Age-dependent changes in rat lacrimal gland anti-oxidant and vesicular related protein expression profiles

Purpose: Anti-oxidation and exocytosis are important for maintaining exocrine tissue homeostasis. During aging, functional and structural alterations occur in the lacrimal gland (LG), including oxidative damage to proteins, lipids, and DNA. The aims of the present study were to determine in the aging LG: a) the effects of aging on LG structure and secretory activity and b) changes in the expression of oxidative stress markers. Methods: To address these goals, tear secretion composition and corneal impression cytology were compared between male Wistar rats of 2 (control) and 24 (aged) months. LG morphology and the expression levels of vitamin E and malonaldehyde (MDA) were evaluated to determine the anti-oxidant activity and lipid peroxidation, respectively. RT-PCR and western blot analysis were used for the analysis of Ras related in brain GTPase protein (Rab) and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins of the secretory machinery (i.e.; Rab 3d, Rab 27, vesicle-associated membrane protein-2 (Vamp-2), and syntaxin). Results: Histological analysis of aged rats revealed a higher frequency of corneal epithelia metaplasia. In the acinar cells, organelles underwent degeneration, and lipofucsin-like material accumulated in the cytoplasm along with declines in the anti-oxidant marker vitamin E. Rab3d and Rab27b mRNA levels fell along with Rab3d protein expression, whereas syntaxin levels increased. Conclusions: These findings indicate that exocytotic and anti-oxidant mechanisms become impaired with age in the rat LG. In parallel with these structural alterations, functional declines may contribute to the pathophysiology caused by tear film modification in dry eye disease.

Anti-inflammatory effect of Spiranthera odoratissima A. St.-Hil. leaves involves reduction of TNF-alpha

Spiranthera odoratissima A. St.-Hil., 'manaca', is a medicinal species used in Brazil, especially in central region, for the treatment of several diseases such as pain and inflammation. In this study, the methanol/aqueous phase of the ethanol extract of the leaves of 'manaca' (MAP), at the doses of 50, 150 and 500 mg/kg was used to evaluate the anti-inflammatory and/or antinociceptive effects and the possible anti-inflammatory mechanism. The antinociceptive and anti-inflammatory activities of MAP were assessed using formalin test, carrageenan-induced paw oedema. The myeloperoxidase activity, capillary permeability, leukocyte migration and tumour necrosis factor alpha (TNF-alpha) levels were evaluated in pleural exudate. The MAP reduced the licking time only in the later phase of formalin test, and showed anti-inflammatory activity by reducing the paw oedema, migration cell, myeloperoxidase activity, capillary permeability and TNF-alpha levels. In conclusion, we confirmed the inflammatory activity of MAP and affirm that this effect involves the reduction of TNF-alpha level.

Differences in early biomechanical properties after myopic and hyperopic lasik using mechanical microkeratome Sub-Bowman's Keratomileusis (SBK)

Purpose: To evaluate biomechanical changes measured with the ORA (Ocular Response Analyzer (R); Reichert Ophthalmic Instruments, Buffalo, New York, USA) after Lasik with the Moria One Use Plus and to compare the biomechanics changes after myopic and hyperopic ablations. Methods: Fourteeneyes for hyperopia (H) and 19 eyes for myopia (M) were evaluated with the ORA preoperatively and 1 month after Lasik with thin flap (100 microns) using SBK-OUP (Sub-Bowman Keratomileusis-One Use Plus, Moria (R)). CH (Corneal Hysteresis), CRF (Corneal Resistance Factor), IOPg (gold-standard, Goldmann correlated Intraocular pressure), IOPcc (Corneal compensated Intraocular pressure) and more 38 variables derived from the corneal biomechanical response signal of the ORA were analyzed. The Wilcoxon test was used to assess differences between the variables before and after surgery for each group and the differences between the pre and postoperative (1 month) myopic eyes were compared with those obtained in hyperopic eyes, using the Mann-Whitney test. Results: There was a significant difference before and after Lasik in myopic and hyperopic eyes in IOPg (Wilcoxon, p<0.05), but not in IOPcc. Only myopic eyes showed a significant difference in CH and CRF measurements before and after LASIK, as well as 9 other biomechanical parameters (aspect1, h1, dive1, path1, p1area1, W11, H11, and w2 path11; Wilcoxon, p<0, 05), 8 of these being related to the first sign of flattening. Five parameters related to the sign of the second applanation showed significant variation only in the eyes before and after hyperopic Lasik (aspect2, h2, dive2, mslew2 and H21; Wilcoxon, p<0,05). There was a difference in both myopic and hyperopic on three parameters related to the applanation signal areas (p1area, and p2area p2area1; Wilcoxon, p<0.05). Differences in IOPg and p1area, before and after surgery were significantly higher in myopic eyes than in hyperopic eyes (Mann-Whitey, p<0.05). Conclusion: There are several significant differences in biomechanical parameters after Lasik with Moria OUP_SBK. Overall, the impact of myopic LASIK on corneal biomechanics is higher than of hyperopic Lasik. The parameters derived from the first sign of the ORA are more affected in myopic LASIK, whereas parameters derived from the second applanation are more affected in hyperopic LASIK.

Mode of Peroxisome Proliferator-Activated Receptor gamma Activation by Luteolin

The peroxisome proliferator-activated receptor gamma (PPAR gamma) is a target for treatment of type II diabetes and other conditions. PPAR gamma full agonists, such as thiazolidinediones (TZDs), are effective insulin sensitizers and anti-inflammatory agents, but their use is limited by adverse side effects. Luteolin is a flavonoid with anti-inflammatory actions that binds PPAR gamma but, unlike TZDs, does not promote adipocyte differentiation. However, previous reports suggested variously that luteolin is a PPAR gamma agonist or an antagonist. We show that luteolin exhibits weak partial agonist/antagonist activity in transfections, inhibits several PPAR gamma target genes in 3T3-L1 cells (LPL, ORL1, and CEBP alpha) and PPAR gamma-dependent adipogenesis, but activates GLUT4 to a similar degree as rosiglitazone, implying gene-specific partial agonism. The crystal structure of the PPAR gamma ligand-binding domain (LBD) reveals that luteolin occupies a buried ligand-binding pocket (LBP) but binds an inactive PPAR gamma LBD conformer and occupies a space near the beta-sheet region far from the activation helix (H12), consistent with partial agonist/antagonist actions. A single myristic acid molecule simultaneously binds the LBP, suggesting that luteolin may cooperate with other ligands to bind PPAR gamma, and molecular dynamics simulations show that luteolin and myristic acid cooperate to stabilize the Omega-loop among H2', H3, and the beta-sheet region. It is noteworthy that luteolin strongly suppresses hypertonicity-induced release of the pro-inflammatory interleukin-8 from human corneal epithelial cells and reverses reductions in transepithelial electrical resistance. This effect is PPAR gamma-dependent. We propose that activities of luteolin are related to its singular binding mode, that anti-inflammatory activity does not require H12 stabilization, and that our structure can be useful in developing safe selective PPAR gamma modulators.

Oral treatment with Hev b 13 prevents experimental arthritis in mice

Hev b 13 is an allergenic esterase obtained from the rubber tree Hevea brasiliensis, which has been shown recently to induce human monocytes to release interleukin (IL)-10 in vitro, and to exert a potent anti-inflammatory effect in vivo. Moreover, Hev b 13 has been shown to reduce clinical signs of inflammation and also histological damage to the distal colon of mice with 2,4,6-trinitrobenze sulphonic acid (TNBS)-induced colitis after its oral administration. The aim of this study was to investigate the effect of Hev b 13 on human mononuclear cells, as well as its therapeutic use in the methylated bovine serum albumin (mBSA) model of antigen-induced arthritis. Five days before the intra-articular challenge, and daily thereafter for 8 days, Hev b 13 was administered by oral gavage. In mice treated with a dose of 0.5 mg/kg of Hev b 13, the severity of oedema, leucocyte infiltration, pannus formation and cartilage erosion were reduced significantly. These findings underscore the anti-inflammatory activity suggested previously for Hev b 13, an activity speculated to be related to its interaction with monocytes/macrophages and the consequent stimulation of IL-10 release and reduction of tumour necrosis factor (TNF) release. The study also opens a wide range of possible applications in the field of immune-mediated inflammatory diseases.

Pachymetry-Guided Intrastromal Air Injection ("Pachy-Bubble") for Deep Anterior Lamellar Keratoplasty

To evaluate an innovative technique for intrastromal air injection to achieve deep anterior lamellar keratoplasty (DALK) with bare Descemet membrane (DM). Thirty-four eyes with anterior corneal pathology, including 27 with keratoconus, underwent DALK. After 400 mm trephination with a suction trephine, ultrasound pachymetry was performed 0.8 mm internally from the trephination groove in the 11 to 1 o'clock position. In this area, a 2-mm incision was created, parallel to the groove, with a micrometer diamond knife calibrated to 90% depth of the thinnest measurement. A cannula was inserted through the incision and 0.5 mL of air was injected to dissect the DM from the stroma. After peripheral paracentesis, anterior keratectomy was carried out to bare the DM. A 0.25-mm oversized graft was sutured in place. Overall, 94.1% of eyes achieved DALK. Bare DM was achieved in 30 eyes, and a pre-DM dissection was performed in 2 eyes. Air injection was successful in detaching the DM (achieving the big bubble) in 88.2% of the eyes. In keratoconus eyes, the rate was 88.9%. All cases but one required a single air injection to achieve DM detachment. Microperforations occurred in 5 cases: 3 during manual layer-by-layer dissection after air injection failed to detach the DM, 1 during removal of the residual stroma after big-bubble formation, and 1 during the diamond knife incision. Two cases (5.9%) were converted to penetrating keratoplasty because of macroperforations. The technique was reproducible, safe, and highly effective in promoting DALK with bare DM.

Structure-function Relationships Using the Cirrus Spectral Domain Optical Coherence Tomograph and Standard Automated Perimetry

Purpose: To evaluate the relationship between glaucomatous structural damage assessed by the Cirrus Spectral Domain OCT (SDOCT) and functional loss as measured by standard automated perimetry (SAP). Methods: Four hundred twenty-two eyes (78 healthy, 210 suspects, 134 glaucomatous) of 250 patients were recruited from the longitudinal Diagnostic Innovations in Glaucoma Study and from the African Descent and Glaucoma Evaluation Study. All eyes underwent testing with the Cirrus SDOCT and SAP within a 6-month period. The relationship between parapapillary retinal nerve fiber layer thickness (RNFL) sectors and corresponding topographic SAP locations was evaluated using locally weighted scatterplot smoothing and regression analysis. SAP sensitivity values were evaluated using both linear as well as logarithmic scales. We also tested the fit of a model (Hood) for structure-function relationship in glaucoma. Results: Structure was significantly related to function for all but the nasal thickness sector. The relationship was strongest for superotemporal RNFL thickness and inferonasal sensitivity (R(2) = 0.314, P < 0.001). The Hood model fitted the data relatively well with 88% of the eyes inside the 95% confidence interval predicted by the model. Conclusions: RNFL thinning measured by the Cirrus SDOCT was associated with correspondent visual field loss in glaucoma.

Sulcus anatomy and diameter in pseudophakic eyes and correlation with biometric data: Evaluation with a 50 MHz ultrasound biomicroscope

PURPOSE: To evaluate the sulcus anatomy and possible correlations between sulcus diameter and white-to-white (WTW) diameter in pseudophakic eyes, data that may be important in the stability of add-on intraocular lenses (IOLs). SETTING: University Eye Hospital, Tuebingen, Germany. DESIGN: Case series. METHODS: In pseudophakic eyes, the axial length (AL) and horizontal WTW were measured by the IOLMaster device. Cross-sectional images were obtained with a 50 MHz ultrasound biomicroscope on the 4 meridians: vertical, horizontal (180 degrees), temporal oblique, and nasal oblique. Sulcus-to-sulcus (STS), angle-to-angle (ATA), and sclera-to-sclera (ScTSc) diameters were measured. The IOL optic diameter (6.0 mm) served as a control. To test reliability, optic measurements were repeated 5 times in a subset of eyes. RESULTS: The vertical ATA and STS diameters were statistically significantly larger than the horizontal diameter (P=.0328 and P=.0216, respectively). There was no statistically significant difference in ScTSc diameters. A weak correlation was found between WTW and horizontal ATA (r = 0.5766, P<.0001) and between WTW and horizontal STS (r = 0.5040, P=.0002). No correlation was found between WTW and horizontal ScTSc (r = 0.2217, P=.1217). CONCLUSIONS: The sulcus anatomy had a vertical oval shape with the vertical meridian being the largest, but it also had variation in the direction of the largest meridian. The WTW measurements showed a weak correlation with STS. In pseudophakic eyes, Soemmerring ring or a bulky haptic may affect the ciliary sulcus anatomy.

Clinical Treatment of Dry Eye Using 0.03% Tacrolimus Eye Drops

Purpose: To report the clinical outcome of the treatment of dry eyes using 0.03% tacrolimus eye drops (olive oil + tacrolimus 0.03%) (Ophthalmos, Sao Paulo, Brazil). Methods: Sixteen eyes of 8 patients with Sjogren syndrome dry eyes (age, 51.13 +/- 9.45 years) were enrolled in this study (prospective noncontrolled interventional case series). Patients were instructed to use topical 0.03% tacrolimus eye drops twice a day (every 12 hours) in the lower conjunctival sac. Schirmer I test, break-up time, corneal fluorescein, and rose bengal staining score were performed in all patients 1 day before, and 14, 28, and 90 days after treatment with 0.03% tacrolimus eye drops. Results: The average fluorescein staining and rose bengal staining scores improved statistically significantly after 14 days of treatment and improved even more after 28 and 90 days. The average Schirmer I test did not improve statistically significantly after 28 days of treatment, although we did observe a significant improvement after 90 days of treatment with 0.03% tacrolimus eye drops. The average break-up time did not improve statistically after 14 days of treatment, although we observed a significant improvement after 28 and 90 days of treatment with 0.03% tacrolimus eye drops. Conclusions: Topical 0.03% tacrolimus eye drops successfully improved tear stability and ocular surface status in patients with dry eyes.

The Role of Dyslipidemia on Ocular Surface, Lacrimal and Meibomian Gland Structure and Function

Purpose: Dyslipidemia is characterized by high lipid blood levels that are risk factors for cardiovascular diseases, which are leading causes of death. However, it is unclear whether dyslipidemia is a cause of the dry eye syndrome (DES). Therefore we determined in transgenic mice models of dyslipidemia, whether there is an association with DES development. Methods: Dyslipidemic models included male and female adult mice overexpressing apolipoprotein CIII (Apo CIII), LDL receptor knockout (LDLR-KO) and ApoE knockout (ApoE-KO). They were compared with age-and gender-matched C57BL/6 mice. Ocular health was evaluated based on corneal slit lamp assessment, phenol red thread test (PRT) and impression cytology. Blood lipid profiles and histology of meibomian and lacrimal glands were also evaluated. Effects of high-fat diet and aging were observed in LDLR-KO and ApoCIII strains, respectively. Results: Body weight and lacrimal gland weight were significantly higher in male mice compared to females of the same strain (P < 0.05). Body weight was significantly lower in LDLRKO mice receiving high lipid diet compared to their controls (P = 0.0043). ApoE-KO were hypercholesterolemic and ApoCIII hypertriglyceridemic while LDLR-KO showed increases in both parameters. The PRT test was lower in male LDLR-KO mice with high-fat diet than control mice with standard diet (P = 0.0273). Aging did not affect lacrimal structural or functional parameters of ApoCIII strain. Conclusions: DES development is not solely dependent on dyslipidemia in relevant mice models promoting this condition. On the other hand, lacrimal gland structure and function are differentially impacted by lipid profile changes in male and female mice. This dissociation suggests that other factors beside dyslipidemia impact on tear film dysfunction and DES development.

In Vitro Effects of Bevacizumab Treatment on Newborn Rat Retinal Cell Proliferation, Death, and Differentiation

PURPOSE. Vascular endothelial growth factor (VEGF) is an important signal protein in vertebrate nervous development, promoting neurogenesis, neuronal patterning, and glial cell growth. Bevacizumab, an anti-VEGF agent, has been extensively used for controlling pathological retinal neovascularization in adult and newborn patients, although its effect on the developing retina remains largely unknown. The purpose of this study was to investigate the effect of bevacizumab on cell death, proliferation, and differentiation in newborn rat retina. METHODS. Retinal explants of sixty 2-day-old Lister hooded rats were obtained after eye enucleation and maintained in culture media with or without bevacizumab for 2 days. Immunohistochemical staining was assessed against proliferating cell nuclear antigen (PCNA, to detect cell proliferation); caspase-3 and beclin-1 (to investigate cell death); and vimentin and glial fibrillary acidic protein (GFAP, markers of glial cells). Gene expressions were quantified by real-time reverse-transcription polymerase chain reaction. Results from treatment and control groups were compared. RESULTS. No significant difference in the staining intensity (on immunohistochemistry) of PCNA, caspase-3, beclin-1, and GFAP, or in the levels of PCNA, caspase-3, beclin-1, and vimentin mRNA was observed between the groups. However, a significant increase in vimentin levels and a significant decrease in GFAP mRNA expression were observed in bevacizumab-treated retinal explants compared with controls. CONCLUSIONS. Bevacizumab did not affect cell death or proliferation in early developing rat retina but appeared to interfere with glial cell maturation by increasing vimentin levels and downregulating GFAP gene expression. Thus, we suggest anti-VEGF agents be used with caution in developing retinal tissue. (Invest Ophthalmol Vis Sci. 2012;53:7904-7911) DOI:10.1167/iovs.12-10283

Trypanosoma cruzi invades host cells through the activation of endothelin and bradykinin receptors: a converging pathway leading to chagasic vasculopathy

BACKGROUND AND PURPOSE Independent studies in experimental models of Trypanosoma cruzi appointed different roles for endothelin-1 (ET-1) and bradykinin (BK) in the immunopathogenesis of Chagas disease. Here, we addressed the hypothesis that pathogenic outcome is influenced by functional interplay between endothelin receptors (ETAR and ETBR) and bradykinin B2 receptors (B2R). EXPERIMENTAL APPROACH Intravital microscopy was used to determine whether ETR/B2R drives the accumulation of rhodamine-labelled leucocytes in the hamster cheek pouch (HCP). Inflammatory oedema was measured in the infected BALB/c paw of mice. Parasite invasion was assessed in CHO over-expressing ETRs, mouse cardiomyocytes, endothelium (human umbilical vein endothelial cells) or smooth muscle cells (HSMCs), in the presence/absence of antagonists of B2R (HOE-140), ETAR (BQ-123) and ETBR (BQ-788), specific IgG antibodies to each GPCRs; cholesterol or calcium-depleting drugs. RNA interference (ETAR or ETBR genes) in parasite infectivity was investigated in HSMCs. KEY RESULTS BQ-123, BQ-788 and HOE-140 reduced leucocyte accumulation in HCP topically exposed to trypomastigotes and blocked inflammatory oedema in infected mice. Acting synergistically, ETAR and ETBR antagonists reduced parasite invasion of HSMCs to the same extent as HOE-140. Exogenous ET-1 potentiated T. cruzi uptake by HSMCs via ETRs/B2R, whereas RNA interference of ETAR and ETBR genes conversely reduced parasite internalization. ETRs/B2R-driven infection in HSMCs was reduced in HSMC pretreated with methyl-beta-cyclodextrin, a cholesterol-depleting drug, or in thapsigargin-or verapamil-treated target cells. CONCLUSIONS AND IMPLICATIONS Our findings suggest that plasma leakage, a neutrophil-driven inflammatory response evoked by trypomastigotes via the kinin/endothelin pathways, may offer a window of opportunity for enhanced parasite invasion of cardiovascular cells.

Isolation, enzymatic characterization and antiedematogenic activity of the first reported rattlesnake hyaluronidase from Crotalus durissus terrificus venom

A hyaluronidase (CdtHya1) from Crotalus durissus terrificus snake venom (CdtV) was isolated and showed to exhibit a high activity on hyaluronan cleavage. However, surveys on this enzyme are still limited. This study aimed at its isolation, functional/structural characterization and the evaluation of its effect on the spreading of crotoxin and phospholipase A(2) (PLA(2)). The enzyme was purified through cation exchange, gel filtration and hydrophobic chromatography. After that, it was submitted to a reverse-phase fast protein liquid chromatography (RP-FPLC) and Edman degradation sequencing, which showed the first N-terminal 44 amino acid residues whose sequence evidenced identity with other snake venom hyaluronidases. CdtHya1 is a monomeric glycoprotein of 64.5 kDa estimated by SDS-PAGE under reducing conditions. It exhibited maximum activity in the presence of 0.2 M NaCl, at 37 degrees C, pH 5.5 and a specificity to hyaluronan higher than that to chondroitin-4-sulphate, chondroitin-6-sulphate or dermatan. Divalent cations (Ca2+ and Mg2+) and 1 M NaCl significantly reduced the enzyme activity. The specific activity of CdtHya1 was 5066 turbidity reducing units (TRU)/mg, against 145 TRU/mg for the soluble venom, representing a 34.9-fold purification. The pure enzyme increased the diffusion of crotoxin and PLA (2) through mice tissues. CdtHya1 (32 TRU/40 mu L) potentiated crotoxin action, as evidenced by mice death, and it decreased the oedema caused by subplantar injections of buffer, crotoxin or PLA(2), thus evidencing the relevance of hyaluronidase in the crotalic envenoming. This work yielded a highly active antiedematogenic hyaluronidase from CdtV, the first one isolated from rattlesnake venoms. (C) 2012 Elsevier Masson SAS. All rights reserved.

Portable light transmission measuring system for preserved corneas

Abstract Background The authors have developed a small portable device for the objective measurement of the transparency of corneas stored in preservative medium, for use by eye banks in evaluation prior to transplantation. Methods The optical system consists of a white light, lenses, and pinholes that collimate the white light beams and illuminate the cornea in its preservative medium, and an optical filter (400–700 nm) that selects the range of the wavelength of interest. A sensor detects the light that passes through the cornea, and the average corneal transparency is displayed. In order to obtain only the tissue transparency, an electronic circuit was built to detect a baseline input of the preservative medium prior to the measurement of corneal transparency. The operation of the system involves three steps: adjusting the "0 %" transmittance of the instrument, determining the "100 %" transmittance of the system, and finally measuring the transparency of the preserved cornea inside the storage medium. Results Fifty selected corneas were evaluated. Each cornea was submitted to three evaluation methods: subjective classification of transparency through a slit lamp, quantification of the transmittance of light using a corneal spectrophotometer previously developed, and measurement of transparency with the portable device. Conclusion By comparing the three methods and using the expertise of eye bank trained personnel, a table for quantifying corneal transparency with the new device has been developed. The correlation factor between the corneal spectrophotometer and the new device is 0,99813, leading to a system that is able to standardize transparency measurements of preserved corneas, which is currently done subjectively.

Keratometry device for surgical support

Abstract Background High astigmatisms are usually induced during corneal suturing subsequent to tissue transplantation or any other surgery which involves corneal suturing. One of the reasons is that the procedure is intimately dependent on the surgeon's skill for suturing identical stitches. In order to evaluate the influence of the irregularity on suturing for the residual astigmatism, a prototype for ophthalmic surgical support has been developed. The final intention of this prototype is to be an evaluation tool for guided suture and as an outcome diminish the postoperative astigmatism. Methods The system consists of hand held ring with 36 infrared LEDs, that is to be projected onto the lachrymal film of the cornea. The image is reflected back through the optics of the ocular microscope and its distortion from the original circular shape is evaluated by developed software. It provides keratometric and circularity measurements during surgery in order to guide the surgeon for uniformity in suturing. Results The system is able to provide up to 23D of astigmatism (32D - 55D range) and is ± 0.25D accurate. It has been tested in 14 volunteer patients intraoperative and has been compared to a commercial keratometer Nidek Oculus Hand-held corneal topographer. The correlation factors are 0.92 for the astigmatism and 0.97 for the associated axis. Conclusion The system is potentially efficient for guiding the surgeon on uniformity of suturing, presenting preliminary data indicating an important decrease on the residual astigmatism, from an average of 8D - for patients not submitted to the prototype guidance - to 1.4D - for patients who have actually been submitted to the prototype guidance - after the first 24 hours post-surgery and in the subsequent weeks. It also indicates that the surgeon should achieve circularity greater or equal to 98% in order to avoid postoperative astigmatisms over 1D. Trial Registration Trial registration number: CAAE - 0212.0.004.000-09.