Andrew D. Pucker

Analysis of Meibum and Tear Lipids

The meibum is a lipid-rich secretion that is the primary component of the external layer of the tear film. The meibomian glands produce the meibum, and meibomian gland dysfunction can lead to degradation of the tear film. Such dysfunction can result in ocular irritation, inflammation, and clinical disease. Understanding this relationship is critical to preventing ocular disease; therefore, a search of peer-reviewed literature focusing on the collection, quantification, and analysis of normal and abnormal meibum and tear lipids was conducted. Numerous collection and quantification techniques are described, including their advantages and disadvantages. Studies indicate that the meibum and tear lipids consist of a large array of polar and nonpolar lipids; individual lipids or their classes can be correlated to pathology. Significant amounts of lipids are deposited on contact lenses, depending on the nature of their polymer chemistry. These findings taken together indicate that normal meibum and tear lipids are essential for normal ocular health. Additional studies are required to provide a better understanding of the meibum and tear film biomolecules so that more effective treatments for blepharitis, dry eye disease, and tear film-related contact lens complications can be devised.

The Presence and Significance of Polar Meibum and Tear Lipids

The ocular tear film is a complex structure composed of a number of elements. While all of these components serve valuable functional and structural roles, the external lipid layer has been a focus because it is known to play a critical role in dry eye. Traditionally, meibomian gland phospholipids have been considered to be the vital amphiphilic molecules needed to create an interphase between the outer nonpolar lipid layer and inner aqueous layers, yet recent work has called this theory into question. The purpose of this review is to clarify the current understanding of the origins, identity, and significance of polar tear lipids. Studies indicate that both phospholipids and ω-hydroxy fatty acids likely play a critical role in tear film stability. Studies also indicate that polar lipids likely originate from multiple sources and that they are integrally involved in ocular surface disease. Additional studies are needed to fully understand the origins and significance of polar tear lipids, because to date only correlational evidence has described their hypothesized origins and functions.

Enzymatic quantification of cholesterol and cholesterol esters from silicone hydrogel contact lenses.

PURPOSE The purpose of this work was to develop an enzymatic method of quantification of cholesterol and cholesterol esters derived from contact lenses, both in vitro and ex vivo. METHODS Lotrafilcon B (O2 Optix; CIBA Vision, Inc., Duluth, GA) and galyfilcon A (Acuvue Advance; Vistakon, Inc., Jacksonville, FL) silicone hydrogel contact lenses were independently incubated in cholesterol oleate solutions varying in concentrations. After incubation, the lenses were removed and underwent two separate 2:1 chloroform-methanol extractions. After in vitro studies, 10 human subjects wore both lotrafilcon B and galyfilcon A contact lenses for 7 days. The lenses also underwent two separate 2:1 chloroform-methanol extractions. All in vitro and ex vivo samples were quantified with a cholesterol esterase enzymatic reaction. RESULTS Calibration curves from quantifications of in vitro contact lens samples soaked in successively decreasing concentrations of cholesterol oleate yielded coefficients of determination (R(2)) of 0.99 (lotrafilcon B) and 0.97 (galyfilcon A). For in vitro contact lens samples, galyfilcon A was associated with an average cholesterol oleate extraction of 39.85 +/- 48.65 microg/lens, whereas lotrafilcon B was associated with 5.86 +/- 3.36 microg/lens (P = 0.05) across both extractions and all incubation concentrations. For ex vivo contact lens samples, there was significantly more cholesterol and cholesterol esters deposited on galyfilcon A (5.77 +/- 1.87 microg/lens) than on lotrafilcon B (2.03 +/- 1.62 microg/lens; P = 0.0005). CONCLUSIONS This is an efficient and simple method of quantifying total cholesterol extracted from silicone hydrogel contact lenses and, potentially, the meibum and/or tear film. Certain silicone hydrogel materials demonstrate more affinity for cholesterol and its esters than do others.

Region-Specific Relationships Between Refractive Error and Ciliary Muscle Thickness in Children

PURPOSE To determine if there is a relationship between refractive error and ciliary muscle thickness in different muscle regions. METHODS An anterior segment optical coherence tomographer was used to measure cycloplegic ciliary muscle thicknesses at 1 mm (CMT1), 2 mm (CMT2), and 3 mm (CMT3) posterior to the scleral spur; maximum (CMTMAX) thickness was also assessed. An autorefractor was used to determine cycloplegic spherical equivalent refractive error (SPHEQ). Apical ciliary muscle fibers were obtained by subtracting corresponding CMT2 values from CMT1 and CMTMAX. Multilevel regression models were used to determine the relationship between ciliary muscle thickness in various regions of the muscle and refractive error. RESULTS Subjects included 269 children with a mean age of 8.71 ± 1.51 years and a mean refractive error of +0.41 ± 1.29 diopters. In linear models with ciliary muscle thicknesses and SPHEQ, SPHEQ was significantly associated only with CMT2 (β = -11.34, P = 0.0008) and CMT 3 (β = -6.97, P = 0.007). When corresponding values of CMT2 were subtracted from CMT1 and CMTMAX, apical fibers at CMT1 (β = 14.75, P < 0.0001) and CMTMAX (β = 18.16, P < 0.0001) had a significant relationship with SPHEQ. CONCLUSIONS These data indicated that in children the posterior ciliary muscle fibers are thicker in myopia (CMT2 and CMT3), but paradoxically, the apical ciliary muscle fibers are thicker in hyperopia (CMTMAX and CMT1). This may be the first evidence that hyperopia is associated with a thicker apical ciliary muscle region.

Impact of a rinse step on protein removal from silicone hydrogel contact lenses.

Purpose. To determine the impact of the rinse step in “no rub” contact lens care systems relative to its ability to assist in removing loosely associated and bound tear film proteins from a worn silicone hydrogel lens. Methods. After informed consent, subjects were fitted with lotrafilcon B contact lenses (CIBA Vision). If the fit was acceptable, subjects were asked to wear the lenses on a daily wear basis for 5 (+2, −0) days for an outcome visit. Subjects were instructed to use AQuify Multi-Purpose Disinfecting Solution (CIBA Vision) following the manufacturer’s “no rub” instructions. At the outcome visit, contact lenses were then collected by a gloved examiner, with a sterile metal forceps, who rinsed the right lens but did not rinse the left lens on removal from the eyes. Protein was extracted with a 50:50 0.2% trifluoroacetic acid-acetonitrile solution and quantified using a Bradford analyses. Results. Twenty contact lens wearers were enrolled in this study. For the non-rinsed lenses, the first extraction yielded 13.4 ± 9.2 &mgr;g/lens of protein, whereas the second extraction yielded 5.8 ± 2.8 &mgr;g/lens of protein. For the rinsed lenses, the first extraction yielded an average of 3.0 ± 1.9 &mgr;g/lens of protein, whereas the second extraction yielded an average of 4.0 ± 2.3 &mgr;g/lens. Repeated measures analysis of variance showed a significant interaction (F-statistic = 18.9, p < 0.0001) between the rinse of a lens and extraction number. Conclusions. Rinsing a contact lens after removal from the eye removes well more than one-half of the protein associated with it. Further, to biochemically recover all protein from a silicone hydrogel lens, it may be important to perform more than one chemical extraction from it.

Associations with Meibomian Gland Atrophy in Daily Contact Lens Wearers.

Purpose To determine associations for contact lenses (CLs) and meibomian gland atrophy in a matched-pair study. Methods Contact lens wearers (case) and age- and sex-matched non–contact lens (NCL) wearers with no history of CL use (control) were recruited for a multicenter study. All subjects were administered the Ocular Surface Disease Index questionnaire and a comprehensive battery of clinical tests (e.g., tear breakup time, bulbar and limbal redness, meibography, etc.) were performed. Upper and lower eyelid meibomian gland atrophy were graded with both digital meibography (percent gland atrophy) and visual meiboscore methods. Conditional logistic regression analyses were then used to determine relationships among CL use, meibomian gland atrophy, and ocular surface signs and symptoms. Results A total of 70 matched pairs were analyzed. The mean (±SD) age of the CL group was 30.6 (±12.4) years, and that of the NCL group was 30.1 (±12.2) years. The subjects were 63% female. The association between CL wear and meiboscore was not significant univariately, but the best-fitting multivariate regression model showed that higher meiboscores were associated with being a CL wearer (odds ratio [OR], 2.45) in a model that included eyelid margin erythema (OR, 0.25) and lissamine green staining (OR, 1.25). Percent gland atrophy was not associated with CL wear in regression analysis (p = 0.31). Conclusions This study determined inconclusive associations with CLs and meibomian gland atrophy. This study also provided a comprehensive assessment of differences between CL and NCL wearers.

A method of imaging lipids on silicone hydrogel contact lenses.

Purpose. To determine whether Nile Red and Oil Red O stains are able to detect tear film lipids deposited on silicone hydrogel contact lenses. Methods. Eight unworn lotrafilcon A lenses were individually soaked in successively decreasing amounts of cholesterol oleate solution (5.6 to 0.00 mg/ml) for 1 day in triplicate for each staining procedure (etafilcon A lenses were also soaked as a control). The sets of lenses were then stained with Nile Red or Oil Red O. The lenses were then individually visualized with a Nikon Eclipse 80i florescent microscope at 100× magnification, and two representative photos were taken of each lens. Both staining procedures were repeated with human worn lotrafilcon A lenses. Results. The Nile Red stain detected variable yet decreasing amounts of lipids when lenses were incubated in lipid concentrations ≥0.09 mg/ml. Oil Red O detected decreasing amounts of lipids when lenses were soaked in lipid concentrations ≥0.35 mg/ml. The Nile Red stain produced considerably more background staining than Oil Red O, and approximately half of the negative control lenses stained with Nile Red while there was minimal staining of lenses stained with Oil Red O. Etafilcon A lenses yielded decreasing amounts of lipid when soaked in successively lower concentrations of lipid when stained with Nile Red. Human-worn lotrafilcon A lenses yielded similar lipid characteristics when compared with in vitro lenses, with variable amounts of lipid detection when comparing individual subjects. Conclusions. Nile Red and Oil Red O are both able to detect lipids on soft lenses in both in vitro and ex vivo conditions. Oil Red O appears to be a better stain for silicone hydrogel lenses as it offers a higher signal to noise ratio.

Guinea pig ciliary muscle development.

Purpose The purpose of this study was to develop a method for quantifying guinea pig ciliary muscle volume (CMV) and to determine its relationship to age and ocular biometric measurements. Methods Six albino guinea pigs’ eyes were collected at each of five ages (n = 30 eyes). Retinoscopy and photography were used to document refractive error, eye size, and eye shape. Serial sections through the excised eyes were made and then labeled with an &agr;-smooth muscle actin antibody. The ciliary muscle was then visualized with an Olympus BX51 microscope, reconstructed with Stereo Investigator (MBF Bioscience), and analyzed using Neurolucida Explorer (MBF Bioscience). Full (using all sections) and partial (using a subset of sections) reconstruction methods were used to determine CMV. Results There was no significant difference between the full and partial volume determination methods (p = 0.86). The mean (±SD) CMV of the 1-, 10-, 20-, 30-, and 90-day-old eyes was 0.40 (±0.16) mm3, 0.48 (±0.13) mm3, 0.67 (±0.15) mm3, 0.86 (±0.35) mm3, and 1.09 (±0.63) mm3, respectively. Ciliary muscle volume was significantly correlated with log age (p = 0.001), ocular length (p = 0.003), limbal circumference (p = 0.01), and equatorial diameter (p = 0.003). It was not correlated with refractive error (p = 0.73) or eye shape (p = 0.60). Multivariate regression determined that biometric variables were not significantly associated with CMV after adjustment for age. Conclusions Three-dimensional reconstruction was an effective means of determining CMV. These data provide evidence that ciliary muscle growth occurs with age in tandem with eye size in normal albino guinea pigs. Additional work is needed to determine the relationship between CMV and abnormal ocular growth.

Ciliary Muscle Cell Changes During Guinea Pig Development.

PURPOSE Guinea pig ciliary muscle (CM) increases robustly in volume, length, and thickness with age. We wanted to characterize CM cells during development to determine the contributions of hypertrophy (cell size increase) and hyperplasia (cell number increase) during development. METHODS Six pigmented guinea pig eyes were collected at each of five ages: 1, 10, 20, 30, and 90 days. Refractive errors and axial lengths were determined. Eyes were temporally marked, enucleated, hemisected, and fixed. Nasal and temporal eye segments were embedded and 30-μm serial sections were collected; the two most central slides from each hemisection were analyzed with an epifluorescence microscope and Stereo Investigator software to determine normal morphologic parameters. RESULTS Refractive errors became less hyperopic (P = 0.0001) while axial lengths and CM lengths, cross-sectional areas, volumes, and cell sizes all increased linearly with log age (all P < 0.00001). Ciliary muscle cell numbers increased only during the first 20 days of life (P = 0.02). Nasal and temporal CM lengths (P = 0.07), cross-sectional areas (P = 0.18), and cell numbers (P = 0.70) were not different, but CM cell sizes were initially larger temporally and became larger nasally after age 30 days. CONCLUSIONS The mechanism of guinea pig CM cell growth during the first 90 days of life was characterized by early hyperplasia combined with hypertrophic cell growth throughout development that results in larger CM lengths, cross-sectional areas, and volumes. Nasal-temporal CM development was generally symmetric, but there was more CM hypertrophy nasally at older ages.