Intraocular lens opacification after corneal endothelial keratoplasty: electron microscopy and x-ray element spectroscopy analysis.

PURPOSE: To assess a newly recognized long-term complication of Descemet-stripping automated endothelial keratoplasty (DSAEK). SETTING: Plymouth Royal Eye Infirmary and Plymouth Electron Microscope Centre, Plymouth, United Kingdom. DESIGN: Retrospective case series. METHODS: This study evaluated cases of intraocular lens (IOL) opacification that developed after uneventful DSAEK. None of the IOLs was previously known to opacify. In 1 case, the opacified IOL was explanted and analyzed using detailed light microscopy, scanning electron microscopic (SEM) analysis, and element x-ray spectroscopy. RESULTS: In all 5 cases, the IOL was hydrophilic acrylic and the eye developed IOL anterior surface opacification 4 to 12 months after DSAEK. In 1 eye, the opacification was symptomatic; thus, an IOL exchange was performed. Light microscopy and SEM analysis of the explanted IOL confirmed opacification on the anterior surface and subsurface areas. X-ray element spectroscopy showed the granules were composed of calcium and phosphorous. CONCLUSIONS: These cases indicate that IOL opacification after DSAEK is a late, although newly recognized, complication of endothelial keratoplasty. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.

Effects of hyperoxia on the permeability of 16HBE14o- cell monolayers--the protective role of antioxidant vitamins E and C.

The use of hyperoxia for critically ill patients is associated with adverse impacts resulting in lung injury accompanied by inflammation. The aim of this study was to evaluate aspects of mechanisms that contribute to hyperoxia-induced disruption of the epithelial permeability barrier, and also the protective effects of the antioxidants α-tocopherol and ascorbate. 16HBE14o- cells were cultured as monolayers at an air-liquid interface for 6 days, after which transepithelial electrical resistance reached 251.2 ± 4.1 Ω.cm(2) (mean ± standard error of the mean). They were then exposed for 24 h to normoxia (21% O2, 5% CO2), hyperoxia (95% O2, 5% CO2), hyperoxia with 10(-7) M α-tocopherol, hyperoxia with 10(-7) M ascorbate, hyperoxia with 10(-6) M ascorbate, and hyperoxia with a combination of α-tocopherol and ascorbate (10(-7) M and 10(-6) M, respectively). Significant reductions (P < 0.05) in transepithelial electrical resistance seen after hyperoxia (with or without antioxidants) were associated with reductions in the levels of zona occludens-1 (ZO-1) observed by immunohistochemistry, and downregulation of ZO-1 expression (P < 0.01) as compared with normoxia. In contrast, the expression levels of interleukin (IL)-8, IL-6 and tumour necrosis factor-α (TNF-α) were increased after hyperoxia (P < 0.01), and marked increases in the levels of these cytokines (ELISA) were seen in the medium (P < 0.001) as compared with normoxia. The antioxidant vitamins E and C had a partial protective effect against the hyperoxia-induced reduction in ZO-1 levels and the increase in levels of the proinflammatory cytokines IL-8, IL-6, and TNF-α. In conclusion, hyperoxia-induced epithelial disruption is associated with tight junction weakening, and induction of a proinflammatory environment.

An in vitro study assessing the infection risk of low-cost polyethylene mosquito net compared with commercial hernia prosthetics.

BACKGROUND: The innovative use of sterilized mosquito net as a cheaper alternative to commercial mesh for hernia repair has gained increasing recognition. Developing health care systems have inherently higher surgical site infection rates, and concerns regarding the introduction of untested prosthetic hernia meshes have been raised. This in vitro study assesses the infection risk of polyethylene (PE) mosquito net mesh compared with commercial hernia prosthetics by assessing the essential (first) step in the pathogenesis of mesh infections. MATERIALS AND METHODS: Individual meshes were inoculated with Staphylococcusepidermidis and Staphylococcusaureus with a bacterial inoculum of 10(2) bacteria. Inoculated meshes were incubated for 18 h in tryptone soy broth and then analyzed using scanning electron microcopy. The final fraction of the bacteria adherent to each of the meshes was compared. One-way analysis of variance was performed on the bacterial counts. The Tukey test was used to determine the difference between the different biomaterials in the event the one-way analysis of variance was significant. RESULTS: There was no significant difference in the mean number of adherent bacteria to PE mosquito net compared with the monofilament polypropylene-based meshes (Prolene and Bard Soft Mesh). Multifilament Vypro mesh had significantly greater mean bacterial adherence compared with PE mosquito net (P < 0.001 with S aureus and P = 0.003 with S epidermidis). CONCLUSIONS: In vitro infection risk of PE mosquito net is not significantly different from commonly used monofilament polypropylene commercial prosthetics and is in fact lower than a commonly used commercial multifilament mesh. This study adds to the growing body of evidence that indicates that these meshes can be safely deployed.

An experimental study exploring the relationship between the size of bacterial inoculum and bacterial adherence to prosthetic mesh.

BACKGROUND: Infection is a major concern with medical implants. Surgical meshes used for the repair of abdominal wall hernias are associated with wound infection rates ranging from 7 to 18 %. Although mesh infection is relatively rare, once a patient shows clinical signs of mesh infection, the surgeon may be required to remove the mesh, resulting in additional surgery, morbidity, and cost. The usual causative organisms associated with cases of mesh infection are Staphylococcus species. The first stage of implant infection is bacterial adherence to the biomaterial. An accurate assessment of adherent bacteria to medical prosthetics is therefore important in order to determine the infection risk associated with surgical implants. METHODS: This experimental study evaluated the relationship between the size of the bacterial inoculum and bacterial adherence to three commonly used hernia prosthetics (polypropylene, polyester, and ePTFE). Tenfold dilutions of S. epidermidis (Evans-ATCC 12228) and S. aureus (Rosenbach-ATCC 25923), created with phosphate-buffered saline, were used to inoculate each of the meshes in 3 ml of tryptone soya broth for 18 h at 37 °C, 95 % air/5 % CO(2). The number of viable bacteria in each dilution was calculated using a spot plate technique. The number of adherent bacteria to the meshes was counted using direct imaging analysis with scanning electron microscopy and expressed as a mean. RESULTS: One hundred eight mesh samples were analysed. The size of the bacterial inoculum of S. epidermidis significantly influenced the number of adherent bacteria to the mesh, and lower rates of adhesion were observed with smaller inoculums for all three meshes (polypropylene, p = 0.02; ePTFE p = 0.03; polyester p = 0.02). A similar, albeit less profound, pattern of results was observed with S. aureus. Bacterial adherence was observed with inoculum sizes as small as <10 bacteria. CONCLUSIONS: The results demonstrate that even a very low number of bacterial inoculums can result in adherence to hernia biomaterials and that the level of adherence is directly related to the size of the inoculum. These in vitro results provide evidence that the size of the inoculum is important in the colonization of hernia biomaterials and demonstrate the importance of minimising the bacterial inoculum in the clinical setting.

Hyperoxia-induced ciliary loss and oxidative damage in an in vitro bovine model: the protective role of antioxidant vitamins E and C.

Although elevated oxygen fraction is used in intensive care units around the world, pathological changes in pulmonary tissue have been shown to occur with prolonged exposure to hyperoxia. In this work a bovine bronchus culture model has been successfully used to evaluate the effects of hyperoxia on ciliated epithelium in vitro. Samples were cultured using an air interface method and exposed to normoxia, 21% O(2) or hyperoxia, 95% O(2). Cilial coverage was assessed using scanning electron microscopy (SEM). Tissue damage (lactate dehydrogenase, LDH, in the medium), lipid peroxidation (thiobarbituric acid reactive substances, TBARS), DNA damage (comet assay), protein oxidation (OxyBlot kit) and antioxidant status (total glutathione) were used to assess whether the hyperoxia caused significant oxidative stress. Hyperoxia caused a time-dependent decline (t(½)=3.4d compared to 37.1d under normoxia) in cilial coverage (P<0.0001). This was associated with a significant increase in the number of cells (2.80 ± 0.27 × 10(6) compared to 1.97 ± 0.23 × 10(6)ml(-1) after 6d), many apparently intact, in the medium (P<0.05); LDH release (1.06 ± 0.29 compared to 0.83 ± 0.36 µmol min(-1)g(-1) after 6d; P<0.001); lipid peroxidation (352 ± 16 versus 247 ± 11 µmol MDA g(-1) for hyperoxia and normoxia, respectively); % tail DNA (18.7 ± 2.2 versus 11.1 ± 1.5); protein carbonyls (P<0.05); and total glutathione (229 ± 20 µmol g(-1) versus 189 ± 15 µmol g(-1)). Vitamins E (10(-7)M) and C (10(-6) or 10(-7)M) alone or in combination (10(-7)M and 10(-6)M, respectively) had a significant protective effect on the hyperoxia-induced reduction in percentage cilial coverage (P<0.05). In conclusion, hyperoxia caused damage to cultured bovine bronchial epithelium and denudation of cilia. The antioxidant vitamins E and C significantly protected against hyperoxia-induced cilia loss.

Design and validation of a novel quantitative method for rapid bacterial enumeration using programmed stage movement scanning electron microscopy.

The adhesion of bacteria to surgical implants is the first stage of implant infection. The method for detecting bound bacteria is an important consideration in the study of bacterial adherence and colonisation. Enumeration of bacteria by direct visualisation techniques is labour intensive and time consuming. We have developed and validated a method for enumerating bacteria on porous material surfaces using programmed stage movement scanning electron microscopy and compared cumulative counts after 1-10 stage movements with absolute bacterial counts. We describe this method with three commercially sourced meshes used for abdominal wall hernia repair and with three different inoculums of Staphylococcus epidermidis. The results demonstrate significant correlation to the absolute count after five cumulative counts for all meshes analysed. The mean time saved by the cumulative counting method was 1h and 9 min per mesh. We conclude that advances in scanning electron microscopy and the advent of precise automated stage control have facilitated rapid data acquisition for bacterial counting purposes and that five cumulative counts at 1000× or 2500× magnification are a valid quantitative method for enumerating S. epidermidis bacteria on porous surfaces (with a pore size of up to 1.3 mm).

Regulation of Schwann cell differentiation and proliferation by the Pax-3 transcription factor.

Pax-3 is a paired domain transcription factor that plays many roles during vertebrate development. In the Schwann cell lineage, Pax-3 is expressed at an early stage in Schwann cells precursors of the embryonic nerve, is maintained in the nonmyelinating cells of the adult nerve, and is upregulated in Schwann cells after peripheral nerve injury. Consistent with this expression pattern, Pax-3 has previously been shown to play a role in repressing the expression of the myelin basic protein gene in Schwann cells. We have studied the role of Pax-3 in Schwann cells and have found that it controls not only the regulation of cell differentiation but also the survival and proliferation of Schwann cells. Pax-3 expression blocks both the induction of Oct-6 and Krox-20 (K20) by cyclic AMP and completely inhibits the ability of K20, the physiological regulator of myelination in the peripheral nervous system, to induce myelin gene expression in Schwann cells. In contrast to other inhibitors of myelination, we find that Pax-3 represses myelin gene expression in a c-Jun-independent manner. In addition to this, we find that Pax-3 expression alone is sufficient to inhibit the induction of apoptosis by TGFß1 in Schwann cells. Expression of Pax-3 is also sufficient to induce the proliferation of Schwann cells in the absence of added growth factors and to reverse K20-induced exit from the cell cycle. These findings indicate new roles for the Pax-3 transcription factor in controlling the differentiation and proliferation of Schwann cells during development and after peripheral nerve injury.

The effect of material choice on biofilm formation in a model warm water distribution system.

Water distribution systems (WDS) are composed of a variety of materials and may harbour potential pathogens within surface-attached microbial biofilms. Biofilm formation on four plumbing materials, viz. copper, stainless steel 316 (SS316), ethylene propylene diene monomer (EPDM) and cross-linked polyethylene (PEX), was investigated using scanning electron microscope (SEM)/confocal microscopy, ATP-/culture-based analysis, and molecular analysis. Material 'inserts' were incorporated into a mains water fed, model WDS. All materials supported biofilm growth to various degrees. After 84 days, copper and SS316 showed no significant overall differences in terms of the level of biofilm formation observed, whilst PEX supported a significantly higher level of biofilm. EPDM exhibited gross contamination by a complex, multispecies biofilm, at a level significantly higher than was observed on the other materials, regardless of the analytical method used. PCR-DGGE analysis showed clear differences in the composition of the biofilm community on all materials after 84 days. The primary conclusion of this study has been to identify EPDM as a potentially unsuitable material for use as a major component in WDS.

Elevated oxygen fraction reduces cilial abundance in explanted human bronchial tissue.

The effect of hyperoxia on ciliary abundance in cultured explants of adult human bronchus was investigated. Bronchus samples were removed during surgery from patients receiving pneumonectomy or lobectomy for malignancy. Part or all of each of these samples was used for measurement of cilial abundance by scanning electron microscopy (SEM); in many cases the remainder was subdivided and cultured at 37 degrees C in DMEM medium, maintaining an air interface at the ciliated surface of each segment. Cultured segments were exposed to normoxia or hyperoxia (95% O(2)), and a segment was removed every other day for quantification of cilial abundance by SEM. There was a significant inverse relationship between smoking history and abundance (p = .017; ANOVA); mean values for nonsmokers, ex-smokers, and smokers were 98.2% (n = 6), 97.0% (n = 17), and 84.02% (n = 9), respectively. There was some loss of cilia on explant segments cultured under normoxia, but the rate of loss from segments cultured under hyperoxia was significantly greater (W test, p = .00011); rate constants (means +/- SE) for cilial loss of 0.0208 +/- 0.0044 day(-1) and 0.0880 +/- 0.0179 day(-1) were found for explant segments exposed to 21 and 95% O2, respectively (n = 20).