence. Expression of TGF- β1, b-FGF, and IL-6 mRNA in the retina was measured by quantitative real-time PCR. ResultsCompared to WT OIR mice, retinal neovascularization was attenuated in TLR2−/− OIR mice. The co-expressions of TLR2 and VEGF were remarkably and consistently increased in WT OIR mice; however, there was no expression of TLR2 and a significant decrease in VEGF expression in TLR2−/− OIR mice. These results suggest that TLR2 plays a central role in OIR model angiogenesis. Expression of TGF- β1, b-FGF, and IL-6 mRNA were reduced in the TLR2−/− OIR mice, suggesting that the inflammatory response induced by TLR2 relates to angiogenesis. ConclusionTLR2 signaling in the retina is associated with neovascularization in mice. Inflammation contributes to the activation of angiogenesis and is partially mediated through the TLR2-VEGF retinal signaling pathway.

Entrapment of Hydrophilic and Hydrophobic Molecules in Beads Prepared from Isolated Denatured Whey Protein.

The oral route of administration is by far the most convenient route, especially in the treatment of chronic conditions. However, many therapeutics present formulation difficulties which make them unsuitable for oral delivery. Recently, we synthesized a denatured whey protein isolate (dWPI) bead entrapped with insulin. Our present goal was to assess the suitability of this delivery system to the delivery of other potential molecules, both hydrophilic and hydrophobic. Beads of 1.2–1.5 mm in diameter were entrapped with four payloads representing a range of solubilities. The water-soluble payloads were sodium fluorescein (SF) and FITC dextran 4000 Da (FD4), while the hydrophobic ones were Fast Green and curcumin. Encapsulation efficiency (EE) was 73%, 84%, 70%, and 83% for SF, FD4, Fast Green, and curcumin-loaded beads, respectively. The corresponding loading capacity for each bead was 0.07%, 1.1%, 0.75%, and 1.1%, respectively. Each payload produced different release profiles in simulated gastric fluid (SGF) and simulated intestinal fluids (SIF). SF released steadily in both SGF and SIF. FD4 and curcumin release was not substantial in any buffers, while Fast Green release was low in SGF and high in SIF. The differences in release behaviour were likely due to the varying properties of the payloads. The effect of proteolysis on beads suggested that enzymatic degradation of the whey bead may promote payload release. The beads swelled rapidly in SGF compared to SIF, which likely contributed to the release from the beads, which was largely governed by solvent diffusion and polymer relaxation. Our results offer a systematic examination of the behaviour of hydrophilic and hydrophobic payloads in a dWPI delivery system. These beads may be further designed to orally deliver poorly permeable macromolecules and poorly soluble small molecules of pharmaceutical interest.

Toll-like receptor 4 mediates blood-brain barrier permeability and disease in C3H mice during Venezuelan equine encephalitis virus infection

ABSTRACT Venezuelan equine encephalitis virus (VEEV) is an encephalitic alphavirus that can cause debilitating, acute febrile illness and potentially result in encephalitis. Currently, there are no FDA-licensed vaccines or specific therapeutics for VEEV. Previous studies have demonstrated that VEEV infection results in increased blood-brain barrier (BBB) permeability that is mediated by matrix metalloproteinases (MMPs). Furthermore, after subarachnoid hemorrhage in mice, MMP-9 is upregulated in the brain and mediates BBB permeability in a toll-like receptor 4 (TLR4)-dependent manner. Here, we demonstrate that disease in C3H mice during VEEV TC-83 infection is dependent on TLR4 because intranasal infection of C3H/HeN (TLR4 WT ) mice with VEEV TC-83 resulted in mortality as opposed to survival of TLR4-defective C3H/HeJ (TLR4 mut ) mice. In addition, BBB permeability was induced to a lesser extent in TLR4 mut mice compared with TLR4 WT mice during VEEV TC-83 infection as determined by sodium fluorescein and fluorescently-conjugated dextran extravasation. Moreover, MMP-9, MMP-2, ICAM-1, CCL2 and IFN-γ were all induced to significantly lower levels in the brains of infected TLR4 mut mice compared with infected TLR4 WT mice despite the absence of significantly different viral titers or immune cell populations in the brains of infected TLR4 WT and TLR4 mut mice. These data demonstrate the critical role of TLR4 in mediating BBB permeability and disease in C3H mice during VEEV TC-83 infection, which suggests that TLR4 is a potential target for the development of therapeutics for VEEV.

Towards the development of a human in vitro model of the blood-brain barrier for virus-associated acute encephalopathy: assessment of the time- and concentration-dependent effects of TNF-α on paracellular tightness.

The pathogenesis of virus-associated acute encephalopathy (VAE) involves brain edema caused by disruption of the blood-brain barrier (BBB). We aimed to develop an in vitro VAE model using an in vitro BBB model, to evaluate the dynamics of vascular dysfunction caused by tumor necrosis factor (TNF)-α. A co-culture model, consisting of Transwell®-grown human brain microvascular endothelial cells and pericytes, was treated with serially diluted TNF-α. Transendothelial electrical resistance (TER) was measured using cellZscope®. A permeability assay, using fluorescein isothiocyanate-conjugated sodium or dextran, was performed. Changes in claudin-5 localization and expression after TNF-α treatment were observed using immunofluorescence staining and western blot analysis. The TER decreased and permeability increased after TNF-α treatment; recovery time was dependent on TNF-α concentration. Claudin-5 was delocalized after TNF-α treatment and recovered in a TNF-α concentration-dependent manner. The expression of claudin-5 decreased 24h after the TNF-α treatment and completely recovered 48h after TNF-α treatment. Claudin-5 delocalization was likely associated with vascular hyperpermeability. To conclude, we evaluated vascular endothelial cell permeability and injury in VAE using an in vitro BBB model treated with TNF-α. This system can be useful for developing novel therapeutic strategies for VAE and designing treatments that target vascular permeability.

Oxidative Stress Product, 4-Hydroxy-2-Nonenal, Induces the Release of Tissue Factor-Positive Microvesicles From Perivascular Cells Into Circulation.

TF (Tissue factor) plays a key role in hemostasis, but an aberrant expression of TF leads to thrombosis. The objective of the present study is to investigate the effect of 4-hydroxy-2-nonenal (HNE), the most stable and major oxidant produced in various disease conditions, on the release of TF+ microvesicles into the circulation, identify the source of TF+ microvesicles origin, and assess their effect on intravascular coagulation and inflammation. Approach and Results: C57BL/6J mice were administered with HNE intraperitoneally, and the release of TF+ microvesicles into circulation was evaluated using coagulation assays and nanoparticle tracking analysis. Various cell-specific markers were used to identify the cellular source of TF+ microvesicles. Vascular permeability was analyzed by the extravasation of Evans blue dye or fluorescein dextran. HNE administration to mice markedly increased the levels of TF+ microvesicles and thrombin generation in the circulation. HNE administration also increased the number of neutrophils in the lungs and elevated the levels of inflammatory cytokines in plasma. Administration of an anti-TF antibody blocked not only HNE-induced thrombin generation but also HNE-induced inflammation. Confocal microscopy and immunoblotting studies showed that HNE does not induce TF expression either in vascular endothelium or circulating monocytes. Microvesicles harvested from HNE-administered mice stained positively with CD248 and α-smooth muscle actin, the markers that are specific to perivascular cells. HNE was found to destabilize endothelial cell barrier integrity. HNE promotes the release of TF+ microvesicles from perivascular cells into the circulation. HNE-induced increased TF activity contributes to intravascular coagulation and inflammation.

Effect of ZO Protein Knockdown on Leak Pathway Pore Size in MDCK Renal Epithelial Cell Populations

The tight junction is the primary barrier to the paracellular movement of water and solutes across epithelial cells (paracellular permeability). Paracellular permeability is divided into two pathways: the Pore Pathway (small solute size, high capacity, charge‐selective) and the Leak Pathway (large solute size, low capacity, charge‐nonselective). Much has been learned about the molecular basis, properties, and regulation of the Pore Pathway. The Leak Pathway is much less well understood. Two basic questions about the Leak Pathway that remain controversial are: 1) does the Leak Pathway have a size limit; and 2) if so, how is the Leak Pathway size limit affected by manipulating specific tight junction proteins. We have initiated an investigation of these two questions by measuring the paracellular permeability of a size range of fluorescein dextrans (4 kDa to 70 kDa) and calculating pore size using the mathematical model, the Renkin sieving equation. In this mathematical model, calculation of pore size is independent of total flux rate, which is also affected by the pore volume fraction and pore length. We first used this approach to examine Leak Pathway permeability in the wild type MDCK renal epithelial cell line. The paracellular movement of all fluorescein dextrans across MDCK cell monolayers was linear with time. The apparent permeability (Papp) decreased with increasing solute Stokes radius. Using the Renkin sieving equation, the Leak Pathway pore size in wild type MDCK cell monolayers was calculated to be ~423 Angstroms. We then examined the effect of knockdown of either of the tight junction‐associated cytoplasmic proteins, ZO‐1 or ZO‐2, on pore size. The paracellular flux rates for all fluorescein dextrans were higher across ZO‐1 knockdown MDCK (ZO‐1 KD) cell populations than across either wild type MDCK cell populations or ZO‐2 knockdown MDCK (ZO‐2 KD) cell populations, which were similar. The paracellular flux of each fluorescein dextran was linear with time across populations of either ZO‐1 KD cells or ZO‐2 KD cells. Papp decreased with increasing Stokes radius for both knockdown cell lines. The calculated pore size for ZO‐2 KD cell populations was similar to that calculated for wild type MDCK cell populations. In contrast, the pore size for ZO‐1 KD cell populations was substantially smaller (~171 Angstroms) than was calculated for wild type MDCK cell populations despite the fact that the total flux rate for all fluorescein dextrans was significantly higher across ZO‐1 KD cell populations. These results indicate that knockdown of ZO‐1 in MDCK cells decreases the Leak Pathway pore size, whereas, knockdown of ZO‐2 does not alter Leak Pathway pore size. In addition, since the flux rate across ZO‐1 KD cell monolayers is higher than across monolayers of the other two cell lines, these results suggest ZO‐1 KD cell populations exhibit either a greater pore volume fraction and/or a shorter pore length than do either wild type MDCK cell populations or ZO‐2 KD cell populations.Support or Funding InformationNYITCOM internal funds

A measure of tear inflow in habitual scleral lens wearers with and without midday fogging.

The purpose of this pilot study was to evaluate tear inflow in a scleral lens system using fluorophotometry, and indirectly assess the exchange of the tear reservoir in habitual scleral lens wearers with the presence or absence of midday fogging (MDF). Habitual scleral lens wearers (n=23) and normal scleral lens neophytes (n=10) were recruited. Of the 23 habitual wearers, 11 of them experienced MDF and 12 did not have a diagnosis of MDF. Contact lens-fitting characteristics were evaluated using ocular coherence tomograpy (OCT) and biomicroscopy. High molecular weight fluorescein (FITC) Dextran was instilled into the tear reservoir beneath the scleral lens, and the tear fluid fluorescein concentration was measured using the Fluorotron fluorophotometer. Calculated fluorescein concentrations were plotted over time to measure the fluorescein decay rate of the tear fluid beneath the scleral lens, which was used to calculate the tear exchange rate. There was no significant difference in tear inflow between the MDF group (mean: 0.111%) and the non-MDF group (mean: 0.417%), and there was a high amount of variability seen in the rates (p = 0.26). In addition, there was no significance between the tear reservoir thickness in the MDF (283um) and non-MDF (326um) groups (p = 0.53). The relationship between the amount of tear exchange during scleral lens wear and the incidence of MDF was not significant. Additional studies are needed to further examine the role of tear exchange in MDF and address the causes of variability to improve measurement techniques with fluorophotometry in the scleral lens system.

Quantification of solute diffusivity in osteoarthritic human femoral cartilage using correlation spectroscopy.

Osteoarthritis is a chronic joint disease characterized by articular cartilage degeneration, pain, and disability. As an avascular tissue, the movement of water and solutes through the tissue is critical to cartilage health and function, and early changes in solute diffusivity due to micro-scale changes in the properties of cartilage's extracellular matrix might precede clinical symptoms. A diagnostic technique for quantifying alteration to the diffusive environment of cartilage that precedes macroscopic changes may allow for the earlier identification of osteoarthritic disease, facilitating earlier intervention strategies. Toward this end, we used two confocal microscopy-based correlation spectroscopy techniques, fluorescence correlation spectroscopy and raster image correlation spectroscopy, to quantify the diffusion of two small solutes, fluorescein and 3k dextran, within human osteoarthritic articular cartilage. Our goal was to determine if these relatively simple optical correlation spectroscopy techniques could detect changes in solute diffusivity associated with increasing cartilage damage as assessed by International Cartilage Repair Society scoring guidelines, and if these measures are correlated with mechanical and compositional measures of cartilage health. Our data show a modest, yet significant increase in solute diffusivity and cartilage permeability with increasing osteoarthritis score (grades 0-2), with a strong correlation between diffusion coefficients, permeability, and cartilage composition. The described correlation spectroscopy techniques are quick, simple, and easily adapted to existing laboratory workflow and equipment. Furthermore, the minimal solute concentrations and laser powers required for analysis, combined with recent advances in arthroscopic microscopy, suggest correlation spectroscopy techniques as translational candidates for development into early OA diagnosis tools. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:3256-3267, 2018.

The effects of ldb1 dosage on the expression of dll1 and histology in the developing vertebrate eye.

Ldb1 is a relatively new gene is the realm of eye development. Its role as a transcriptional cofactor is well-known, but its targets and direct effects on the developing optic cup, retina, and lens are still to be understood. This project uses Xenopus laevis as a model system of the vertebrate eye in studying the effects of knocking down or overexpressing the active truncation of the ldb1 gene to the eye. Ldb1 RNA or morpholino oligonucleotide, together with fluorescein dextran (FLDX), was microinjected into one dorsal cell at the 4-cell or 8-cell embryonic stages. This allows for an internally controlled mechanism of tracking the injection among the two sides of the embryos’ developing heads.In situ hybridization and histological sectioning was used to visualize the effects of the ldb1 manipulation on dll1, a candidate target of ldb1, and the structures of the eye. In preliminary results, dll1 expression in both the retina and neural tube of Stage 28-30 embryos appears to show a shift in expression in the injected areas. Altering the levels of ldb1 in the dorsal, animal hemisphere of the embryo appears to affect normal eye development; increase in ldb1 dosage appears to result in advanced eye development and an increase in neural tube and retinal dll1 expression. Further results of dll1 expression in ectoderm explanted from ldb1 RNA-injected zygotes will also give insight specifically to confirm the suggestion that dll1 is a possible direct target of ldb1 in the developing embryo.

Adenosine receptor agonist NECA increases cerebral extravasation of fluorescein and low molecular weight dextran independent of blood-brain barrier modulation.

Conventional methods for therapeutic blood-brain barrier (BBB) disruption facilitate drug delivery but are cumbersome to perform. A previous study demonstrated that adenosine receptor (AR) stimulation by 5′-N-ethylcarboxamide adenosine (NECA) increased the extravasation of intravascular tracers into the brain and proposed that AR agonism may be an effective method for therapeutic BBB disruption. We attempted to confirm the extravasation of tracers into the brain and also investigated tracer extravasation into peripheral organs and tracer retention in the blood. We found that NECA not only increased the extravasation of intravascular fluorescein and low molecular weight dextran into the brain of mice but also increased the concentrations of these tracers in the blood. In fact, the brain:blood ratio-normalized BBB permeability for either tracer is actually decreased by NECA administration. Elevated blood urea nitrogen levels in mice following NECA treatment suggested that renal function impairment was a probable cause of tracer retention. Therefore, NECA has almost no effect on the extravasation of intravascular Evans blue dye (EBD), an albumin-binding tracer with little renal clearance. Rather than inducing BBB disruption, our study demonstrated that NECA increased tracer extravasation into the brain by increasing the concentration gradient of the tracer across the BBB.

Neuroprotective Effect of VEGF-Mimetic Peptide QK in Experimental Brain Ischemia Induced in Rat by Middle Cerebral Artery Occlusion.

We investigated the effect of the VEGF-mimetic peptide, QK, on ischemic brain damage and on blood-brain barrier permeability in the rat. QK administered by the intracerebroventricular, intravenous, or intranasal route caused a 40% decrease in ischemic brain damage induced by permanent occlusion of the middle cerebral artery relative to that in controls. No increase in the volume of the ischemic hemisphere compared to that of the contralateral nonischemic hemisphere was observed in rats treated with QK, suggesting that this peptide did not cause brain edema. The effect of QK on vessel permeability was evaluated by intravital pial microvessel videoimaging, a technique that allows the pial vessels to be visualized through a surgically prepared open cranial window. The results showed that QK did not cause any leakage of intravenously injected fluorescein-dextran conjugates after intracarotid administration or topical application to the brain cortex. Collectively, these data suggest that QK may exert neuroprotective activity in the context of stroke without promoting any increase in vascular permeability. Because VEGF's neuroprotective activity may be overshadowed by the appearance of brain edema and microbleeds, QK could represent a significant step forward in stroke treatment.

Diffusion profile of macromolecules within and between human skin layers for (trans)dermal drug delivery

Delivering a drug into and through the skin is of interest as the skin can act as an alternative drug administration route for oral delivery. The development of new delivery methods, such as microneedles, makes it possible to not only deliver small molecules into the skin, which are able to pass the outer layer of the skin in therapeutic amounts, but also macromolecules. To provide insight into the administration of these molecules into the skin, the aim of this study was to assess the transport of macromolecules within and between its various layers. The diffusion coefficients in the epidermis and several locations in the papillary and reticular dermis were determined for fluorescein dextran of 40 and 500kDa using a combination of fluorescent recovery after photobleaching experiments and finite element analysis. The diffusion coefficient was significantly higher for 40kDa than 500kDa dextran, with median values of 23 and 9µm2/s in the dermis, respectively. The values only marginally varied within and between papillary and reticular dermis. For the 40kDa dextran, the diffusion coefficient in the epidermis was twice as low as in the dermis layers. The adopted method may be used for other macromolecules, which are of interest for dermal and transdermal drug delivery. The knowledge about diffusion in the skin is useful to optimize (trans)dermal drug delivery systems to target specific layers or cells in the human skin.

Transport, resealing, and re-poration dynamics of two-pulse electroporation-mediated molecular delivery

Electroporation is of interest for many drug-delivery and gene-therapy applications. Prior studies have shown that a two-pulse-electroporation protocol consisting of a short-duration, high-voltage first pulse followed by a longer, low-voltage second pulse can increase delivery efficiency and preserve viability. In this work the effects of the field strength of the first and second pulses and the inter-pulse delay time on the delivery of two different-sized Fluorescein–Dextran (FD) conjugates are investigated. A series of two-pulse-electroporation experiments were performed on 3T3-mouse fibroblast cells, with an alternating-current first pulse to permeabilize the cell, followed by a direct-current second pulse. The protocols were rationally designed to best separate the mechanisms of permeabilization and electrophoretic transport. The results showed that the delivery of FD varied strongly with the strength of the first pulse and the size of the target molecule. The delivered FD concentration also decreased linearly with the logarithm of the inter-pulse delay. The data indicate that membrane resealing after electropermeabilization occurs rapidly, but that a non-negligible fraction of the pores can be reopened by the second pulse for delay times on the order of hundreds of seconds. The role of the second pulse is hypothesized to be more than just electrophoresis, with a minimum threshold field strength required to reopen nano-sized pores or defects remaining from the first pulse. These results suggest that membrane electroporation, sealing, and re-poration is a complex process that has both short-term and long-term components, which may in part explain the wide variation in membrane-resealing times reported in the literature.

Changes in extra- and intracellular pH in hepatocytes exposed to gabexate mesilate.

Gabexate mesilate (GM) is a synthetic inhibitor of plasmatic and pancreatic serine proteases licensed for the treatment of pancreatitis. Here we show that in suspensions of isolated hepatocytes, profound changes in extracellular, cytoplasmic, and vesicular pH occur after addition of GM. Isolated hepatocytes obtained by collagenase perfusion of rat liver were pre-incubated with 1, 2, and 4 mM GM. Extracellular pH (pH in the incubation medium) was measured by a conventional pH electrode, cytosolic and vesicular pH were measured by fluorescence changes of 2',7'-biscarboxyethyl-5,6-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and fluorescein dextran, respectively. Incubation of hepatocytes with GM resulted in a dose-dependent decrease of extracellular pH. Cytosolic pH decreased rapidly and markedly in a dose-dependent manner during the first minutes and gradually returned towards baseline. Simultaneously, GM induced a rapid alkalinization of acidic vesicles. The presence of bis-(p-nitrophelyl) phosphate (BNPP), an esterase inhibitor, reduced the extent of extracellular acidification. Incubation of hepatocytes in the presence of dimethylamiloride, an Na+/H+ exchanger inhibitor, or in a sodium-free medium, did not modify the rate and extent of extracellular acidification. GM, a commercially available pharmacological agent, could be useful to manipulate extra- and intracellular pH.

Effect of VEGF and anti-VEGF Compounds on Retinal Pigment Epithelium Permeability: An in Vitro Study

To evaluate the effect of 2 vascular endothelial growth factor (VEGF) isoforms (121 and 165) and 2 anti-VEGF compounds (ranibizumab and pegaptanib sodium) on the permeability of human retinal pigment epithelium (RPE) cells in vitro. The RPE permeability was assessed on ARPE19 cells grown onto inserts of polytetrafluoroethylene previously treated with ammonia gas plasma. Paracellular permeability to ions was measured by mean of transepithelial electrical resistance (TEER). Permeability to non-ionic molecules was gathered by the amount of fluorescein dextran (FD) passing across the monolayer within 2 hours. Only VEGF165 applied at the apical side of the monolayer induced a statistically significant decrease of TEER (p<0.001). No changes in TEER were observed when pegaptanib sodium or ranibizumab were apically administered together with VEGF165. Both VEGF isoforms significantly increased permeability to 4 kDa dextran (p<0.01). Apical administration of ranibizumab or pegaptanib sodium as well as coadministration of pegaptanib sodium with VEGF121 or VEGF165 induced a statistically significant increase of permeability to 4 kDa FD. Both VEGF isoforms and anti-VEGF compounds exert an effect on human RPE permeability in vitro.

Attenuation of streptozotocin-induced diabetic retinopathy with low molecular weight fucoidan via inhibition of vascular endothelial growth factor

Diabetic retinopathy (DR) is a hyperglycemia-induced ischemic disorder characterized by microvascular dysfunction and neovascularization. It is a leading cause of blindness in many countries, yet efficient drugs are limited now for prevention and treatment of DR. Low molecular weight fucoidan (LMWF), extract from brown algae, has been shown to possess multiple biological activities like anti-inflammation, anti-oxidation and anti-aggregation, which all could be beneficial for attenuating ischemia-induced tissue damages. Here, by comparing with calcium dobesilate, the potent antioxidant compound currently used for the treatment of DR, we investigated the protective effect of LMWF against DR in streptozotocin-induced diabetic mice and high glucose-promoted vascular endothelial growth factor (VEGF) production and cell proliferation in microvascular endothelial cells. One week after diabetes induction, the mice were administered with LMWF (50, 100 or 200 mg/kg/day) or calcium dobesilate (200 mg/kg/day) for four months, then the retinal pathological changes and neovascularization were detected by hematoxylin-eosin staining and fluorescein dextran angiography, respectively. Immunofluorescence staining, ELISA and RT-PCR were used to examine the expression levels of hypoxia-inducible factor-1α (HIF-1α) and VEGF in retina and endothelial cells. Here, we found that LMWF resembled calcium dobesilate, in alleviating retinal pathological change and hindering neovascularization due to diabetes in vivo. The relative levels of VEGF expression and HIF-1α induction were also less in retinas of LMWF- or calcium dobesilate-treated diabetic mice than those in retinas of control mice. Furthermore, high glucose-induced VEGF overexpression and cell proliferation in primary cultured vascular endothelial cells were also inhibited by LMWF in a dose-dependent manner. Therefore, this study demonstrated that LMWF alleviates diabetic retinal neovascularization and damage likely through lowering HIF-1α and VEGF expressions, providing a potential candidate drug for prevention and treatment of diabetic retinopathy.

Brain edema in acute liver failure: Role of neurosteroids

Brain edema is a major neurological complication of acute liver failure (ALF) and swelling of astrocytes (cytotoxic brain edema) is the most prominent neuropathological abnormality in this condition. Elevated brain ammonia level has been strongly implicated as an important factor in the mechanism of astrocyte swelling/brain edema in ALF. Recent studies, however, have suggested the possibility of a vasogenic component in the mechanism in ALF. We therefore examined the effect of ammonia on blood–brain barrier (BBB) integrity in an in vitro co-culture model of the BBB (consisting of primary cultures of rat brain endothelial cells and astrocytes). We found a minor degree of endothelial permeability to dextran fluorescein (16.2%) when the co-culture BBB model was exposed to a pathophysiological concentration of ammonia (5mM). By contrast, lipopolysaccharide (LPS), a molecule well-known to disrupt the BBB, resulted in an 87% increase in permeability. Since increased neurosteroid biosynthesis has been reported to occur in brain in ALF, and since neurosteroids are known to protect against BBB breakdown, we examined whether neurosteroids exerted any protective effect on the slight permeability of the BBB after exposure to ammonia. We found that a nanomolar concentration (10nM) of the neurosteroids allopregnanolone (THP) and tetrahydrodeoxycorticosterone (THDOC) significantly reduced the ammonia-induced increase in BBB permeability (69.13 and 58.64%, respectively). On the other hand, we found a marked disruption of the BBB when the co-culture model was exposed to the hepatotoxin azoxymethane (218.4%), but not with other liver toxins commonly used as models of ALF (thioacetamide and galactosamine, showed a 29.3 and 30.67% increase in permeability, respectively). Additionally, THP and THDOC reduced the effect of TAA and galactosamine on BBB permeability, while no BBB protective effect was observed following treatment with azoxymethane. These findings suggest that ammonia does not cause a significant BBB disruption, and that the BBB is intact in the TAA or galactosamine-induced animal models of ALF, likely due to the protective effect of neurosteroids that are synthesized in brain in the setting of ALF. However, caution should be exercised when using azoxymethane as an experimental model of ALF as it caused a severe breakdown of the BBB, and neurosteriods failed to protect against this breakdown.

Folic acid-tethered Pep-1 peptide-conjugated liposomal nanocarrier for enhanced intracellular drug delivery to cancer cells: conformational characterization and in vitro cellular uptake evaluation

BackgroundA novel dual ligand–modified liposome, folic acid-tethered Pep-1 peptide-conjugated liposomal nanocarrier (FP-Lipo), was designed to overcome the nonselectivity of conventional penetrating peptide-tagged nanoparticulates and to provide the advantage of selective targeting of the folic acid receptor, which is frequently overexpressed on epithelial cancer cells.MethodsFP-Lipo was prepared by a sequential process of formation of a maleimide-derivatized small unilamellar vesicle, postinsertion of distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000–folate to the vesicle, and Pep-1 peptide conjugation via thiol-maleimide linkage. Conformational and physical characteristics of the FP-Lipo nanocarriers were investigated for the extent of Pep-1 peptide and folic acid on the surface, vesicle size, and zeta potential. In vitro cellular uptake behaviors of the novel carrier containing a fluorescein dextran isothiocyanate probe were examined by spectrophotometry or by confocal laser scanning microscopy.ResultsA novel nanocarrier bearing approximately 750 folate ligands and 100 penetrating peptides per vesicle was successfully prepared. The physical properties were as follows: 140 nm in size; 5 mV in zeta potential; less than 0.3 in polydispersity index. An in vitro cellular uptake study revealed that the FP-Lipo nanocarrier system exhibited more than twofold enhanced translocation into the folic acid receptor–positive HeLa cells compared with the single Pep-1 peptide–modified liposome. Meanwhile, its cellular association and internalization into the folic acid receptor–negative normal HaCaT cells was comparable with that of Pep-1 peptide–modified liposome.ConclusionAn advanced dual ligand-modified liposome is potentially useful for the treatment of folic acid receptor–positive tumors with high translocation capability of the penetrating peptide–modified liposome.

Fluorescein-dextran sequestration in the reproductive tract of the migratory grasshopper Melanoplus sanguinipes (Orthoptera, Acridiidae)

The fluid dynamics of the reproductive system of the migratory grasshopper, Melanoplus sanguinipes F. (Orthoptera: Acrididae) was examined by the introduction of fluorescein-dextran (FD) into the hemocoel and observing its tissue specific sequestration. Male grasshoppers were observed to sequester FD first in the apical end of each sperm tube. FD then moved into the vasa deferentia and ejaculatory duct. This suggests that materials, that transit the hemolymph could be a component of the spermatophore in M. sanguinipes. Female grasshoppers were observed to sequester hemolymph FD into vitellogenic oocytes and to sometimes reuptake the FD during the resorption of oocytes in the formation of yellow bodies or corpora lutea. Female M. sanguinipes who performed long duration flight sequestered FD in their oocytes to a greater degree than controls as determined by fluorescence intensity data collected as the mean gray value of the flourescein emission channel. Transfer of hemolymph FD from males to females was observed at the pores along the margin of the operculum of eggs in the female common oviduct following mating. FD has the potential to be an effective tracker of male reproductive secretions and as a tool for the observation of insect reproductive tract development.