Rabbit Tear Research Articles

Simultaneous determination of fluconazole and ofloxacin in rabbit tear fluid by LC-MS/MS: Application to ocular pharmacokinetic studies

The expansion of polymicrobial keratomycosis (PMK) requires dynamic pharmacotherapy of antimycotics along with antibacterial agents such as fluconazole (FCZ) and ofloxacin (OFX). To effective clinical cure, different microbes require different dosage regimens. A responsive, selective, and fast method for estimation of FCZ and OFX in rabbit tears using high-performance liquid chromatography together with tandem mass spectrometry (LC-MS/MS) was established and validated using ketoconazole as an internal standard (IS). An isocratic separation was achieved using a C18 column with methanol and aqueous 0.2% formic acid (80:20, v/v) as a mobile phase with a total run time and flow rate of 4 min and 400 µL/ min, respectively. The FCZ and OFX were detected utilizing positive ion electrospray ionization (ESI) in multiple reactions monitoring mode. The tear sample extraction was carried out using simple deproteination using methanol. The systematic method validation was carried out according to USFDA regulatory guidelines for selectivity, linearity (r2>0.99), intra-day and inter-day precision and accuracy, matrix effect, dilution integrity, and stability. The validated bioanalytical method was successfully pertained to determine the pharmacokinetics profile of FCZ and OFX marketed formulation in preclinical rabbit tears.

Effects of electroacupuncture on conjunctival cell apoptosis and the expressions of apoptosis-related proteins Caspase-3, Fas and Bcl-2 in rabbits with dry eye syndrome

By observing the effects of electroacupuncture (EA) on the apoptosis of conjunctival cells of rabbits with dry eye syndrome (DES) and the expressions of apoptosis-related proteins Caspase-3, Fas and Bcl-2, to discuss the mechanism of EA in the treatment of DES from the perspective of cell apoptosis. Male New Zealand rabbits were randomly divided into a normal group (NG), a model group (MG), an EA group (EAG) and a sham EA group (SEAG). DES rabbit model was developed by eye drop of 0.1% benzalkonium chloride. The rabbit tear secretion and tear film break-up time (BUT) were measured; terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was used to detect the apoptosis of conjunctival cells; the expressions of Caspase-3, Fas and Bcl-2 proteins in conjunctival cells were detected by immunohistochemistry. Compared with the NG, the rabbit tear secretion decreased and the BUT was shortened in the MG (both P<0.01); compared with the MG and the SEAG, the rabbit tear secretion increased and the BUT was prolonged in the EAG (all P<0.05). Compared with the NG, the apoptosis of rabbit conjunctival cells increased (P<0.01), the expressions of Caspase-3 and Fas proteins increased (both P<0.05), and the expression of Bcl-2 protein decreased (P<0.01) in the MG; compared with the MG and the SEAG, the apoptosis of rabbit conjunctival cells decreased (both P<0.01), the expressions of Caspase-3 and Fas proteins decreased (all P<0.05), and the expression of Bcl-2 protein increased (both P<0.01) in the EAG. EA can inhibit the apoptosis of rabbit conjunctival cells, down-regulate the expressions of apoptosis-related proteins Caspase-3 and Fas, and up-regulate the expression of Bcl-2 protein, which may be one of the mechanisms of EA in treatment of DES.

Characterization of Polyethersulfone Membranes for Tear Fluid Collection.

The sampling of tear fluids is essential for the reproducibility of tear assays. But, an operable, efficient and stable method for tear collection has not been widely established. This study evaluated the utility of polyethersulfone membranes (PESms) for tear collection and compared it with two frequently employed approaches: the use of Schirmer test paper (STP) and capillary tube (CaT). STP and PESms (0.45 and 0.65 µm) were examined using scanning electron microscopy(SEM), applied to soak up water, bovine albumin and cellular lysates, and employed to collect tear fluids from rabbits. The proteins in the cellular lysates and tear fluid were characterized through band profiling of SDS-polyacrylamide electrophoresis (PAGE) gels. Western blot analyses with antibodies against vinculin and lysozyme C were used to compare rabbit tear fluids sampling with CaT, STP and PESm. Rabbit ocular surfaces and eye blinking were evaluated under different conditions. The SEM examination showed that PESms exhibited much smoother surfaces and smaller pores than STP. PESm65 exhibited the highest water absorption and water recovery in vitro. Although SDS-PAGE revealed no obvious differences in the cellular or tear protein band patterns, the highest Dice's similarity coefficient and lowest variance in the differences in peak heights(i.e. band intensities) were observed in the PESm65 samples. The Western blot showed that the band intensities of vinculin protein in tear fluids obtained using CaT, STP and PESm65 are in order of CaT<STP=PESm65, while that of lysozyme C is in order of CaT>PESm65>STP. PESm65 exhibited less blinks and conjunctival injuries than STP during sampling-rabbit tears. Tear collection using PESm has less variance in protein components and is easily operable than traditional STP. PESm possesses the potential for clinical utilization in diagnostic tear assays.

Rapid Quantification of Free Cholesterol in Tears Using Direct Insertion/Electron Ionization–Mass Spectrometry

To establish a simple and rapid analytical method, based on direct insertion/electron ionization-mass spectrometry (DI/EI-MS), for measuring free cholesterol in tears from humans and rabbits. A stable-isotope dilution protocol employing DI/EI-MS in selected ion monitoring mode was developed and validated. It was used to quantify the free cholesterol content in human and rabbit tear extracts. Tears were collected from adult humans (n = 15) and rabbits (n = 10) and lipids extracted. Screening, full-scan (m/z 40-600) DI/EI-MS analysis of crude tear extracts showed that diagnostic ions located in the mass range m/z 350 to 400 were those derived from free cholesterol, with no contribution from cholesterol esters. DI/EI-MS data acquired using selected ion monitoring (SIM) were analyzed for the abundance ratios of diagnostic ions with their stable isotope-labeled analogues arising from the D6-cholesterol internal standard. Standard curves of good linearity were produced and an on-probe limit of detection of 3 ng (at 3:1 signal to noise) and limit of quantification of 8 ng (at 10:1 signal to noise). The concentration of free cholesterol in human tears was 15 ± 6 μg/g, which was higher than in rabbit tears (10 ± 5 μg/g). A stable-isotope dilution DI/EI-SIM method for free cholesterol quantification without prior chromatographic separation was established. Using this method demonstrated that humans have higher free cholesterol levels in their tears than rabbits. This is in agreement with previous reports. This paper provides a rapid and reliable method to measure free cholesterol in small-volume clinical samples.

Efficacy of Acupuncture and Identification of Tear Protein Expression Changes Using iTRAQ Quantitative Proteomics in Rabbits

Purpose: To evaluate the effect of acupuncture on rabbit tear secretion and compare the difference in tear protein expression caused by acupuncture.Materials and Methods: Ten male New Zealand White rabbits were enrolled in this study. The following acupoints around the left eye, Extra 1 (Taiyang), BL 2 (Zanzhu) and SJ 23 (Sizhukong), were selected for acupuncture therapy. Each rabbit received 10 acupuncture sessions of 30 min, three times per week. A quantity of 50 μl rabbit tear was collected at the pre- and post-acupuncture stage in every subject, respectively. Total protein content analysis, one-dimensional gel electrophoresis and quantitative proteomics analysis (iTRAQ) were performed and the results were compared.Results: Generally, the tear protein expression after acupuncture was different from that before acupuncture though to some extent they were similar. The time spent collecting rabbit tear after acupuncture was shorter than that before acupuncture. The total protein content in rabbit tear pre- and post-acupuncture was 7.12 μg/μl versus 11.28 μg/μl, respectively. One-dimensional gel electrophoresis showed that tear proteins collected before acupuncture were substantially different than post-acupuncture proteins. In total, twenty-eight tear proteins were identified by iTRAQ. Associated with acupuncture were six up-regulated proteins (tear lipocalin, α-1-antiproteinase, histidine-rich glycoprotein, hemopexin, Vitamin D-binding protein, α-2-HS-glycoprotein) and five down-regulated proteins (Annexin A1, serum amyloid A-3 protein, Helicase-like transcription factor, 15 kDa protein A, protein S100-A9).Conclusions: The rabbit tear protein expression difference caused by acupuncture indicates that acupuncture not only stimulates lacrimal gland secretion function but also induces the quantitative change of some proteins in rabbit tear, which may support a positive effect of acupuncture in the treatment of dry eye.

Topical application of nucleotides increase lysozyme levels in tears

The present work studies the effects of topical application of nucleotides on rabbit tear lysozyme levels. Lysozyme values were determined by the diffusion in Agar method described by van Bijsterveld in 1974, and the protein amount was obtained by measuring the inhibitory halos around a Whatman n degrees 1 paper disc of 5 mm in diameter. The tested nucleotides were UTP, Ap(4)A and Up(4)U. These compounds were topically instilled in a single-dose in one eye (with the contralateral eye as a control) and the lysozyme halos were measured along 5 hours. The obtained results showed an increase in the lysozyme concentrations of 67%, 93%, and 119% for UTP, Ap(4)A, and Up(4)U, respectively, over the basal levels of lysozyme. For this reason, we suggest these molecules as a potential treatment for the reinforcement of the tear film barrier against ocular infection.

Ocular distribution, bactericidal activity and settling characteristics of TobraDex® ST ophthalmic suspension compared with TobraDex® ophthalmic suspension

TobraDex ophthalmic suspension (tobramycin 0.3%, dexamethasone 0.1%; Alcon Laboratories Inc, Fort Worth, Tex) is frequently used for inflammatory ocular conditions where a risk of bacterial ocular infection exists. A new formulation, TobraDex ST ophthalmic suspension (tobramycin 0.3%, dexamethasone 0.05%, Alcon), utilises a novel suspension technology to reduce viscosity and help prevent settling in the container. A rabbit model that closely mimics the human eye and a clinical study with cataract patients was used to compare the pharmacokinetics and tissue permeability of TobraDex ST and TobraDex. An in-vitro model was used to assess the bactericidal activity using the rabbit tear concentrations of tobramycin 10 minutes after a single topical dose. Concentrations of both tobramycin and dexamethasone were greater in the tear film and ocular tissues of rabbits treated with TobraDex ST. There was an 8.3-fold increase in tobramycin concentration in the rabbit tear film 10 minutes after dosing with TobraDex ST compared with TobraDex. Concentrations of tobramycin and dexamethasone in ocular tissues from rabbits exposed to TobraDex ST were up to 12.5-fold greater relative to TobraDex. The in-vitro bactericidal activity (>99.9% kill, 3-log reduction) of TobraDex ST toward tobramycin-resistant and methicillin-resistant Staphylococcus aureus occurred in 90 minutes. TobraDex ST killed Streptococcus pneumoniae 3-log in 5 minutes. TobraDex had no activity toward tobramycin-resistant, methicillin-resistant S. aureus and required approximately 120 minutes for 3-log reduction of S. pneumoniae. In humans, the mean ratio of dexamethasone levels in the aqueous humour at 1 hour was 1.17 in favour of TobraDex ST. TobraDex ST demonstrated improved suspension formulation characteristics, enhanced pharmacokinetic distribution and improved bactericidal characteristics, and may provide a useful alternative as compared to TobraDex.

Single eye analysis and contralateral eye comparison of tear proteins in normal and dry eye model rabbits by MALDI-ToF mass spectrometry using wax-coated target plates.

A study of rabbit tear protein expression in a dry eye rabbit model was performed to determine if a pattern in expressed proteins could be identified. The uniqueness of the model allows the comparison of normal (control) eye tear protein expression with surgically induced dry eye tear protein expression in individual animals. The sensitivity of the method allows for single eye analysis. One-dimensional mini-gel electrophoresis of the tear proteins did not show substantial differences between band patterns of the normal versus the dry eye, but was used to assess the molecular weight ranges of the major proteins. Specific assignments of some of the predominant proteins were obtained by tandem mass spectrometry (MS) which showed that the lower molecular weight lipid-binding proteins (approximately 10 kDa to 36 kDa) constitute a considerable amount of the observed protein, followed in lesser quantities by the transferrins which have higher molecular weights ranging from 70 kDa to 85 kDa. Enhancement of matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) MS linear mode analysis of intact proteins in tear fluid was demonstrated through the use of wax-coated MALDI plates and spot washing. MALDI-ToF MS analysis of the expressed tear proteins illustrates that differences between normal eye tear and dry eye tear protein content are manifested in changes in the lower molecular weight lipid-binding proteins such as lipophilin which exhibits an increase in concentration in the dry eye, and beta-2 microglobulin which undergoes a decrease.

Identification and Functional Expression of a Carrier-Mediated Riboflavin Transport System on Rabbit Corneal Epithelium

Purpose: To investigate the functional expression of a carrier-mediated transport mechanism for riboflavin (vitamin B2) across cultured rabbit primary corneal epithelial cells (rPCECs) and intact rabbit cornea. The secondary objective was to understand the physiological significance behind the presence of such a transport system for riboflavin on the apical side of the corneal epithelium. Methods: rPCECs and freshly excised rabbit corneas were selected as in vitro and ex vivo models, respectively. Transport and uptake characteristics of [3H]riboflavin were determined at various time points, concentrations, temperatures, and pH. Substrate specificity, energy, and ion dependence studies were carried out to characterize the translocation mechanism. Rabbit tear analysis was done with liquid chromatography/tandem mass spectrometry (LC-MS/MS) to understand the physiological relevance of this transporter. Results: The uptake process in rPCECs was found to be concentration dependent and saturable at higher concentrations. The process was also independent of pH, Na+, and Cl− but dependent on energy and temperature. Unlabeled riboflavin and its structural analogues caused significant inhibition, whereas unrelated vitamins did not interfere with the process. Transport of [3H]riboflavin across rabbit cornea was also saturable at higher concentration and energy dependent but Na+ independent. Substrate specificity studies across intact rabbit cornea produced results similar to the uptake studies in cultured rPCECs. LC-MS/MS analysis of rabbit tears showed the presence of riboflavin. Conclusions: Results suggest the presence of a specialized, high-affinity transport mechanism for riboflavin that is expressed on the apical side of rabbit corneal epithelium and may in turn be responsible for influx of riboflavin from tears to cornea.

Identification, quantification and comparison of major non‐polar lipids in normal and dry eye tear lipidomes by electrospray tandem mass spectrometry

Millions of individuals suffer from a health condition known as keratoconjunctivitis sicca (KCS, also known as 'dry eye'). Studies have indicated that the lipids in the tear film layer, which covers the outer portion of the eye, may be directly correlated with the existence of dry eye syndrome. By identifying and comparing the major, non-polar lipids in normal eye tears with a dry eye model, it may be possible to identify a symptom of, or a contributing factor to, dry eye. Electrospray tandem mass spectrometry (ES-MS/MS) was used to identify and compare the non-polar lipids, detected as lithium adducts, from normal and dry eye tear samples obtained from rabbits. A limited number of normal human tear samples were also examined for lipid content, and a close resemblance to rabbit was observed. Three distinct regions were delineated in the ES mass spectra of the non polar lipids, m/z 20-500, 500-800 and 800-1100. A common feature noted among identified lipid components was a glycerol backbone with fatty acyl substituents attached. Product ion spectra were obtained for lithiated monoacyl-, 1,2- and 1,3-diacyl- and triacylglyceride standards. Newly proposed structures and fragmentation pathways for the major product ions are presented for the 1,2- and 1,3-diglycerides, and also for the monoglyceride. New approaches to distinguishing asymmetric 1,2-diglycerides and 1,2- from 1,3-diglycerides are proposed. For the rabbit tear samples, the m/z 20-500 range contains monoester diols with empirical formulas C(n)H(2n)O(4), the m/z 500-800 range includes diesters with empirical formulas C(n)H(2n-2)O(5) and the m/z 800-1100 range contains triesters with empirical formulas C(n)H(2n-4)O(6). Also found in the extracts were three isoprene acetals (terpenoids).

Development of a rabbit model of tear film instability and evaluation of viscosity of artificial tear preparations.

The purposes of this study were to establish a quantitative method for evaluating rabbit tear film status and investigate the efficacy of artificial tear preparations through ocular surface bathing or eye drop application. The rabbit tear film was evaluated using a noninvasive specular reflection video recording system. The appearance of a tear break area (TBA) on the tear film images (7.4 mm2/mm) after 30 seconds of eye opening was quantified by image analysis. To induce disruption of the rabbit tear film, the ocular surface was challenged for 60 minutes with 1 ppm hypochloric acid (HOCl). Immediately after irrigation, artificial tear preparations composed of viscosity agents sodium hyaluronate (SH), hydroxypropylmethycellulose (HPMC), hydroxyethylcellulose (HEC), or chondroitin sulfate (CS) were applied to the rabbit eye through ocular surface bathing or eye drop application, and the recovery of the disrupted tear film was compared for each preparation. A dramatic increase in TBA was observed immediately after the ocular surface was challenged with HOCl, and it returned to the initial level after 6 hours. Immediately after ocular surface bathing and eye drop application, a dramatic recovery of TBA was observed in all the test solution-treated eyes. One hour after treatments, prolonged amelioration of the tear film instability was observed after ocular surface bathing, but not by eye drop application, with the artificial tear preparations composed of HPMC or SH. Ocular surface bathing with artificial tear preparations composed of a suitable viscosity agents could be useful in managing tear film instability.

Analysis of rabbit tear proteins by high-pressure liquid chromatography/electrospray ionization mass spectrometry.

The aim of this study was to develop a fast and reliable analytical procedure for the display of the protein components of tears that can be used to differentiate the status of the ocular surface. Using this new procedure, we analyzed the tear protein components following a corneal wound in the rabbit. Calibrated 10-microL glass, fire-polished capillary micropipettes were used to collect tears from New Zealand White rabbits prior to and daily for 9 days following a unilateral 6-mm diameter centrally placed anterior keratectomy. Tear proteins were eluted by a reversed-phase high-performance liquid chromatography (RP-HPLC) column and the tear protein profile was monitored by electrospray ionization (ESI) mass spectrometry positive total ion current (TIC) chromatography. Tear proteins were reliably separated into 17 peaks, each of which contained one or a number of protein components. The molecular weight of each protein component was determined by on-line ESI. Major tear protein components, lactoferrin, lysozyme (minimally detectable in rabbit tears), albumin, lipocalin, lipophilin and beta2-microglobulin, were tentatively identified by this method. Based on the mass spectrometric data, beta2-microglobulin was found to be glycosylated with N-acetylhexosamine. ESI-positive TIC chromatograms and mass spectra revealed comparative differences in the tear protein spectra after corneal wounding. One day after wounding, rabbit lysozyme with a molecular weight of 14,717 Da was found to be 8-fold higher in the tears of wounded eyes when compared with tears from unwounded eyes. It dropped back to normal 3 days after wounding. The expression of an unidentified tear protein with the molecular weight of 16,060 Da was also elevated after corneal wounding and returned to normal level by day 5. In this study, LC/ESI-MS was developed as a fast, reproducible and simple method for the identification and analysis of many of the protein components of the tears. Importantly, this technique also allows quantification of each component resolved in the chromatogram. This method is very suitable for mapping peptides and proteins (<80 kDa) in tears.

Electrophoretic polymorphism in rabbit tear lysozyme

Rabbit tears were found to contain two lysozymes which differed in their electrophoretic mobility and were designated tear lysozymes 1 and 2. Rabbit tear lysozyme 1 was purified to homogeneity by conventional purification methods. It was found to be distinct from other known mammalian c-type lysozymes, rabbit tear lysozyme 2 and the major rabbit gastrointestinal lysozyme. The activity profile is centered around the neutral region with an optimum of 7 which is slightly lower than that for chicken lysozyme. The thermal stability as well as inhibition profiles by the substrate analogues, N-acetylglucosamine (NAG) and chitotetraose (NAG)4 are comparable to those of chicken lysozyme. Based on its molecular weight and catalytic properties this isozyme is classified as a c-type lysozyme.

Increased corneal permeability induced by the dual effects of transient tear film acidification and exposure to benzalkonium chloride

A number of common eyedrop solutions were found to be at acid pH and to have considerable acidic buffering capacity. Rabbit tears were shown to have weak buffering capacity at physiologic pH; a single drop of acidic ophthalmic preparation markedly reduced tear film pH for 10 to 15 min. One drop of pH 4 buffer eyedrop without other components reduced rabbit tear film pH in vivo by the same magnitude as the ophthalmic formulations; depressed pH persisted for at least 5 min. To assess the integrity of the corneal epithelial barrier after exposure to transiently acidified tear film, trace amounts of [ 14C]inulin were added to buffer solutions and the inulin concentrations in cornea and aqueous humor determined as 14C (d/min)/mg. Even 15 min after a pH 4 buffer eyedrop, mean corneal inulin concentration was three times that in pH 7 buffer-treated eyes. A concomitant increase in aqueous humor inulin concentration was observed. Corneal permeability was also assayed with the [ 14C]inulin instilled 15 min after application of a buffered eyedrop containing either 0·01 or 0·02% benzalkonium chloride, a common eyedrop preservative. In neutral pH solution, 0·01% benzalkonium chloride did not alter corneal permeability significantly, but the 0·02% solution increased corneal inulin concentration by a factor of seven compared to controls. In pH 4 buffer, however, both the 0·01% and 0·02% benzalkonium chloride solutions increased corneal inulin penetration—by tenfold and eighteenfold, respectively. Thus, the combined effects of acid buffering and benzalkonium chloride on corneal permeability were more than double the effects of either one alone.