University of Groningen
Self-assembling nanofiber hydrogels to attenuate epithelial mesenchymal transition in lens
epithelial cells
da Cruz Barros, Raquel Sofia
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2018
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da Cruz Barros, R. S. (2018). Self-assembling nanofiber hydrogels to attenuate epithelial mesenchymal
transition in lens epithelial cells. University of Groningen.
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CHAPTER 5
Investigation of friction-induced
damage to the pig cornea
Re-production with permission of the Ocular Surface: Barros RC, Van Kooten TG, Veeregowda DH. Investigation of Friction-induced Damage to the Pig Cornea. The Ocular Surface. 2015; 13:315-20.
ABSTRACT
Mechanism of friction and damage to cornea is important to the ocular health. A friction measurement device with minimal intervention with the pig eye cornea tear film revealed a low friction coefficient of 0.011 in glycerine solution. Glycerine molecules presumably bind to water, mucins and epithelial cells and therewith improve both squeeze film and boundary lubrication. Consequently the damage to epithelial cells is reduced by 50% compared with phosphate buffer saline, determined using confocal microscopy.
KEYWORDS
5
Re-production with permission of the Ocular Surface: Barros RC, Van Kooten TG, Veeregowda DH. Investigation of Friction-induced Damage to the Pig Cornea. The Ocular Surface. 2015; 13:315-20.
ABSTRACT
Mechanism of friction and damage to cornea is important to the ocular health. A friction measurement device with minimal intervention with the pig eye cornea tear film revealed a low friction coefficient of 0.011 in glycerine solution. Glycerine molecules presumably bind to water, mucins and epithelial cells and therewith improve both squeeze film and boundary lubrication. Consequently the damage to epithelial cells is reduced by 50% compared with phosphate buffer saline, determined using confocal microscopy.
KEYWORDS
Chapter 5
INTRODUCTION
Tear films comprised of lipid, aqueous and mucin layers provide lubrication, improve optical quality and comfort to our eyes [1]. Damage to the tear film affects its stability against eyelid pressure and increases friction, resulting dry eyes [2]. Often, artificial tears composed of lubricants, viscosity modifiers, buffer and electrolytes are used to improve the tear film stability, reduce friction and prevent damage to the dry eyes [3]. However, the effect of artificial tear on corneal friction and resulting cell damage has yet to be explored.
Very few in vitro studies on corneal friction and damage have been reported. Dunn et
al [4] and Angeline et al [5]used custom-made friction device to determine the friction coefficient of murine cornea against a hydrophilic glass probe. Samsom et al [6] used a friction device with a specialized holder to accommodate cornea, which has to be surgically removed from the eyes. Such an intervention could damage the tear film and the cornea before the friction measurements. Therefore, a minimal intervention setup that measures the friction directly on the eyes is needed.
Usually, murine cornea is used in ocular friction studies to relate with human eye cornea. However, unlike murine cornea, the pig eye cornea is considered to be a potential substitute to damaged human eye cornea [7]. Pig eye cornea is similar to human cornea in thickness [8], refractive power and biomechanical properties [9]. Even the anatomy of tear film producing lacrimal glands in pig cornea and human eye cornea is similar [10]. Despite these similarities, the pig eye cornea has not been exploited for in vitro friction experiments.
In this study, we have developed a setup with a portable tribometer to measure friction directly on the pig cornea obtained after sacrificing the animal in the slaughterhouse. Furthermore, a qualitative and quantitative assessment of damage to the pig eye cornea after imposing friction was made using fluorescent staining and subsequent confocal imaging. Glycerine, a common ingredient in artificial tears, was instilled to the pig cornea to evaluate the relationship between friction and damage to the cornea.
MATERIALS AND METHODS
Portable tribometer (Ducom Instruments, the Netherlands) was used to measure friction between glass and pig eye cornea (Figure 1A). First, the optic nerves connected to the pig eyes were cut with scissors. Then, the eyes were manually pushed and separated from the muscles without the use of any instruments, thereby preserving and avoiding any damage to the eye and cornea. After harvesting, the pig eyes were kept hydrated by immersion in a phosphate buffer saline (PBS) at room temperature. Pig eyes were studied within 45 min after harvesting.
Pig eye was placed in a six well plate, 4-6 drops of PBS buffer (saline) or 1 % v/v glycerine i.e. 0.1 mL of 99.9 % pure glycerol (Sigma Aldrich, Germany) was mixed in 10 mL of PBS solution and was added to the cornea. The entire pig eye with the cornea facing the tribometer was placed and not fixed to the well plate. Later the cleaned microscope glass specimen (or slide) was pressed over the cornea at 450 mN. Microscope glass slides (Thomas Scientific, NJ, USA) used in this study were cleaned by sonication in a 2% surfactant RBS 35 (Fluka Chemie, Buchs, Switzerland), followed by thorough rinsing with hot tap water and subsequently alternate rinsing with methanol and demineralized water. The average surface roughness of the cleaned glass slides was 1.2 ± 0.4 nm, as measured using colloidal probe atomic force microscopy. Glass slide pressed against cornea was shearing or reciprocating linearly over the cornea at 0.5mm/s and at a length of 5 mm per stroke (Figure 1B and attached Movie S1). As shown in the movie, there was no slippage between the pig eye and well plate during the shearing.
Friction force was acquired through a bidirectional load cell during forward and reverse stroke for up to 600 sec or 12 cycles. Friction force data from the load cell was acquired at a rate of 1000 samples per second. Every 50 milliseconds, one data array of 50 samples is taken and average value of this array is calculated, converting it to one data point for every 50 samples. This averaged data point is run to a running average filter set at 20% for new and old value respectively. The square root of the mean of the averaged friction force data points for 12 cycles is calculated by the WinDucom software (Ducom Instruments, the Netherlands) to display a RMS friction force value. RMS friction coefficient is determined by dividing the RMS friction force with the applied load of 450 mN. For each type of solution, RMS friction coefficient was averaged over the 5 measurements. For each measurement, a fresh pig eye, clean glass slide, and freshly prepared solutions were used.
. Corneas after friction measurement or without friction (controls) were cut at their border with the sclera and were kept hydrated with PBS during this process. Samples were
then fixed with 3.7 % paraformaldehyde for 15 min and washed with PBS. No detergents were used for staining in order to analyze the real damage made by the friction measurements. Antibodies were diluted in 1% bovine serum albumin – BSA (Sigma-Aldrich, Zwijndrecht, Netherlands), diluted in PBS (1% PBSA) and incubated at room temperature for 2 hr. DAPI (4',6-diamidino-2-phenylindole) of 2 μg/mL was used for nucleic staining (blue color) and TRITC-labelled phalloidin of 2 μg/mL (Sigma-Aldrich, Zwijndrecht, The Netherlands) for staining of the actin cytoskeleton (red color). Samples were visualized by confocal laser scanning microscope (LEICA TCS SP2, Germany) with an UV, Argon and Helium laser using a 40x water-immersion objective lens to allowed permanent hydration of the samples.
In order to allow a semi-quantitative analysis the laser intensities remained equal between the samples. The number of nuclei was analysed by Image J.
5
INTRODUCTION
Tear films comprised of lipid, aqueous and mucin layers provide lubrication, improve optical quality and comfort to our eyes [1]. Damage to the tear film affects its stability against eyelid pressure and increases friction, resulting dry eyes [2]. Often, artificial tears composed of lubricants, viscosity modifiers, buffer and electrolytes are used to improve the tear film stability, reduce friction and prevent damage to the dry eyes [3]. However, the effect of artificial tear on corneal friction and resulting cell damage has yet to be explored.
Very few in vitro studies on corneal friction and damage have been reported. Dunn et
al [4] and Angeline et al [5]used custom-made friction device to determine the friction coefficient of murine cornea against a hydrophilic glass probe. Samsom et al [6] used a friction device with a specialized holder to accommodate cornea, which has to be surgically removed from the eyes. Such an intervention could damage the tear film and the cornea before the friction measurements. Therefore, a minimal intervention setup that measures the friction directly on the eyes is needed.
Usually, murine cornea is used in ocular friction studies to relate with human eye cornea. However, unlike murine cornea, the pig eye cornea is considered to be a potential substitute to damaged human eye cornea [7]. Pig eye cornea is similar to human cornea in thickness [8], refractive power and biomechanical properties [9]. Even the anatomy of tear film producing lacrimal glands in pig cornea and human eye cornea is similar [10]. Despite these similarities, the pig eye cornea has not been exploited for in vitro friction experiments.
In this study, we have developed a setup with a portable tribometer to measure friction directly on the pig cornea obtained after sacrificing the animal in the slaughterhouse. Furthermore, a qualitative and quantitative assessment of damage to the pig eye cornea after imposing friction was made using fluorescent staining and subsequent confocal imaging. Glycerine, a common ingredient in artificial tears, was instilled to the pig cornea to evaluate the relationship between friction and damage to the cornea.
MATERIALS AND METHODS
Portable tribometer (Ducom Instruments, the Netherlands) was used to measure friction between glass and pig eye cornea (Figure 1A). First, the optic nerves connected to the pig eyes were cut with scissors. Then, the eyes were manually pushed and separated from the muscles without the use of any instruments, thereby preserving and avoiding any damage to the eye and cornea. After harvesting, the pig eyes were kept hydrated by immersion in a phosphate buffer saline (PBS) at room temperature. Pig eyes were studied within 45 min after harvesting.
Pig eye was placed in a six well plate, 4-6 drops of PBS buffer (saline) or 1 % v/v glycerine i.e. 0.1 mL of 99.9 % pure glycerol (Sigma Aldrich, Germany) was mixed in 10 mL of PBS solution and was added to the cornea. The entire pig eye with the cornea facing the tribometer was placed and not fixed to the well plate. Later the cleaned microscope glass specimen (or slide) was pressed over the cornea at 450 mN. Microscope glass slides (Thomas Scientific, NJ, USA) used in this study were cleaned by sonication in a 2% surfactant RBS 35 (Fluka Chemie, Buchs, Switzerland), followed by thorough rinsing with hot tap water and subsequently alternate rinsing with methanol and demineralized water. The average surface roughness of the cleaned glass slides was 1.2 ± 0.4 nm, as measured using colloidal probe atomic force microscopy. Glass slide pressed against cornea was shearing or reciprocating linearly over the cornea at 0.5mm/s and at a length of 5 mm per stroke (Figure 1B and attached Movie S1). As shown in the movie, there was no slippage between the pig eye and well plate during the shearing.
Friction force was acquired through a bidirectional load cell during forward and reverse stroke for up to 600 sec or 12 cycles. Friction force data from the load cell was acquired at a rate of 1000 samples per second. Every 50 milliseconds, one data array of 50 samples is taken and average value of this array is calculated, converting it to one data point for every 50 samples. This averaged data point is run to a running average filter set at 20% for new and old value respectively. The square root of the mean of the averaged friction force data points for 12 cycles is calculated by the WinDucom software (Ducom Instruments, the Netherlands) to display a RMS friction force value. RMS friction coefficient is determined by dividing the RMS friction force with the applied load of 450 mN. For each type of solution, RMS friction coefficient was averaged over the 5 measurements. For each measurement, a fresh pig eye, clean glass slide, and freshly prepared solutions were used.
. Corneas after friction measurement or without friction (controls) were cut at their border with the sclera and were kept hydrated with PBS during this process. Samples were
then fixed with 3.7 % paraformaldehyde for 15 min and washed with PBS. No detergents were used for staining in order to analyze the real damage made by the friction measurements. Antibodies were diluted in 1% bovine serum albumin – BSA (Sigma-Aldrich, Zwijndrecht, Netherlands), diluted in PBS (1% PBSA) and incubated at room temperature for 2 hr. DAPI (4',6-diamidino-2-phenylindole) of 2 μg/mL was used for nucleic staining (blue color) and TRITC-labelled phalloidin of 2 μg/mL (Sigma-Aldrich, Zwijndrecht, The Netherlands) for staining of the actin cytoskeleton (red color). Samples were visualized by confocal laser scanning microscope (LEICA TCS SP2, Germany) with an UV, Argon and Helium laser using a 40x water-immersion objective lens to allowed permanent hydration of the samples.
In order to allow a semi-quantitative analysis the laser intensities remained equal between the samples. The number of nuclei was analysed by Image J.
Chapter 5
Figure 1: Portable tribometer used for friction measurements in this study, (A) portable tribometer with stepper
motor drive (1), linear guide bearing (2), loading unit (3) and well plate (4). (B) loading unit with specially designed holder for plain glass specimen (5) in direct contact on pig cornea in a well plate. Fluid is squeezed and held due to capillary forces between the glass and cornea.
RESULTS
The friction coefficient during forward (negative friction coefficient) and reverse shearing (positive friction coefficient) on the cornea in the presence of PBS (or saline solution) was higher compared with glycerine (Figure 2A and 2B). The average root mean square value of the friction coefficient in PBS and glycerine-containing medium was 0.026 ± 0.013 and 0.011 ± 0.003 (Figure 2C).
A
B
Figure 2: Real time changes in the friction coefficient during forward (positive) and reverse (negative) stroke
during reciprocating motion of glass on pig cornea in (A) PBS buffer and (B) 1% glycerine; (C) Error bars represent measurements on five pig eye cornea. Statistically significant difference (p<0.1, Two tailed, Student’s t-test) with the friction coefficient in PBS is indicated by # sign.
A
B
5
Figure 1: Portable tribometer used for friction measurements in this study, (A) portable tribometer with stepper
motor drive (1), linear guide bearing (2), loading unit (3) and well plate (4). (B) loading unit with specially designed holder for plain glass specimen (5) in direct contact on pig cornea in a well plate. Fluid is squeezed and held due to capillary forces between the glass and cornea.
RESULTS
The friction coefficient during forward (negative friction coefficient) and reverse shearing (positive friction coefficient) on the cornea in the presence of PBS (or saline solution) was higher compared with glycerine (Figure 2A and 2B). The average root mean square value of the friction coefficient in PBS and glycerine-containing medium was 0.026 ± 0.013 and 0.011 ± 0.003 (Figure 2C).
A
B
Figure 2: Real time changes in the friction coefficient during forward (positive) and reverse (negative) stroke
during reciprocating motion of glass on pig cornea in (A) PBS buffer and (B) 1% glycerine; (C) Error bars represent measurements on five pig eye cornea. Statistically significant difference (p<0.1, Two tailed, Student’s t-test) with the friction coefficient in PBS is indicated by # sign.
A
B
Chapter 5
Post friction analysis using the confocal microscopy could qualitatively and quantitatively differentiate the damage to the cornea. In a normal fluorescent staining the use of detergents (e.g. Triton X-100) is needed to open the cell membrane and to allow the infiltration of stainings with low permeability (e.g. phalloidin staining). Nonetheless in this study, no detergents were used. The membrane will become permeable if there is a sufficient physical damage caused by the friction measurements. Also, when there is no damage on the cornea the outer layer of epithelial cells is still highly connected and therefore not penetrable for the dyes. The cell membranes impermeable for the dyes yield a low detection for the cytoskeleton (red) and nucleus (blue). Therefore, the cytoskeleton on fresh cornea – without friction was barely visible and the quantity of dyed nuclei was low (Figure 3A). Upon damage both a loosening of cell-cell-contact and an increased permeability of cell membranes occur. This is reflected in corneas after friction measurements in PBS, where the cytoskeleton and nucleus are extremely visible indicating high cellular damage (Figure 3B). Corneas after friction measurements in glycerine look similar to the fresh corneas; however there is a slight increase of nucleus staining (Figure 3C). The number of nuclei exposed to staining on fresh cornea was 124 ± 16 and this increased to 449 ± 50 and 210 ± 23 after friction measurements in PBS and glycerine medium (Figure 3D). Overall, the glycerine medium decreased the number of stained nuclei by 50% compared with PBS.
Figure 3: Confocal images for DAPI (nuclei- blue) and phalloidin-TRITC (cytoskeleton - red) of pig cornea. A Before friction measurements; B After friction measurements in PBS; C After friction measurements in 1% glycerine; D Number of nuclei exposed to staining in pig cornea before friction, after friction in PBS, and 1% glycerine. Error bars represent measurements at three different spots for five pig cornea. Statistically significant difference (p<0.05, one-way ANOVA model) with the fresh corneas is indicated by *.
5
Post friction analysis using the confocal microscopy could qualitatively and quantitatively differentiate the damage to the cornea. In a normal fluorescent staining the use of detergents (e.g. Triton X-100) is needed to open the cell membrane and to allow the infiltration of stainings with low permeability (e.g. phalloidin staining). Nonetheless in this study, no detergents were used. The membrane will become permeable if there is a sufficient physical damage caused by the friction measurements. Also, when there is no damage on the cornea the outer layer of epithelial cells is still highly connected and therefore not penetrable for the dyes. The cell membranes impermeable for the dyes yield a low detection for the cytoskeleton (red) and nucleus (blue). Therefore, the cytoskeleton on fresh cornea – without friction was barely visible and the quantity of dyed nuclei was low (Figure 3A). Upon damage both a loosening of cell-cell-contact and an increased permeability of cell membranes occur. This is reflected in corneas after friction measurements in PBS, where the cytoskeleton and nucleus are extremely visible indicating high cellular damage (Figure 3B). Corneas after friction measurements in glycerine look similar to the fresh corneas; however there is a slight increase of nucleus staining (Figure 3C). The number of nuclei exposed to staining on fresh cornea was 124 ± 16 and this increased to 449 ± 50 and 210 ± 23 after friction measurements in PBS and glycerine medium (Figure 3D). Overall, the glycerine medium decreased the number of stained nuclei by 50% compared with PBS.
Figure 3: Confocal images for DAPI (nuclei- blue) and phalloidin-TRITC (cytoskeleton - red) of pig cornea. A Before friction measurements; B After friction measurements in PBS; C After friction measurements in 1% glycerine; D Number of nuclei exposed to staining in pig cornea before friction, after friction in PBS, and 1% glycerine. Error bars represent measurements at three different spots for five pig cornea. Statistically significant difference (p<0.05, one-way ANOVA model) with the fresh corneas is indicated by *.
Chapter 5
DISCUSSION
The process of fixing the cornea before the friction measurements is challenging, and such an attempt can damage the tear film and cornea before the friction tests. We have overcome this problem with a portable tribometer and well-plate setupthat can measure friction on the cornea without removing it from the eye. This minimal intervention setup has been applied to demonstrate the role of glycerine in lubrication and damage prevention of cornea.The friction coefficient between glass against the pig eye cornea measured in our study is 10 times lower than the friction coefficient between the human eye lid and cornea measured in vitro[6]. The contact pressure in this study is ~12 kPa, and it was of similar order applied in our friction measurements i.e. ~11 kPa (calculated by dividing the applied load 450 mN with the squeezed film contact area of 41 mm2 ). Note that the contact area
was calculated by measuring the circular diameter of the squeezed film shown in the Figure 1B, before the test.Therefore, the higher friction reported by Samsom et al [6] can be due to rougher eye lid surface compared with glass plate and also potential damage to tear films during surgery and mounting the cornea for in vitro friction measurements. However, the friction coefficient in our study was comparable with the friction coefficient between glass and murine cornea measured in vivo [4]. In both the studies, the test setup had minimal intervention to the cornea before friction measurements.
Based on our results, we hypothesize and illustrate the structure and function of tear films; however, the tear films in our study are not the same as in vivo tear films (Figure 4). Tear film is composed of lipid, aqueous and mucin layers. Glycosylated mucin layers bind to the water molecules in PBS and increase the viscosity of water. During shearing by the glass slide, the aqueous layer exhibits a squeezed film lubrication partially separating the glass and cornea. However, exhaustion of squeezed film lubrication due to viscous dissipation is possible, and hence results in a full contact between glass and cornea, leading to a boundary friction condition. In the boundary friction condition, due to unavailability of stable surface-active lubricating molecules, the cornea epithelial cells suffer severe damage (Figure 4B). However, introducing the glycerine molecules onto the cornea improves lubrication due to certain changes in the lubrication mechanism. Glycerine molecules can interact with water molecules and mucins through hydrogen bonding (Figure 4C). Therefore, the glycerine – water mucin mixture improves the viscosity of the aqueous layer and reduces the viscous dissipation of the aqueous layer during the shearing. Subsequently, it can retain the separation between the glass and pig cornea longer, hence exhibiting a longer duration of squeeze film lubrication before transitioning into a boundary friction condition.Furthermore, it is known that glycerine molecules bind the tear film to the epithelial cells and improve the tear film stability on the cornea [11]. Such stability in the tear film is useful for preventing corneal damage during the boundary friction condition occurring after exhaustion of squeeze film lubrication (Figure 4D). Overall, the glycerine-based aqueous lubrication mechanism is 50% more effective than aqueous-only lubrication mechanism in preventing damage to the cornea.
Figure 4. Illustration of tear film architecture on cornea. (A) Before friction measurements in PBS buffer, (B) after friction measurement in PBS buffer, (C) before friction measurement in 1% glycerine, (D) after friction measurement in 1% glycerine.
5
DISCUSSION
The process of fixing the cornea before the friction measurements is challenging, and such an attempt can damage the tear film and cornea before the friction tests. We have overcome this problem with a portable tribometer and well-plate setupthat can measure friction on the cornea without removing it from the eye. This minimal intervention setup has been applied to demonstrate the role of glycerine in lubrication and damage prevention of cornea.The friction coefficient between glass against the pig eye cornea measured in our study is 10 times lower than the friction coefficient between the human eye lid and cornea measured in vitro[6]. The contact pressure in this study is ~12 kPa, and it was of similar order applied in our friction measurements i.e. ~11 kPa (calculated by dividing the applied load 450 mN with the squeezed film contact area of 41 mm2 ). Note that the contact area
was calculated by measuring the circular diameter of the squeezed film shown in the Figure 1B, before the test.Therefore, the higher friction reported by Samsom et al [6] can be due to rougher eye lid surface compared with glass plate and also potential damage to tear films during surgery and mounting the cornea for in vitro friction measurements. However, the friction coefficient in our study was comparable with the friction coefficient between glass and murine cornea measured in vivo [4]. In both the studies, the test setup had minimal intervention to the cornea before friction measurements.
Based on our results, we hypothesize and illustrate the structure and function of tear films; however, the tear films in our study are not the same as in vivo tear films (Figure 4). Tear film is composed of lipid, aqueous and mucin layers. Glycosylated mucin layers bind to the water molecules in PBS and increase the viscosity of water. During shearing by the glass slide, the aqueous layer exhibits a squeezed film lubrication partially separating the glass and cornea. However, exhaustion of squeezed film lubrication due to viscous dissipation is possible, and hence results in a full contact between glass and cornea, leading to a boundary friction condition. In the boundary friction condition, due to unavailability of stable surface-active lubricating molecules, the cornea epithelial cells suffer severe damage (Figure 4B). However, introducing the glycerine molecules onto the cornea improves lubrication due to certain changes in the lubrication mechanism. Glycerine molecules can interact with water molecules and mucins through hydrogen bonding (Figure 4C). Therefore, the glycerine – water mucin mixture improves the viscosity of the aqueous layer and reduces the viscous dissipation of the aqueous layer during the shearing. Subsequently, it can retain the separation between the glass and pig cornea longer, hence exhibiting a longer duration of squeeze film lubrication before transitioning into a boundary friction condition.Furthermore, it is known that glycerine molecules bind the tear film to the epithelial cells and improve the tear film stability on the cornea [11]. Such stability in the tear film is useful for preventing corneal damage during the boundary friction condition occurring after exhaustion of squeeze film lubrication (Figure 4D). Overall, the glycerine-based aqueous lubrication mechanism is 50% more effective than aqueous-only lubrication mechanism in preventing damage to the cornea.
Figure 4. Illustration of tear film architecture on cornea. (A) Before friction measurements in PBS buffer, (B) after friction measurement in PBS buffer, (C) before friction measurement in 1% glycerine, (D) after friction measurement in 1% glycerine.
Chapter 5
CONCLUSION
For the first time friction was measured on pig eye cornea with minimal intervention to the cornea and tear film prior to the test by using a portable tribometer and well plate setup. Corneal cytoskeleton and number of DAPI-stained nuclei were used as damage indicators, as determined by confocal laser scanning microscopy. Glycerine-based aqueous lubrication is more effective than aqueous lubrication in reducing friction, due to its contribution in the squeeze film and boundary film lubrication. We have demonstrated that the lower friction produced less damage to cornea. We speculate that this study provides valuable useful information in the development of artificial tears for patients suffering from dry eyes.
ACKNOWLEDGEMENTS
We acknowledge financial support for the Tribochemist from the Netherlands Organization for Scientific Research (ZonMW91113014).
Dr. Deepak H. Veeregowda is also a Manager of Ducom Instruments in the Netherlands.
Supplementary video data associated with this article can be found, in the online version, at http://dx.doi.org.proxy-ub.rug.nl/10.1016/j.jtos.2015.05.004.
REFERENCES
[1] Ohashi Y, Dogru M and Tsubota K. Laboratory findings in tear fluid analysis. Clinica Chimica Acta. 2006;369:17-28.
[2] Gayton JL. Etiology, prevalence, and treatment of dry eye disease. Clinical Ophthalmology. 2009;3, 405-12.
[3] Wang J, Simmons P, Aquavella J, Vehige J, Palakuru J, Chung S and Feng C. Dynamic distribution of artificial tears on the ocular surface. Archives Ophthalmology. 2008;126:619-25. [4] Dunn AC, Urueña JM, Puig E, Perez VL and Sawyer WG. Friction coefficient measurement of an in vivo murine cornea. Tribology Letters. 2013;49:145-49.
[5] Angelini TE, Dunn AC, Urueña JM, Dickrell DJ, Burris DL and Sawyer WG. Cell friction. Faraday Discussions. 2012;31:156-9.
[6] Samsom ML, Morrison S, Masala N, Sullivan BD, Sullivan DA, Sheardown H and Schmidt TA. Characterization of full-length recombinant human proteoglycan 4 as an ocular surface boundary lubricant. Experimental Eye Research. 2014;127:14-9.
[7] Zeng Y, Yang J, Huang K, Lee Z and Lee X. A comparison of biomechanical properties between human and porcine cornea. Journal of Biomechanics. 2001;34:533-7.
[8] Faber C, Scherfig E and Prause JU. Corneal thickness in pigs measured by ultrasound pachymetry in vivo. Scandinavian Journal of Laboratory Animal Science. 2008;35:39-43. [9] Hara H and Cooper DKC. Xenotransplantation-the future of corneal transplantation? Cornea. 2011;30:371-8.
[10] Henker R, Scholz M, Gaffling S, Asano N, Hampel U, Garreis F and Paulsen F. Morphological features of the porcine lacrimal gland and its compatibility for human lacrimal gland xenografting. Plos One. 2013;8:e74046.
[11] Gensheimer WG, Kleinman DM, Gonzalez MO, Sobti D, Cooper ER, Smits G and Aquavella JV. Novel formulation of glycerin 1% artificial tears extends tear film break-up time compared with Systane lubricant eye drops. Journal Ocular Pharmacology and Therapeutics. 2012;28:473-8.
5
CONCLUSION
For the first time friction was measured on pig eye cornea with minimal intervention to the cornea and tear film prior to the test by using a portable tribometer and well plate setup. Corneal cytoskeleton and number of DAPI-stained nuclei were used as damage indicators, as determined by confocal laser scanning microscopy. Glycerine-based aqueous lubrication is more effective than aqueous lubrication in reducing friction, due to its contribution in the squeeze film and boundary film lubrication. We have demonstrated that the lower friction produced less damage to cornea. We speculate that this study provides valuable useful information in the development of artificial tears for patients suffering from dry eyes.
ACKNOWLEDGEMENTS
We acknowledge financial support for the Tribochemist from the Netherlands Organization for Scientific Research (ZonMW91113014).
Dr. Deepak H. Veeregowda is also a Manager of Ducom Instruments in the Netherlands.
Supplementary video data associated with this article can be found, in the online version, at http://dx.doi.org.proxy-ub.rug.nl/10.1016/j.jtos.2015.05.004.
REFERENCES
[1] Ohashi Y, Dogru M and Tsubota K. Laboratory findings in tear fluid analysis. Clinica Chimica Acta. 2006;369:17-28.
[2] Gayton JL. Etiology, prevalence, and treatment of dry eye disease. Clinical Ophthalmology. 2009;3, 405-12.
[3] Wang J, Simmons P, Aquavella J, Vehige J, Palakuru J, Chung S and Feng C. Dynamic distribution of artificial tears on the ocular surface. Archives Ophthalmology. 2008;126:619-25. [4] Dunn AC, Urueña JM, Puig E, Perez VL and Sawyer WG. Friction coefficient measurement of an in vivo murine cornea. Tribology Letters. 2013;49:145-49.
[5] Angelini TE, Dunn AC, Urueña JM, Dickrell DJ, Burris DL and Sawyer WG. Cell friction. Faraday Discussions. 2012;31:156-9.
[6] Samsom ML, Morrison S, Masala N, Sullivan BD, Sullivan DA, Sheardown H and Schmidt TA. Characterization of full-length recombinant human proteoglycan 4 as an ocular surface boundary lubricant. Experimental Eye Research. 2014;127:14-9.
[7] Zeng Y, Yang J, Huang K, Lee Z and Lee X. A comparison of biomechanical properties between human and porcine cornea. Journal of Biomechanics. 2001;34:533-7.
[8] Faber C, Scherfig E and Prause JU. Corneal thickness in pigs measured by ultrasound pachymetry in vivo. Scandinavian Journal of Laboratory Animal Science. 2008;35:39-43. [9] Hara H and Cooper DKC. Xenotransplantation-the future of corneal transplantation? Cornea. 2011;30:371-8.
[10] Henker R, Scholz M, Gaffling S, Asano N, Hampel U, Garreis F and Paulsen F. Morphological features of the porcine lacrimal gland and its compatibility for human lacrimal gland xenografting. Plos One. 2013;8:e74046.
[11] Gensheimer WG, Kleinman DM, Gonzalez MO, Sobti D, Cooper ER, Smits G and Aquavella JV. Novel formulation of glycerin 1% artificial tears extends tear film break-up time compared with Systane lubricant eye drops. Journal Ocular Pharmacology and Therapeutics. 2012;28:473-8.
