Intravitreal Injection of Normal Saline Induces Retinal Degeneration in the C57BL/6J Mouse

Purpose: To investigate the adverse effect of intravitreal injection of normal saline (NS) and phosphate buffered saline (PBS) in mouse eyes.

Methods: NS or PBS was injected intravitreally into C57BL/6J mouse eyes. Retinal lesions were monitored by fundus imaging, spectral-domain optical coherence tomography (SD-OCT), and histological investigations. Retinal immune gene expression was determined by real-time polymerase chain reaction (PCR). The toxic effect of NS and PBS or retinal protein from NS- or PBS-injected eyes on retinal pigment epithelium (RPE) was tested in B6-RPE-07 mouse RPE cell cultures.

Results: Intravitreal injection of NS dose-dependently induced localized retinal lesion in mice. Histological investigations revealed multiple vacuoles in photoreceptor outer segments and RPE cells. The lesions recovered over time and by 3 weeks post injection the majority of lesions vanished in eyes receiving 1 μl NS. Inflammatory genes, including TNF-α, IL-1β, IL-6, iNOS, and VEGF were upregulated in NS injected eyes. Intravitreal injection of PBS did not cause any pathology. The treatment of B6-RPE07 cells with 30% PBS or 30% NS did not affect RPE viability. However, incubation of 1-μg/ml retinal protein from NS-injected eyes, but not PBS-injected eyes induced RPE cell death.

Conclusion: NS is toxic to the C57BL/6J mouse retina and should not be used as a vehicle for intraocular injection. PBS is not toxic to the retina and is a preferred vehicle.

Translational Relevance: NS is not a physiological solution for intraocular injection in the C57BL/6J mice and questions its suitability for intraocular injection in other species, including human.

Synthetic peptides interacting with the 67-kd laminin receptor can reduce retinal ischemia and inhibit hypoxia-induced retinal neovascularization.

The high-affinity 67-kd laminin receptor (67LR) is expressed by proliferating endothelial cells during retinal neovascularization. The role of 67LR has been further examined experimentally by administration of selective 67LR agonists and antagonists in a murine model of proliferative retinopathy. These synthetic 67LR ligands have been previously shown to stimulate or inhibit endothelial cell motility in vitro without any direct effect on proliferation. In the present study, a fluorescently labeled 67LR antagonist (EGF33–42) was injected intraperitoneally into mice and its distribution in the retina was assessed by confocal scanning laser microscopy. Within 2 hours this peptide was localized to the retinal vasculature, including preretinal neovascular complexes, and a significant amount had crossed the blood retinal barrier. For up to 24 hours postinjection, the peptide was still present in the retinal vascular walls and, to a lesser extent, in the neural retina. Non-labeled EGF33–42 significantly inhibited pre-retinal neovascularization in comparison to controls treated with phosphate-buffered saline or scrambled peptide (P <0.0001). The agonist peptide (Lamß1925–933) also significantly inhibited proliferative retinopathy; however, it caused a concomitant reduction in retinal ischemia in this model by promoting significant revascularization of the central retina (P <0.001). Thus, 67LR appears to be an important target receptor for the modulation of retinal neovascularization. Agonism of this receptor may be valuable in reducing the hypoxia-stimulated release of angiogenic growth factors which drives retinal angiogenesis.

The resistance of polyvinylpyrrolidone–Iodine–poly(e-caprolactone) blends to adherence of Escherichia coli

In this study, the resistance of biodegradable biomaterials, composed of blends of poly(e-caprolactone) (PCL) and the polymeric antimicrobial complex, polyvinylpyrrolidone–iodine (PVP-I) to the adherence of a clinical isolate of Escherichia coli is described. Blends of PCL composed of a range of high (50,000 g mol1) to low (5000 g mol1) molecular weight ratios of polymer and either
devoid of or containing PVP-I (1% w/w) were prepared by solvent evaporation. Following incubation (4 h), there was no relationship between m. wt. ratio of PCL in ?lms devoid of PVP-I and adherence ofE. coli. Conversely, microbial adherence to PCL containing PVP-I decreased as the ratio of high:low m. wt. polymer was decreased and was approximately 1000 fold lower than that to comparator ?lms devoid of PVP-I. Following periods of immersion of PVP-I containing PCL ?lms under sink conditions in phosphate buffered saline, subsequent adherence of E. coli was substantially reduced for 2 days (40:60 m. wt. ratio) and 6 days (100:0 m. wt. ratio). Concurrent exposure of PCL and E. coli to sub-minimum inhibitory concentrations (sub-MIC) of PVP-I signi?cantly reduced microbial adherence to the biomaterial; however, the molecular weight ratio of PCL did not affect this outcome. Pretreatment of PCL with similar sub-MIC of PVP-I prior to inclusion within the microbial adherence assay signi?cantly decreased the subsequent adherence of E. coli. Greatest reduction in adherence was observed following treatment of PCL (40:60 m. wt. ratio) with 0.0156% w/w PVP-I. In conclusion, this study has illustrated the utility of PVP-I as a suitable therapeutic agent for incorporation within PCL as a novel biomaterial. Due to the combined antimicrobial and biodegradable properties, these biomaterials offer a promising strategy for the reduction in medical device related infection. © 2004 Elsevier Ltd. All rights reserved.

Characterisation and evaluation of novel surfactant bacterial anti-adherent coatings for endotracheal tubes designed for the prevention of ventilator-associated pneumonia.

It is accepted that ventilator-associated pneumonia is a frequent cause of morbidity and mortality in intensive care patients. This study describes the physicochemical properties of novel surfactant coatings of the endotracheal tube and the resistance to microbial adherence of surfactant coated endotracheal tube polyvinylchloride (PVC). Organic solutions of surfactants containing a range of ratios of cholesterol and lecithin (0:100, 25:75, 50:50, 75:25, dissolved in dichloromethane) were prepared and coated onto endotracheal tube PVC using a multiple dip-coating process. Using modulated temperature differential scanning calorimetry it was confirmed that the binary surfactant systems existed as physical mixtures. The surface properties of both surfactant-coated and uncoated PVC, following treatment with either pooled human saliva or phosphate-buffered saline (PBS), were characterised using dynamic contact angle analysis. Following treatment with saliva, the contact angles of PVC decreased; however, those of the coated biomaterials were unaffected, indicating different rates and extents of macromolecular adsorption from saliva onto the coated and uncoated PVC. The advancing and receding contact angles of the surfactant-coated PVC were unaffected by sonication, thereby providing evidence of the durability of the coatings. The cell surface hydrophobicity and zeta potentials of isolates of Staphylococcus aureus and Pseudomonas aeruginosa, following treatment with either saliva or PBS, and their adherence to uncoated and surfactant-coated PVC (that had been pre-treated with saliva) were examined. Adherence of S. aureus and Ps. aeruginosa to surfactant-coated PVC at each successive time period (0.5, 1, 2, 4, 8 h) was significantly lower than to uncoated PVC, the extent of the reduction frequently exceeding 90%. Interestingly, the microbial anti-adherent properties of the coatings were dependent on the lecithin content. Based on the impressive microbial anti-adherence properties and durability of the surfactant coating on PVC following dip coatings, it is proposed that these systems may usefully reduce the incidence of ventilator-associated pneumonia when employed as luminal coatings of the endotracheal tube.

Moisture-Activated Rheological Structuring of Nonaqueous Poloxamine–Poly(Acrylic Acid) Systems Designed as Novel Biomedical Implants

This study reports the formulation/characterisation of novel polymeric platforms designed to behave as low-viscosity systems in the nonaqueous state, however, following uptake of aqueous ?uids, exhibit rheological structuring and mucoadhesion. The rheological/mechanical and mucoadhesive properties of platforms containing poly(acrylic acid) (PAA, 1%, 3%, 5%, w/w) and poloxamines (Tetronic 904, 901, 704, 701, 304), both in the absence and presence of phosphate buffered saline (PBS, pH 7.4) are described. With the exception of Tetronic 904, all formulations exhibited Newtonian ?ow in the nonaqueous state, whereas, all aqueous formulations displayed pseudoplastic ?ow. The consistency and viscoelastic properties were dependent on the concentrations of PAA and PBS and Tetronic grade. PBS signi?cantly increased the consistency, viscoelasticity and mucoadhesion, reaching a maximum at a de?ned concentration of PBS that was dependent on PAA concentration and Tetronic grade. Formulations containing Tetronic 904 exhibited greatest consistency and elasticity both prior to and after dilution with PBS. Increasing PAA concentration enhanced the mucoadhesive properties. Prolonged drug release of metronidazole was observed from formulations containing 10% (w/w) PBS, 3% and, particularly, 5% (w/w) PAA. It is suggested that the physicochemical properties of formulations containing 3% or 5% (w/w) PAA and Tetronic 904, would render them suitable platforms for administration to body cavities.

Microcomputed tomography imaging in a rat model of delayed union/non-union fracture

We aimed to develop a clinically relevant delayed union/non-union fracture model to evaluate a cell therapy intervention repair strategy. Histology, three-dimensional (3D) micro-computed tomography (micro-CT) imaging and mechanical testing were utilized to develop an analytical protocol for qualitative and quantitative assessment of fracture repair. An open femoral diaphyseal osteotomy, combined with periosteal diathermy and endosteal excision, was held in compression by a four pin unilateral external fixator. Three delayed union/non-union fracture groups established at 6 weeks-(a) a control group, (b) a cell therapy group, and (c) a group receiving phosphate-buffered saline (PBS) injection alone-were examined subsequently at 8 and 14 weeks. The histological response was combined fibrous and cartilaginous non-unions in groups A and B with fibrous non-unions in group C. Mineralized callus volume/total volume percentage showed no statistically significant differences between groups. Endosteal calcified tissue volume/endosteal tissue volume, at the center of the fracture site, displayed statistically significant differences between 8 and 14 weeks for cell and PBS intervention groups but not for the control group. The percentage load to failure was significantly lower in the control and cell treatment groups than in the PBS alone group. High-resolution micro-CT imaging provides a powerful tool to augment characterization of repair in delayed union/non-union fractures together with outcomes such as histology and mechanical strength measurement. Accurate, nondestructive, 3D identification of mineralization progression in repairing fractures is enabled in the presence or absence of intervention strategies. (c) 2007 Orthopaedic Research Society.

Standardisation and comparison of methods employed for microbial cell surface hydrophobicity and charge determination

Whilst there are a number of methods available to characterise the cell surface hydrophobicity (CSH) and cell surface charge (CSC) of microorganisms, there is still debate concerning the correlation of results between individual methods. In this study, the techniques of bacterial adherence to hydrocarbons (BATH) and hydrophobic interaction chromatography (HTC) were used to measure CSH. Electrostatic interaction chromatography (ESIC) and zeta potential (ZP) measurements were used to determine CSC. To allow meaningful comparisons between the BATH and HIC tests, between ESIC and ZP and also between CSH and CSC, the buffer systems employed in each test were standardised (phosphate buffered saline, pH 7.3, 0.01 mM). Isolates of Staphylococcus epidermidis derived from microbial biofilm were used as the test organism in this study. The isolates examined exhibited primarily medium to high CSH and a highly negative CSC. Good correlation of CSH measurement was observed between the BATH and HIC tests (r = 0.89). Good correlation was observed between ESIC (anionic exchange column) and ZP measurements. No correlations were observed between isolate CSC and either increased or decreased CSH. It is recommended that whenever comparisons of various methods to determine either CSC or CSH (by partitioning methods), the buffer systems should remain constant throughout to achieve consistency of results.

Development of a Broad-Specificity Monoclonal Antibody-Based Immunoaffinity Chromatography Cleanup for Organophosphorus Pesticide Determination in Environmental Samples

An immunoaffinity chromatographic (IAC) method for the selective extraction and concentration of 13 organophosphorus pesticides (OPs, including coumaphos, parathion, phoxim, quinalphos, dichlofenthion, triazophos, azinphos-ethyl, phosalone, isochlorthion, parathion-methyl, cyanophos, disulfoton, and phorate) prior to analysis by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed. The IAC column was prepared by covalently immobilizing a monoclonal antibody with broad specificity for OPs on CNBr-activated Sephrose 4B. The column capacity ranged from 884 to 2641 ng/mL of gel. The optimum elution solvent was 0.01 M phosphate-buffered saline containing 80% methanol. The breakthrough volume of the IAC column was found to be 400 mL. Recoveries of OPs from spiked environmental samples by IAC cleanup and HPLC-MS/MS analysis ranged from 60.2 to 107.1%, with a relative standard deviation below 11.1%. The limit of quantitation for 13 OPs ranged from 0.01 to 0.13 ng/mL (ng/g). The application of IAC cleanup coupled to HPLC-MS/MS in real environmental samples demonstrated the potential of this method for the determination of OP residues in environmental samples at trace levels.

Mesenchymal stem cells enhance survival and bacterial clearance in murine Escherichia coli pneumonia

Rationale: Bacterial pneumonia is the most common infectious cause of death worldwide and treatment is increasingly hampered by antibiotic resistance. Mesenchymal stem cells (MSCs) have been demonstrated to provide protection against acute inflammatory lung injury; however, their potential therapeutic role in the setting of bacterial pneumonia has not been well studied.

Objective: This study focused on testing the therapeutic and mechanistic effects of MSCs in a mouse model of Gram-negative pneumonia.

Methods and results: Syngeneic MSCs from wild-type mice were isolated and administered via the intratracheal route to mice 4 h after the mice were infected with Escherichia coli. 3T3 fibroblasts and phosphate-buffered saline (PBS) were used as controls for all in vivo experiments. Survival, lung injury, bacterial counts and indices of inflammation were measured in each treatment group. Treatment with wild-type MSCs improved 48 h survival (MSC, 55%; 3T3, 8%; PBS, 0%; p<0.05 for MSC vs 3T3 and PBS groups) and lung injury compared with control mice. In addition, wild-type MSCs enhanced bacterial clearance from the alveolar space as early as 4 h after administration, an effect that was not observed with the other treatment groups. The antibacterial effect with MSCs was due, in part, to their upregulation of the antibacterial protein lipocalin 2.

Conclusions: Treatment with MSCs enhanced survival and bacterial clearance in a mouse model of Gram-negative pneumonia. The bacterial clearance effect was due, in part, to the upregulation of lipocalin 2 production by MSCs

Antibacterial Effect of Human Mesenchymal Stem Cells is Mediated in Part from Secretion of the Antimicrobial Peptide LL-37

Recent in vivo studies indicate that mesenchymal stem cells (MSCs) may have beneficial effects in the treatment of sepsis induced by bacterial infection. Administration of MSCs in these studies improved survival and enhanced bacterial clearance. The primary objective of this study was to test the hypothesis that human MSCs possessed intrinsic antimicrobial properties. We studied the effect of human MSCs derived from bone marrow on the bacterial growth of Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) bacteria. MSCs as well as their conditioned medium (CM) demonstrated marked inhibition of bacterial growth in comparison with control medium or normal human lung fibroblasts (NHLF). Analysis of expression of major antimicrobial peptides indicated that one of the factors responsible for the antimicrobial activity of MSC CM against Gram-negative bacteria was the human cathelicidin antimicrobial peptide, hCAP-18/LL-37. Both m-RNA and protein expression data showed that the expression of LL-37 in MSCs increased after bacterial challenge. Using an in vivo mouse model of E. coli pneumonia, intratracheal administration of MSCs reduced bacterial growth (in colony-forming unit) in the lung homogenates and in the bronchoalveolar lavage (BAL) fluid, and administration of MSCs simultaneously with a neutralizing antibody to LL-37 resulted in a decrease in bacterial clearance. In addition, the BAL itself from MSC-treated mice had a greater antimicrobial activity in comparison with the BAL of phosphate buffered saline (PBS)-treated mice. Human bone marrow-derived MSCs possess direct antimicrobial activity, which is mediated in part by the secretion of human cathelicidin hCAP-18/ LL-37.

Effect of short-term macrophage depletion in the development of posterior capsule opacification in rodents

Aim: To evaluate the role of macrophages in the development of posterior capsule opacification (PCO). Methods: For this purpose, an extracapsular lens extraction was performed in 18 consecutive Sprague-Dawley rats. Animals were treated with liposomal clodronate (Cl MDP-lip-treated group, n = 10) or phosphate-buffered saline (PBS) (control group, n = 8) 1 day preoperatively and on the first day postoperatively, and sacrificed 3 days postoperatively. Masked clinical, light microscopy and immunohistochemistry studies were conducted. The Fisher exact test and randomisation test were used to assess statistically differences between groups. Results: A statistically significant reduction in the number of macrophages (ED1+, ED7+, ED8+) was found in the Cl MDP-lip-treated group compared with the PBS-lip-treated group (p = 0.048, p = 0.004, p = 0.027, respectively). There were no statistically significant differences with regards to the presence/absence of central opacification (p = 0.29) and capsular wrinkling (p = 0.21) as detected clinically between groups. Similarly, a qualitative evaluation of the degree of PCO with regards to lens epithelial cell (LEC) proliferation, capsular wrinkling and Soemmerring ring formation showed no statistically significance between groups (p = 0.27, p = 0.061, p = 1.0, respectively). However, a statistically significant reduction in the number of lens epithelial cells (LEC) counted in the centre of the posterior capsule was found in the Cl MDP-lip- treated group (p = 0.009). Conclusion: Depletion of macrophages was accompanied by a reduction in LEC in the centre of the posterior capsule in rodents.

Effect of TGF-β2 and anti-TGF-β2 antibody in a new in vivo rodent model of posterior capsule opacification

PURPOSE. This study evaluated the effect of transforming growth factor (TGF)-ß2 and anti-TGF-ß2 antibody in a rodent model of posterior capsule opacification (PCO). METHODS. An extracapsular lens extraction (ECLE) was performed in 72 Sprague-Dawley rats. At the end of the procedure, 10 µL TGF-ß2 (TGF-ß2-treated group), fetal calf serum (FCS)/phosphate- buffered saline (PBS; FCS/PBS-treated control group), a human monoclonal TGF-ß2 antibody (anti-TGF-ß2-treated group), or a null control IgG4 antibody (null antibody-treated control group) was injected into the capsule. Animals were killed 3 and 14 days postoperatively. Eyes were evaluated clinically prior to euthanatization, then enucleated and processed for light microscopy and immunohistochemistry afterward. PCO was evaluated clinically and histopathologically. Student's t-test and ? were used to assess differences between groups. RESULTS. There were no statistically significant clinical or histopathological differences in degree of PCO between the TGF-ß2- and FCS/PBS-treated groups at 3 and 14 days after ECLE. Nor were there differences between the anti-TGF-ß2- and the null antibody-treated groups, with the exception of the histopathology score for capsule wrinkling 3 days after ECLE (P = 0.02). a-Smooth-muscle actin staining was observed in the lens capsular bag only in areas where there was close contact with the iris. CONCLUSIONS. No sustained effect of TGF-ß2 or anti-TGF-ß2 antibody on PCO was found in rodents at the dose and timing administered in this study. Iris cells may play a role in the process of epithelial mesenchymal transition linked to PCO. Copyright © Association for Research in Vision and Ophthalmology.

Lymphocyte subsets in conjunctival mucosa-associated-lymphoid-tissue after exposure to retinal-S-antigen

Purpose: To evaluate the immune cell subsets in conjunctival mucosa-associated-lymphoid-tissue (C-MALT) following challenge with antigen. Methods: Ten adult female Lewis rats were studied. Five rats received one drop (5 µL) of retinal S-antigen (500 µg/mL in phosphate buffered saline, PBS) instilled into the lower fornix twice daily for 10 consecutive days. Five rats received PBS only and served as controls for the experiment. Two days after the last instillation the animals were sacrificed and the orbital contents prepared for immunohistological staining. A panel of monoclonal antibodies was used: CD5, CD4, CD8, CD25, and CD45RA. The number of positive cells were counted in sections of epibulbar, forniceal, and tarsal conjunctiva. Results: There was a significant increase in the number of CD8 T lymphocytes in the conjunctiva of animals receiving retinal S-antigen when compared to control animals. Conclusion: Conjunctival instillation of retinal S-antigen causes an immune response in the C-MALT with a significant increase in the CD8 T lymphocyte subset in this tissue. This response may be involved in the induction of tolerance to the encountered antigen.

Role of vascular endothelial growth factor and placental growth factors during retinal vascular development and hyaloid regression

PURPOSE. Vascular endothelial growth factor (VEGF)-A and placental growth factor (PIGF) are members of a large group of homologous peptides identified as the VEGF family. Although VEGF-A is known to act as a potent angiogenic peptide in the retina, the vasoactive function of PIGF in this tissue is less well defined. This study has sought to elucidate the expression patterns and modulatory role of these growth factors during retinal vascular development and hyaloid regression in the neonatal mouse. METHODS. C57BL6J mice were killed at postnatal days (P)1, P3, P5, P7, P9, and P11. The eyes were enucleated and processed for in situ hybridization and immunocytochemistry and the retinas extracted for total protein or RNA. Separate groups of neonatal mice were also injected intraperitoneally daily from P2 through P9 with either VEGF-neutralizing antibody, PIGF-neutralizing antibody, isotype immunoglobulin (Ig)-G, or phosphate-buffered saline (PBS). The mice were then perfused with fluorescein isothiocyanate (FITC)-dextran, and the eyes were subsequently embedded in paraffin wax or flat mounted. RESULTS. Quantitative (real-time) reverse transcription-polymerase chain reaction (RT-PCR) demonstrated similar expression patterns of VEGF-A and PIGF mRNA during neonatal retinal development, although the fluctuation between time periods was greater overall for VEGF-A. The localization of VEGF-A and PIGF in the retina, as revealed by in situ hybridization and immunohistochemistry, was also similar. Neutralization of VEGF-A caused a significant reduction in the hyaloid and retinal vasculature, whereas PIGF antibody treatment caused a marked persistence of the hyaloid without significantly affecting retinal vascular development. CONCLUSIONS. Although having similar expression patterns in the retina, these growth factors appear to have distinct modulatory influences during normal retinal vascular development and hyaloid regression.

Toxicity of mildly modified low-density lipoproteins to cultured retinal capillary endothelial cells and pericytes

To investigate the role of modified low-density lipoproteins (LDL) in the pathogenesis of diabetic retinopathy, we studied the cytotoxicity of normal and mildly modified human LDL to bovine retinal capillary endothelial cells and pericytes in vitro. Pooled LDL was incubated (in phosphate-buffered saline-EDTA, 3 days, 37 degrees C) under 1) nitrogen with additional chelating agents and 2) air, to prepare normal and minimally oxidized LDL, respectively. Similar conditions, but with the addition of 50 mM D-glucose, were used to prepare glycated and glycoxidized LDL. None of the LDL preparations was recognized by the macrophage scavenger receptor, confirming limited modification. Retinal capillary endothelial cells and pericytes were grown to confluence and then exposed for 2 or 3 days to serum-free medium (1% albumin) supplemented with normal or modified LDL (100 mg/l) or to serum-free medium alone. Cytotoxicity was assessed by cell counting (live and total cells) and by cell protein determination. Compared with normal LDL, modified LDL were cytotoxic to both cell types at both time points, causing highly significant decreases in live and total cell counts (P <0.001) (analysis of variance). Reductions in cell protein also were significant for pericytes at day 3 (P = 0.016) and of borderline significance for endothelial cells at day 2 (P = 0.05) and day 3 (P = 0.063). Cytotoxicity increased as follows: normal <glycated <or = minimally oxidized <glycoxidized LDL. We conclude that, in diabetes, mild modification of LDL resulting from separate or combined processes of glycation and oxidation may contribute to chronic retinal capillary injury and thus to the development of diabetic retinopathy.