[Show abstract][Hide abstract] ABSTRACT: Purpose
To explore the potential of spectral-domain optical coherence tomography (SD-OCT) using the en-face technology for the imaging of ocular surface diseases and to correlate the findings with in vivo confocal microscopy (IVCM) images.
Patients and methods
113 eyes of 75 subjects with various ocular surface diseases were investigated with the RTVue® anterior-segment en face OCT. En face OCT images were compared to B-scans OCT and IVCM images. Results: Patients with corneal dystrophies, corneal deposits, keratitis, pterygium, conjunctivochalasis, or ocular surface squamous neoplasia and patients who underwent lamellar corneal surgeries were included. En face OCT images showed ocular surface tissue changes that were not discernible using conventional B-scans OCT. Nevertheless, there was a good correlation with IVCM analysis. Compared with IVCM, the major advantages of en-face OCT included easy operation and rapid image acquisition, with minimal operator experience required. In addition, the non-contact method avoided patient discomfort and external pressure on the globe, which was especially useful in patients with corneal dystrophies, ulcers, or corneal abscesses. Although the resolution of en-face OCT was lower than that of IVCM, it allowed useful overall visualization of corneal lesions due to the larger areas analyzed. Conclusion: En-face SD-OCT is a novel, valuable tool to assess a wide variety of ocular surface diseases. It can provide additional information and new insight into different ocular surface conditions with no corneal contact.
[Show abstract][Hide abstract] ABSTRACT: Demodex mites are implicated in several ocular surface diseases such as blepharitis, ocular rosacea and dry eye syndrome. Demodex eyelid infestation is classically diagnosed by analysing depilated eyelashes under the light microscope. The use of in vivo confocal microscopy (IVCM) could be an easy way to improve its diagnosis. The ability of IVCM to identify Demodex was evaluated and compared with the classic depilation method.
[Show abstract][Hide abstract] ABSTRACT: PURPOSE: To compare in vitro, on the human reconstituted corneal epithelial SkinEthics model, and in vivo, using an acute rabbit toxicological model, the effects of a benzalkonium chloride (BAK)-preserved solution of latanoprost and a preservative-free (PF) latanoprost solution. METHODS: In vitro, the 3D reconstituted human corneal epithelia (HCE) were treated with: phosphate-buffered saline (PBS), BAK-latanoprost, PF-latanoprostor 0.02% BAK for 24h followed or not followed by a 24-h postincubation recovery period. Cellular viability was evaluated using the MTT test at 24h and the apoptotic cells were counted using TUNEL labeling on frozen sections at 24h and 24h+24h. In vivo, rabbits received 50 μl of the same solutions, which were applied at 5-min intervals a total of 15 times. Ocular surface toxicity was investigated using slit lamp biomicroscopy examination, conjunctival impression cytology (CIC), and corneal in vivo confocal microscopy (IVCM). Standard immunohistology also assessed inflammatory CD45-positive cells. RESULTS: In vitro, BAK-latanoprost and 0.02% BAK induced significant apoptosis in the apical layers that correlated with the significant decrease of cell viability as assessed by the MTT test. PF-latanoprost slightly decreased cell viability to 83% and few apoptotic cells were found in the superficial layers, without reaching statistical significance compared to PBS. In vivo, clinical observation and IVCM images showed the lowest ocular surface toxicity with PBS and PF-latanoprost, while BAK-latanoprost and BAK induced abnormal corneoconjunctival aspects. PF-latanoprost showed the lowest CIC score, close to the PBS score and induced fewer CD45-positive cells in both the limbus and the conjunctiva compared with BAK and latanoprost, as assessed by immunohistology. CONCLUSIONS: We confirm that rabbit corneoconjunctival surfaces presented better tolerance when treated with PF-latanoprost compared with the standard BAK-latanoprost preparation or the BAK solution.
[Show abstract][Hide abstract] ABSTRACT: PURPOSE:: Cationic emulsions (CEs), developed as vehicles for lipophilic drugs, have been shown to be safe and effective for the treatment of dry eye. The aim of this study was to investigate the effects of a preservative-free latanoprost 0.005% CE (latanoprost-CE) in in vitro and in vivo models of corneal wound healing. METHODS:: An in vitro wound was made by scraping through a confluent layer of human corneal epithelial cells. Cytotoxicity, cell migration, and proliferation were analyzed after an exposure to phosphate-buffered saline, CE, latanoprost-CE, 0.02% benzalkonium chloride (0.02%BAK), and Xalatan (latanoprost). In vivo, the recovery and integrity of corneal wound healing were assessed in rat eyes instilled twice a day for 5 days with the above treatments after deepithelialization of the superior cornea. RESULTS:: In vitro wound distances decreased at 2 and 24 hours for human corneal epithelial cells exposed to CE, latanoprost-CE, and phosphate-buffered saline, whereas they progressively increased for 0.02%BAK-treated and latanoprost-treated cells. The greater wound closure was associated with a higher number of Ki67-positive cells. In CE- and latanoprost-CE-treated rats, reepithelialization of the cornea was enhanced, restoring normal appearance and function. In contrast, 0.02%BAK or latanoprost delayed corneal healing, induced inflammation, and decreased MUC5-AC expression. CONCLUSIONS:: Both models effectively evaluated the cytotoxicity and dynamic recovery of corneal wound healing, and their correlation supports the potential of the in vitro model as a reliable alternative to in vivo ocular toxicity tests. Both models demonstrated that in the face of corneal injury, CEs favored corneal healing, whereas BAK was deleterious.
[Show abstract][Hide abstract] ABSTRACT: The study used a validated acute in vivo model to compare a new formulation of travoprost 0.004% ophthalmic solution(travoprost PQ), preserved with polyquaternium-1 (PQ), with commercially available formulations of benzalkonium-chloride(BAK)-preserved travoprost 0.004% ophthalmic solution(travoprost BAK) and BAK-preserved latanoprost 0.005%ophthalmic solution (latanoprost BAK). Adult male New Zealand albino rabbits (n = 36) were randomly divided into 6 groups. Phosphate-buffered saline (PBS), 0.001% PQ, 0.015% BAK, travoprost PQ, travoprost BAK or latanoprost BAK were applied onto rabbit eyes as 1 drop, for 15 times at 5-min intervals.The ocular surface reactions were investigated at hour 4 and day 1 using slitlamp examination; in vivo confocal microscopy (IVCM) for cornea, limbus and conjunctiva/conjunctiva-associated lymphoid tissue, conjunctival impression cytology and standard immunohistology in cryosections for detecting CD45+ infiltrating cells and MUC-5AC-labeled cells. PBS, PQ and travoprost PQ did not induce obvious irritation by clinical observation, changes in microstructures of the whole ocular surface as measured by IVCM analysis,inflammatory infiltration or cell damage as measured by impression cytology, altered levels of goblet cell counts or numerous CD45+ cells in the cornea. In contrast, all BAK-containing products induced diffuse conjunctival hyperemia and chemosis, abnormal changes in the ocular surface microstructure,significant total ocular surface toxicity scores,damaged epithelial cells, inflammatory cell infiltration and decreased goblet cell density. Travoprost PQ did not elicitocular surface toxicity when administered to rabbit eyes.These results suggest a greater safety advantage for the ocular surface of patients receiving chronic glaucoma treatment with PQ-preserved drugs.
Ophthalmic Research 04/2012; 48(2):89-101. · 1.56 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We investigated in a rabbit model, the eye distribution of topically instilled benzalkonium_(BAK) chloride a commonly used preservative in eye drops using mass spectrometry imaging. Three groups of three New Zealand rabbits each were used: a control one without instillation, one receiving 0.01%BAK twice a day for 5 months and one with 0.2%BAK one drop a day for 1 month. After sacrifice, eyes were embedded and frozen in tragacanth gum. Serial cryosections were alternately deposited on glass slides for histological (hematoxylin-eosin staining) and immunohistological controls (CD45, RLA-DR and vimentin for inflammatory cell infiltration as well as vimentin for Müller glial cell activation) and ITO or stainless steel plates for MSI experiments using Matrix-assisted laser desorption ionization time-of-flight. The MSI results were confirmed by a round-robin study on several adjacent sections conducted in two different laboratories using different sample preparation methods, mass spectrometers and data analysis softwares. BAK was shown to penetrate healthy eyes even after a short duration and was not only detected on the ocular surface structures, but also in deeper tissues, especially in sensitive areas involved in glaucoma pathophysiology, such as the trabecular meshwork and the optic nerve areas, as confirmed by images with histological stainings. CD45-, RLA-DR- and vimentin-positive cells increased in treated eyes. Vimentin was found only in the inner layer of retina in normal eyes and increased in all retinal layers in treated eyes, confirming an activation response to a cell stress. This ocular toxicological study confirms the presence of BAK preservative in ocular surface structures as well as in deeper structures involved in glaucoma disease. The inflammatory cell infiltration and Müller glial cell activation confirmed the deleterious effect of BAK. Although these results were obtained in animals, they highlight the importance of the safety-first principle for the treatment of glaucoma patients.
PLoS ONE 01/2012; 7(11):e50180. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Conjunctiva-associated lymphoid tissue (CALT) is closely associated with ocular surface immunity. This study investigated the effects of antiglaucoma prostaglandin analogs with or without benzalkonium chloride (BAK) preservative on organized CALT using an acute toxic model. A total of 48 albino rabbits were used and seven groups of treatments were constituted. Solutions (50 µl) of PBS, 0.02%BAK, (0.02%BAK+)latanoprost, (0.015%BAK+)travoprost, (0.005%BAK+)bimatoprost, (BAK-free)travoprost preserved with the SofZia® system or (BAK-free)tafluprost were instilled 15 times at 5-min intervals in both eyes. CALT changes were analyzed using in vivo confocal microscopy (IVCM), immunohistology in cryosections for detecting MUC-5AC+ mucocytes and CD45+ hematopoietic cells. Antiglaucoma eye drops stimulated inflammatory cell infiltration in the CALT, and seemed to be primarily related to the concentration of their BAK content. The CALT reaction after instillation of BAK-containing eye drops was characterized by inflammatory cell infiltration in the dome and intrafollicular layers and by cell circulation inside the lymph vessels. CD45 was strongly expressed in the CALT after instillation of all BAK-containing solutions at 4 h and decreased at 24 h. The number of MUC-5AC+ mucocytes around the CALT structure decreased dramatically after instillation of BAK-containing solutions. This study showed for the first time the in vivo aspect of rabbit CALT after toxic stimuli, confirming the concentration-dependent toxic effects of BAK. IVCM-CALT analysis could be a pertinent tool in the future for understanding the immunotoxicologic challenges in the ocular surface and would provide useful criteria for evaluating newly developed eye drops.
PLoS ONE 01/2012; 7(3):e33913. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Topical preparations of cyclosporine (CsA) are common therapeutics for the treatment of dry eye. However, they are not devoid of side effects, such as allergy and irritation. The present study aimed at evaluating the safety profile of a new CsA formulation in cationic emulsion (CEm) in vitro with a dynamic corneal wound healing assay using human corneal epithelial (HCE) cells, and in vivo in a rabbit acute toxicity model.
Three different csa formulations were tested: 1) 0.05%CsA-CEm, 2) commercial 0.05%CsA-Anionic emulsion (CsA-AEm, Restasis®), and 3) 0.05%CsA-Oil solution. Phosphate buffered saline (PBS) was used as negative control and 0.02% benzalkonium chloride (BAK) as the toxic control. In vitro, a wound was created by scratching through a confluent HCE cell layer and exposed 30 min to 1/10 dilutions of the different formulations. Cytotoxicity, cell migration, and proliferation were performed to analyze the recovery at days 1, 2, and 3. In vivo, the eye drops were applied to rabbit eyes 15 times at 5-min intervals. The ocular surface structures were examined with a slit-lamp and by corneal in vivo confocal microscopy (IVCM) for detailed examination of corneal epithelium, stroma, limbus, and conjunctiva-associated lymphoid tissue (CALT) structures.
The in vitro study confirmed that a 0.02% BAK solution delayed the corneal healing process (-57%) by severely damaging the remaining HCE cells. The other formulations maintained a normal healing rate with a similar behavior for CsA-CEm, CsA-AEm, and PBS with no significant differences (at D3, 66%-74% closure). In the rabbit, 0.02%BAK showed the highest toxicity, inducing redness, chemosis with damaged corneal epithelium, and inflammatory cell infiltrations. CsA-AEm and CsA-Oil induced moderate infiltrations of inflammatory cells around the CALT. CsA-CEm presented the lowest toxicity with patterns similar to PBS.
The combination of these in vitro and in vivo models evaluated the tolerance/cytotoxicity and the dynamic wound healing potential of CsA in different formulations. While CsA-AEm, CsA-CEm, and CsA-Oil are generally well tolerated, only CsA-CEm appeared to maintain the HCE cells' normal healing rate in vitro and low levels of inflammation in vivo.
[Show abstract][Hide abstract] ABSTRACT: To compare the toxicological profile of a new formulation of travoprost 0.004% ophthalmic solution (travoprost PQ), containing the preservative polyquaternium-1(PQ, polyquad), with the commercially available formulation of benzalkonium chloride (BAK)-preserved travoprost 0.004% ophthalmic solution (travoprost BAK) and BAK-preserved latanoprost 0.005% ophthalmic solution (latanoprost BAK).
Human conjunctival epithelial cells were incubated with phosphate-buffered saline (PBS), BAK 0.015%, BAK 0.020%, PQ 0.001%, travoprost PQ preserved with PQ 0.001%, travoprost preserved with BAK 0.015%, or latanoprost preserved with BAK 0.020%. Six toxicological assays were used to assess: cell viability (neutral red, Alamar blue), apoptosis (YO-PRO-1, Hoechst 33342), and oxidative stress (H(2)DCF-DA, hydroethidine). Apoptosis and oxidative stress were each reported according to cell viability as observed with neutral red and Alamar blue for a total of 10 analyses per treatment depending on the cell viability test used to interpret apoptosis and oxidative stress responses.
There were no significant differences in toxicity between cells exposed to PBS and cells exposed to travoprost PQ (10/10 analyses) or PQ 0.001% (9/10 analyses). Ten out of 10 analyses revealed that travoprost PQ produced significantly less cytotoxicity than latanoprost BAK (p < 0.0001). Travoprost PQ produced significantly better cell viability and less apoptosis than travoprost BAK (6/6 analyses, p < 0.0001). Travoprost BAK was significantly less cytotoxic than latanoprost BAK in 7 of 10 analyses (p < 0.0001). All 10 of the analyses revealed that BAK 0.015%, BAK 0.020%, and latanoprost BAK produced significantly more cytotoxicity than PBS (p < 0.0001). Travoprost BAK was significantly less cytotoxic than its corresponding BAK 0.015% preservative solution in 9 of 10 analyses (p < 0.0001).
A panel of in vitro toxicity analyses supports the safety of travoprost PQ. Travoprost PQ may be better for ocular surface health than BAK-preserved formulations of latanoprost or travoprost but clinical studies are required to validate these comparisons.
Current eye research 11/2011; 36(11):979-88. · 1.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To compare, in vitro, the cytotoxicity profile of a new formulation of travoprost 0.004%/timolol 0.5% fixed combination ophthalmic solution preserved with polyquaternium-1 0.001% (travoprost/timolol PQ) instead of benzalkonium chloride (BAK) with (1) commercially available travoprost 0.004%/timolol 0.5% fixed combination ophthalmic solution (travoprost/timolol BAK), (2) commercially available latanoprost 0.005%/timolol 0.5% fixed combination ophthalmic solution (latanoprost/timolol BAK), and (3) their associated BAK concentrations.
Compounds tested on Wong-Kilbourne-derived human conjunctival epithelial cells: (1) phosphate-buffered saline, (2) polyquaternium-1 0.001% (Polyquad(®), PQ), (3) travoprost/timolol PQ, (4) travoprost/timolol BAK with 0.015% BAK (DuoTrav(®)), (5) BAK 0.015%, (6) latanoprost/timolol BAK with 0.020% BAK (Xalacom(®)), and (7) BAK 0.020%. Toxicological assays were used to assess cell viability [neutral red (NR), Alamar blue (AB)], apoptosis (YO-PRO-1, Hoechst 33342), and oxidative stress (H(2)DCF-DA, hydroethidine). The apoptosis and oxidative stress assays were each reported according to cell viability as observed with NR and AB (totaling 10 analyses per treatment).
The NR and AB assays demonstrated that cells incubated with travoprost/timolol PQ had significantly better viability than cells incubated with latanoprost/timolol BAK, travoprost/timolol BAK, BAK 0.015%, and BAK 0.020% (P<0.0001 for all). As assessed with YO-PRO-1 and Hoechst 33342 relative to cell viability determined with NR or AB, travoprost/timolol PQ produced significantly less apoptosis than travoprost/timolol BAK and latanoprost/timolol BAK and their respective BAK concentrations alone (P<0.0001 for all). Also, travoprost/timolol BAK induced less apoptosis than latanoprost/timolol BAK (P<0.0001). As assessed with H(2)DCF-DA as a ratio to NR or AB, all of the compounds without BAK (phosphate-buffered saline, PQ 0.001%, and travoprost/timolol PQ) and travoprost/timolol BAK produced significantly less reactive oxygen species than latanoprost/timolol BAK (P<0.0001 for all). As assessed with hydroethidine as a ratio to NR or AB, travoprost/timolol PQ produced significantly fewer superoxide anions than latanoprost/timolol BAK (P<0.0001). In contrast, release of superoxide anions (hydroethidine method) after incubation with travoprost/timolol BAK was not significantly different from incubation with latanoprost/timolol BAK or travoprost/timolol PQ.
Travoprost/timolol PQ may be better for ocular surface health than either BAK preserved formulations of latanoprost/timolol or travoprost/timolol.
Journal of ocular pharmacology and therapeutics: the official journal of the Association for Ocular Pharmacology and Therapeutics 03/2011; 27(3):273-80. · 1.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to use a validated acute rabbit model to test the toxicity of a novel formulation of fixed-combination travoprost 0.004%/timolol 0.5% ophthalmic solution, which contains the antimicrobial preservative polyquaternium-1 (PQ), compared with the commercial formulation of fixed combinations travoprost 0.004%/timolol 0.5% ophthalmic solution and latanoprost 0.005%/timolol 0.5% ophthalmic solution, which both contain the preservative benzalkonium chloride (BAK).
Adult male New Zealand albino rabbits (n=24) were randomly divided into four groups. Phosphate-buffered saline (PBS), travoprost/timolol PQ, travoprost/timolol BAK, or latanoprost/timolol BAK were instilled onto rabbit eyes one drop, 15 times at 5 minute intervals. The ocular surface reactions were investigated at hour 4 and day 1 using slit lamp examination; in-vivo confocal microscopy (IVCM) for cornea, limbus, and conjunctiva-associated lymphoid tissue (CALT); conjunctival impression cytology; and standard immunohistology in cryosections for detecting CD45+ infiltrating cells and MUC-5AC-labeled cells.
Travoprost/timolol PQ was better tolerated than travoprost/timolol BAK or latanoprost/timolol BAK. This improved tolerance was evident via clinical observation under slit lamp, IVCM in different layers of the cornea and conjunctiva, conjunctival impression cytology of superficial epithelium aspects, and immunohistochemistry for inflammatory infiltration of CD45+ cells in the cornea and goblet cell distribution. Travoprost/timolol PQ was similar to PBS in regards to in-vivo findings, the Draize test for ocular irritation, and epithelial and limbal aspects as evaluated with IVCM. Treatment with either travoprost/timolol PQ or PBS produced no obvious inflammatory infiltration inside and outside the CALT follicles, yielded similar IVCM toxicity scores and CD45+ cell counts, and eyes treated with either solution had normal goblet cells.
The fixed combination of travoprost/timolol with 0.001% PQ had decreased ocular surface toxicity relative to the BAK-containing solutions. The potential benefit to the human ocular surface with oncedaily dosing needs to be evaluated clinically.
Advances in Therapy 03/2011; 28(4):311-25. · 2.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Using an established three-dimensional (3D) toxicological model based on reconstituted human corneal epithelium (HCE), this study investigated the tolerability of four topical intraocular-pressure-lowering agents: the commercial solutions of benzalkonium chloride (BAC)-containing 0.005% latanoprost, 0.004% travoprost, 0.03% bimatoprost containing 0.02%, 0.015% and 0.005% BAC, respectively, and the preservative-free (PF) tafluprost. Solutions of 0.01% and 0.02% BAC alone were also evaluated for comparison.
The 3D-HCEs were treated with solutions for 24 h followed or not by a 24 h recovery period. We used a modified MTT (3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) procedure to assess cell viability in the HCE. Frozen sections of HCE were analysed using fluorescence microscopy for the evaluation of apoptosis (terminal deoxynucleotidyl transferase mediated dUTP nick end labelling), inflammation (ICAM-1) and proliferation (Ki67). Corneal epithelial tight junctions (occludin and tight junction protein 1 (zona occludens 1)) were also assessed by en face confocal microscopy in response to the different eye-drops.
The MTT test revealed that the cytotoxicity of antiglaucoma eye-drops was primarily related to the concentration of their common BAC preservative (0.02% BAC-latanoprost>0.015% BAC-travoprost>0.005% BAC-bimatoprost). PF-tafluprost did not induce any obvious cytotoxicity, showed the least expression of inflammatory or apoptotic markers and revealed preservation of membrane immunostaining of tight junction proteins in comparison with BAC-containing solutions.
The toxicological model of the 3D reconstructed corneal epithelia model confirmed the ocular surface cytotoxicity of BAC-containing antiglaucomatous solutions. Compared with the formulations containing the toxic preservative BAC, PF-tafluprost was well tolerated without inducing significant corneal epithelium deterioration.
The British journal of ophthalmology 03/2011; 95(6):869-75. · 2.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: There is a large body of evidence from experimental and clinical studies showing that the long-term use of topical drugs may induce ocular surface changes, causing ocular discomfort, tear film instability, conjunctival inflammation, subconjunctival fibrosis, epithelial apoptosis, corneal surface impairment, and the potential risk of failure for further glaucoma surgery. Subclinical inflammation has also been described in patients receiving antiglaucoma treatments for long periods of time. However, the mechanisms involved, i.e., allergic, toxic, or inflammatory, as well as the respective roles of the active compound and the preservative in inducing the toxic and/or proinflammatory effects of ophthalmic solutions, is still being debated. The most frequently used preservative, benzalkonium chloride (BAK), has consistently demonstrated its toxic effects in laboratory, experimental, and clinical studies. As a quaternary ammonium, this compound has been shown to cause tear film instability, loss of goblet cells, conjunctival squamous metaplasia and apoptosis, disruption of the corneal epithelium barrier, and damage to deeper ocular tissues. The mechanisms causing these effects have not been fully elucidated, although the involvement of immunoinflammatory reactions with the release of proinflammatory cytokines, apoptosis, oxidative stress, as well as direct interactions with the lipid components of the tear film and cell membranes have been well established. Preservative-induced adverse effects are therefore far from being restricted to only allergic reactions, and side effects are often very difficult to identify because they mostly occur in a delayed or poorly specific manner. Care should therefore be taken to avoid the long-term use of preservatives, otherwise a less toxic alternative to BAK should be developed, as this weakly allergenic but highly toxic compound exerts dose- and time-dependent effects. On the basis of all these experimental and clinical reports, it would be advisable to use benzalkonium-free solutions whenever possible, especially in patients with the greatest exposure to high doses or prolonged treatments, in those suffering from preexisting or concomitant ocular surface diseases, and those experiencing side effects related to the ocular surface. Indeed, mild symptoms should not be underestimated, neglected, or denied, because they may very well be the apparent manifestations of more severe, potentially threatening subclinical reactions that may later cause major concerns.
Progress in Retinal and Eye Research 03/2010; 29(4):312-34. · 9.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Conjunctiva-associated lymphoid tissue (CALT) plays an important role in ocular surface immunity. No study until now has been able to show its in vivo aspects, and few have demonstrated its reactions after pathologic patterns. The authors investigated in rabbit eyes the cell reactions occurring in the organized CALT during conjunctival inflammation models.
Using in vivo confocal microscopy (IVCM), the authors analyzed, for the first time in vivo inflammatory cell infiltration and circulation inside the lymph vessels in rabbit CALT after inflammatory stimuli (LPS, TNFalpha, LPS+/-anti-TNF). Cresyl violet staining was performed to observe the morphology of CALT, and immunohistology was performed in whole mount conjunctiva and cryosections for detecting CD45(+) lymphocytes. Human CALT in vivo aspects were also explored in a patient with vernal keratoconjunctivitis (VKC).
The conjunctivitis model induced by LPS was characterized by inflammatory cell infiltration in the dome and intrafollicular layers of CALT and cell circulation inside the lymph vessels. TNF alone induced moderate inflammatory infiltration in CALT. However anti-TNF antibodies could significantly decrease LPS/TNFalpha-induced inflammation. CD45(+) lymphocytes were strongly expressed in the CALT after injection of LPS or TNFalpha at 4 hours and decreased with injection of anti-TNFalpha. The authors also showed the presence of the CALT pattern in VKC.
The authors showed for the first time the in vivo aspects of normal and pathologic cell reactions in rabbit CALT after inflammatory stimuli. IVCM-CALT could be a pertinent tool in the future for the comprehension of ocular surface defense mechanisms, and inflammatory cell analysis in CALT could constitute a new criterion for evaluating ocular surface inflammation.
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to investigate high-molecular-weight hyaluronan (HA-HMW) corneal protection against sodium lauryl sulfate (SLS)-induced toxic effects with in vitro and in vivo experimental approaches.
In vitro experiments consisted of a human corneal epithelial cell line incubated with HA-HMW, rinsed, and incubated with SLS. Cell viability, oxidative stress, chromatin condensation, caspase-3, -8, -9, and P2X7 cell death receptor activation, interleukin-6, and interleukin-8 production were investigated. In vivo experiments consisted of 36 New Zealand white rabbits treated for 3 days, 3 times per day, with HA-HMW or phosphate-buffered salt solution. At day 4, eyes were treated with SLS. Clinical observation and in vivo confocal microscopy using the Rostock Cornea Module of the Heidelberg Retina Tomograph-II were performed to evaluate and to compare SLS-induced toxicity between eyes treated with HA-HMW and eyes treated with phosphate-buffered salt solution.
In vitro data indicate that exposure of human corneal epithelial cells to HA-HMW significantly decreased SLS-induced oxidative stress, apoptosis, and inflammation cytokine production. In vivo data indicate that SLS cornea injuries, characterized by damaged corneal epithelium, damaged anterior stroma, and inflammatory infiltrations, were attenuated with HA-HMW treatment.
A good correlation was seen between in vitro and in vivo findings showing that HA-HMW decreases SLS-induced toxic effects and protects cornea.
[Show abstract][Hide abstract] ABSTRACT: Using an established rabbit toxicological model, this in vivo study compared the ocular cytotoxicity of four topical intraocular pressure (IOP)-lowering agents: the commercial benzalkonium chloride (BAC)-containing solutions of 0.005% latanoprost, 0.004% travoprost, 0.03% bimatoprost (containing 0.02%, 0.015%, and 0.005% BAC, respectively), and 0.005% latanoprost in a new cationic emulsion (LCEm) formulation.
Thirty adult male New Zealand albino rabbits were used in this study. They were randomly divided into five groups: 50 microl of sterile phosphate-buffered saline (PBS) along with each formulation was applied onto rabbit eyes 15 times at 5 min intervals. The ocular surface changes were investigated using slit-lamp examination, corneal in vivo confocal microscopy (IVCM) for cornea, limbus, conjunctiva/conjunctiva-associated lymphoid tissue (CALT) investigations, and conjunctival imprints for cytology and flow cytometry (FCM) analyses.
Antiglaucoma eye drops induced an ocular surface cytotoxicity primarily related to the concentration of their common BAC preservative ((0.02%BAC+)latanoprost> (0.015%BAC+)travoprost> (0.005%BAC+)bimatoprost). LCEm did not induce any obvious signs of toxicity on the rabbit ocular surface with results similar to those of PBS; moreover, the conjunctiva/CALT and cornea had almost normal aspects.
These in vivo and ex vivo toxicological procedures performed in an acute stress model confirmed the ocular surface cytotoxicity of BAC-containing antiglaucomatous eye drop solutions. The new formulation, LCEm, was well tolerated without inducing ocular surface damage or CALT activation. The cationic emulsion of latanoprost will most likely have fewer long-term adverse effects on the ocular surface than formulations containing toxic preservative BAC and may improve long-term tolerance over BAC-containing antiglaucomatous topical treatments.
[Show abstract][Hide abstract] ABSTRACT: Olive oil and fish oils are known to possess beneficial properties for human health. We investigated whether different oils and fatty acids alone were able to decrease oxidative stress induced on corneal cells.
In our in vivo study, rats were fed with marine oils rich in polyunsaturated fatty acids (PUFA) or refined olive oil during 28 days. At the end of the protocol, corneas were analysed for their fatty acids composition to study the incorporation of fatty acids in cell membranes. In our in vitro study, a human corneal cell line was incubated with marine oils or refined olive oil and subjected to oxidative stress (tBHP 50 muM, 1 hour). Effects on reactive oxygen species generation, mitochondria and caveolin-1 expression were studied using microcytofluorometry, flow cytometry and confocal microscopy.
Our results indicate that dietary oils changed the fatty acids composition of corneal cell membranes. According to our results, PUFA-rich oils and refined olive oil (free of antioxidants) blocked reactive oxygen species production. Oleic acid, the major fatty acid of olive oil, also decreased oxidative stress. Moreover, oleic acid modified caveolin-1 expression. Antioxidant properties of oleic acid could be due to disruption of membrane microdomains such as caveolae.
Oleic acid, a potential potent modulator of oxidative stress, could be added to PUFA-rich oils to prevent oxidative stress-linked corneal pathology.
[Show abstract][Hide abstract] ABSTRACT: To propose an in vivo confocal microscopic scoring system for evaluation of irritant-induced corneal changes.
Rat corneas were instilled with 0.01-0.5% benzalkonium chloride (BAC) and examined using high-resolution in vivo confocal microscopy (HRT-II) to measure corneal thickness and characterize corneal damage patterns. Severity scores were given for each predefined evaluation parameter and then totaled.
The scoring system revealed a dose-dependent effect of BAC and discriminated between high- and low-dose treatments.
This HRT-II scoring standardizes damage evaluation at the cellular level, even when assessing irritating compounds at low concentrations.
Current eye research 11/2008; 33(10):826-38. · 1.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The purpose of this study was to investigate responses to toxic cellular stresses in different human ocular epithelia.
Reactivity with a specific anti-P2X7 antibody was studied using confocal fluorescence microscopy on conjunctival, corneal, lens, and retinal cell lines as well as using impression cytology on human ocular cells. Activation of the P2X7 receptor by selective agonists (ATP and benzoylbenzoyl-ATP) and inhibition by antagonists (oATP, KN-62, and PPADS) were evaluated using the quinolinium,4-[(3-methyl-2-(3H)-benzoxazolylidene) methyl]-1-[3-(triethylammonio)propyl]di-iodide (YO-PRO-1) test in cytofluorometry. Different specific stresses were then induced by a chemical toxin (benzalkonium chloride) and a chemical oxidant (tert-butyl hydroperoxide) to assess the role of the P2X7 receptor. Modulation of P2X7 receptor activation was performed with several ionic solutions.
Our data show that four cell lines express the P2X7 cell death purinergic receptor as judged by reactivity with a specific anti-P2X7 antibody, activation by the selective P2X7 agonist benzoylbenzoyl-ATP and to a lesser extent by ATP (YO-PRO-1 dye uptake), and inhibition by three antagonists (oATP, KN-62, and PPADS). Benzalkonium chloride, a widely used preservative, induced dramatic membrane permeabilization through P2X7 pore opening on conjunctival and corneal epithelia. Reactive oxygen species, induced by tert-butyl hydroperoxide, lead to P2X7 receptor activation on retinal pigment epithelium. Modulation of P2X7 receptor activation was obtained with extracellular Ca(2+) and Mg(2+) and with a controlled ionization marine solution rich in different divalent cations. This marine solution could be proposed as a new ophthalmic solution.
Our observations reveal a novel pathway for epithelial cells apoptosis/cytolysis by inducing different toxic stresses and their modulation by using ionic solutions.
[Show abstract][Hide abstract] ABSTRACT: To investigate the expression of CCR5 and CCR4, two chemokine receptors, as markers of the T helper (Th) 1 and Th2 pathways, respectively, and class II antigen HLA-DR as a hallmark of inflammation on conjunctival cells obtained from patients receiving long-term glaucoma treatment.
A total of 18 normal subjects and 70 glaucoma patients treated with topical antiglaucoma drugs for more than 1 year: 14 receiving a beta-blocker as monotherapy, 38 treated with a prostaglandin analog alone (19 with latanoprost, 6 with travoprost, 13 with bimatoprost), and 18 receiving multiple treatments.
Impression cytologic specimens (ICSs) were obtained from 1 eye of the patients and processed for flow cytometry. Conjunctival cells were extracted and incubated with monoclonal antibodies against CCR4, CCR5, HLA-DR, or their specific controls to measure, in a masked manner, the percentages of conjunctival cells positive for the 3 markers.
HLA-DR and chemokine receptors (CCR4 and CCR5) in ICSs.
Compared with all other groups, HLA-DR expression was raised significantly in the multitreatment group, whereas all monotherapies showed slight and nonsignificant increases. Both CCR4 and CCR5 were increased significantly in all 5 glaucoma groups compared with normal subjects, with no between-group differences.
This study demonstrates the overexpression of 2 chemokine receptors in the conjunctival epithelium of glaucoma patients treated over the long term. These results show the simultaneous overexpression of CCR4 and CCR5, suggesting that the chronic use of topical treatments may stimulate both the Th1 and Th2 systems simultaneously. These results also suggest that inflammatory mechanisms combining allergy with toxicity are at work and illustrate the complexity of inflammatory reactions occurring in the ocular surface of glaucoma patients.