Ruozhong Xie

Normal development of refractive state and ocular dimensions in guinea pigs

PURPOSE This study investigated changes in refraction, corneal curvature, axial components and weight of posterior sclera in guinea pig eyes during the normal development from birth. METHODS Sixty-four guinea pigs were assigned to eight groups (n=8 each). Each group underwent a series of ocular measurements at one of the eight time-points (0, 1, 2, 3, 5, 7, 9 and 11 weeks), including refraction (streak retinoscopy), corneal radius of curvature (CRC; keratometry), anterior segment length (AS: corneal thickness and depth of the anterior chamber), thickness of the crystalline lens (CL), vitreous chamber length (VC; all A-scan ultrasonography) and dry weight of a circular 6mm diameter punch in the posterior sclera (electronic balance). Results of all the measurements were statistically compared between right eye and left eye, male and female and among different age groups. Artifacts of retinoscopy due to small eye artifact were also estimated at different ages. RESULTS The refraction in guinea pig eyes was +5.22+/-0.23 D (Mean, SE) at birth. This value decreased rapidly during the first 3 weeks followed by a slow decline. The overall decrease in refraction was highly significant from birth to 11 weeks (p<0.001 one way ANOVA). The small eye artifact was approximately 4.00 D at birth, which reduced to 2.76 D at 11 weeks. The guinea pig eyes were emmetropic by 3 weeks of age when the small eye artifact was taken into account. The CRC (3.24+/-0.01 mm at birth), AS (1.20+/-0.01 mm at birth), CL (2.72+/-0.03 mm at birth) and VC (3.28+/-0.01 mm at birth) increased within the first 3 weeks despite a transient decrease in the CRC within the first week. The increase in CRC, CL and VC continued after 3 weeks, however, the AS remained constant after this age. The increase in VC was better correlated to the decline of hyperopia (R(2)=0.70) than the other components (R(2)=0.33-0.39). Dry weight of the posterior sclera increased linearly from birth (p<0.001 between any two close time-points from 3 to 9 weeks) and had a moderately linear correlation with the VC (R(2)=0.60). There were no significant differences between the right eye and left eye or between male and female in all the measurements. CONCLUSIONS In guinea pigs, the hyperopia present at birth rapidly reduces to emmetropia within the first 3 weeks of age. The emmetropization process in guinea pigs is mainly related to the increase in the vitreous chamber length. This relationship in guinea pigs is similar to that in chickens, tree shrews, primates and humans. The axial development of the vitreous chamber in guinea pigs appears to be associated with tissue growth of the posterior sclera.

Correlation between myopia and major biometric parameters of the eye: a retrospective clinical study.

Purpose. To investigate the relationship between myopia and changes in ocular biometry and macular thickness in young adults. Methods. Two hundred sixteen eyes from 108 adults (23.3 ± 6.3 years old, mean ± SD) were measured for refractive status, corneal curvature, and axial components of the eye. Macular thickness was measured in 118 eyes (59 subjects) by optical coherence tomography. All eyes were categorized into emmetropia, low, moderate, or high myopia based on the refractive status. Biometric results from right eyes of all subjects were compared between sub-groups with the linear correlation analyzed between refraction and other parameters for each group. Results. The vitreous chamber depth was longest in high myopia, followed by the moderate myopia group, the low myopia group and finally the emmetropic group (p ≤ 0.004). Average thickness of the inner and outer ring macula in all the myopia groups was significantly thinner than in the emmetropia group (p ≤ 0.021). Among different macular regions, the inferior quadrant of the outer ring was consistently the thinnest in myopia. Corneal curvature, anterior chamber depth, and lens thickness measures were not associated with myopia. Conclusions. Myopia in young adults is associated with an increase in vitreous length and a decrease in para-foveal thickness. The thinness in the retinal region inferior to the fovea appears to be more highly correlated with myopia than any other retinal region.

The presence of m1 to m5 receptors in human sclera: evidence of the sclera as a potential site of action for muscarinic receptor antagonists.

Purpose: The aim of this study was to identify the presence of muscarinic acetylcholine receptors (mAChRs) in human sclera in order to determine whether the sclera is a potential site of action for mAChR antagonists. Methods: Cell lines of human scleral fibroblasts were cultured in Dulbecco modified Ealges medium. Reverse transcription–polymerase chain reaction (RT-PCR) and Northern blot analysis were used to detect mRNA expression of muscarinic acetylcholine receptors in the cell lines of the fibroblasts. Western blot analysis and immunocytochemistry were used to detect proteins of mAChRs in the cell lines. Immunohistochemical study was used to further detect the presence of mAChR proteins in the frozen scleral sections. Results: The cultured fibroblasts demonstrated mRNA expression of five mAChRs (m1 to m5) in RT-PCR and Northern blot analysis. The molecular size of mRNA expression was largest for the m3 receptor, followed by the m2, m4, m5, and m1 in both RT-PCR and Northern blot analysis. Proteins of the m1 to m5 receptors were present in cell line fibroblasts under Western blot analysis and immunocytochemistry with a range of molecular weight from 80 kDa (m5) to 60 kDa (m1) in Western blot analysis. The presence of these five receptors was also detected in scleral tissues with immunohistochemistry. Conclusions: This study demonstrated the presence of mAChR subtypes (m1 to m5) in human scleral fibroblasts at both mRNA and protein levels. This finding indicates that the sclera is a potential site of action for the currently used mAChR antagonists in prevention of human myopia.

Axial myopia induced by hyperopic defocus in guinea pigs: A detailed assessment on susceptibility and recovery

This study investigated whether adolescent guinea pigs can develop myopia induced by negative lenses, and whether they can recover from the induced myopia. Forty-nine pigmented guinea pigs (age of 3 weeks) were randomly assigned to 4 groups: 2-week defocus (n=16), 4-week defocus (n=9), 2-week control (n=15) and 4-week control (n=9). A -4.00D lens was worn in the defocus groups and a plano lens worn in the control groups monocularly. The lenses were worn from 3 weeks to 5 weeks of age in the 2-week treatment groups with the biometry measured at 2, 4, 6, 10 and 14 days of lens wear. The lenses were worn from 3 weeks to 7 weeks of age in the 4-week treatment groups with the biometry measured immediately and at 2, 4, 6, 10 and 14 days after lens removal. Refractions in the defocused eyes developed towards myopia rapidly within 2 days of lens wear, followed by a slower development. The defocused eyes were at least 3.00D more myopic with a greater increase in vitreous length by 0.08 mm compared to the fellow eyes at 14 days (p<0.05). The estimated choroidal thickness of the defocused eyes decreased rapidly within 2 days of lens wear, followed by a slower decrease over the next 4 days. Relative myopia induced by 4 weeks of negative-lens treatment declined rapidly following lens removal. A complete recovery occurred 14 days after lens removal when compared to the fellow controls. The refractive changes during the recovery corresponded to a slower vitreous lengthening and a rapid thickening of the choroid. The plano-lens wearing eyes showed a slight but significant myopic shift (<-0.80D) with no associated biometrical changes. Guinea pigs aged 3 weeks can still develop negative lens induced myopia and this myopia is reversible after removal of the lens. The myopia and recovery are mainly due to changes in vitreous length and choroidal thickness.

Recovery from axial myopia induced by a monocularly deprived facemask in adolescent (7-week-old) guinea pigs

PURPOSE Guinea pigs have been increasingly used as an animal model for experimental myopia. Infant guinea pigs are susceptible to recovery from myopia within 2 weeks of form deprivation. This study investigated whether adolescent guinea pigs are susceptible to recovery from myopia after a longer period of form deprivation. METHOD Twenty-two guinea pigs (age of 3 weeks) were randomly assigned to two groups: MDF (monocularly deprived facemask, n=11) and normal control (free of form deprivation, n=11). All animals underwent biometric measurement (refraction, corneal curvature and axial length) prior to the experiment. Animals in the MDF group wore a facemask that covered the right eye for 4 weeks. The MDF was then removed and biometric measurement was performed immediately and at 2, 6, 10 and 14 days. The same measurement was performed in the normal control group at time-points matching those of the MDF group. RESULTS The MDF eyes were approximately 4D more myopic with a greater increase in vitreous length by 0.12 mm compared to either the fellow or the normal control eyes after form deprivation (p<0.01). This relative myopia shifted rapidly towards hyperopia within 2 days after removal of the MDF, followed by a more gradual recovery. A complete recovery occurred by 6 days after removal of the MDF compared to the fellow and normal control eyes (p>0.05). Vitreous length in the MDF eyes slightly reduced within 2 days after removal of the MDF and then remained steady. The MDF eyes were similar to both the fellow and normal control eyes in vitreous length (p>0.05) 6 days after removal of the MDF. There was no significant difference between the MDF, fellow and normal control eyes in the other axial components during the form deprivation and recovery period. CONCLUSION Adolescent guinea pigs are susceptible to recovery from MDF-induced myopia. The refractive recovery is mainly correlated to the inhibited axial elongation of the vitreous chamber of the previously deprived eyes.

Activation of dopamine D2 receptor is critical for the development of form-deprivation myopia in the C57BL/6 mouse.

PURPOSE This study used dopamine D2 receptor (D2R) knockout (KO) mice to investigate the role of D2R activity in the development of form-deprivation myopia (FDM). Sulpiride, a D2R antagonist, was administered systemically into wild-type (WT) mice to validate the involvement of D2R in FDM development. METHODS The D2R KO and WT C57BL/6 mice were subjected to FDM. Wild-type mice received daily intraperitoneal injections of sulpiride, 8 μg/g body weight, for a period of 4 weeks. The body weight, refraction, corneal radius of curvature, and ocular axial components were measured at week 4 of the experiment. Differences in all ocular parameters between the experimental and control groups were compared statistically. RESULTS Form-deprivation myopia in D2R KO mice (FD-KO) was significantly reduced compared with their WT littermates (interocular difference, -2.12 ± 0.91 diopter [D] in FD-KO versus -5.35 ± 0.83 D in FD-WT, P = 0.014), with a smaller vitreous chamber depth (0.008 ± 0.006 vs. 0.026 ± 0.006 mm, P = 0.044) and axial length (-0.001 ± 0.007 vs. 0.027 ± 0.008 mm, P = 0.007). Furthermore, FDM was attenuated in animals treated with sulpiride (-2.01 ± 0.31 D in FD-sulpiride versus -4.06 ± 0.30 D in FD-DMSO, P < 0.001) compared with those treated with vehicle, with a retardation in growth of vitreous chamber depth (-0.001 ± 0.006 vs. 0.022 ± 0.004 mm, P = 0.003) and axial length (-0.004 ± 0.007 vs. 0.027 ± 0.005 mm, P = 0.001). CONCLUSIONS Genetic and pharmacological inactivation of D2R attenuates FDM development in mice, suggesting that dopamine acting on D2R appears to promote the development of FDM in C57BL/6 mice. Further studies are required to confirm these results using animal models in which retinal D2R is selectively blocked.

Spontaneous high myopia in one eye will affect the development of form deprivation myopia in the fellow eye.

Purpose: Whether there is an interaction between eyes of individual subjects in refractive development is an important issue to guide experimental designs and help understand mechanisms involved in development of refractive errors. This study investigated whether spontaneous high myopia in one eye will affect refractive development of the fellow eye treated with form deprivation. Methods: Thirty-four guinea pigs were divided into four groups: MD (monocularly form-deprived animals with a pre-treatment anisometropia ≤ 2D, n = 8), anisometropic MD (monocular form deprivation on a relatively hyperopic eye in animals with a pre-treatment anisometropia ≥ 10D, n = 9), normal control (non-form deprivation in animals with a pre-treatment anisometropia ≤ 2D, n = 8), and anisometropic control (non-form deprivation in animals with a pre-treatment anisometropia ≥ 10D, n = 9). All eyes in different groups underwent biometric measurements on days 0, 12, 24, and 36 of the experiment. Results: High myopia in one eye reduced form deprivation myopia in the fellow treated eye. The change in refraction from 0 to 36 days in the deprived eyes was -3.07D for the MD group, but -1.22D for the anisometropic MD group (-3.07D vs. -1.22D: p = 0.009, independent sample t-test). The amount of vitreous chamber lengthening over the same period in the deprived eyes was 0.19 mm for the MD group, but 0.12 mm for the anisometropic MD group (0.19 mm vs. 0.12 mm: p = 0.038, independent sample t-test). Myopic development in the anisometropic animals is mainly inhibited within the first 12 days compared to normal MD animals. Conclusions: These results indicate that an interaction in refractive development may exist temporarily between two eyes of a highly anisometropic animal if the visual environment has been changed.

Feasibility of Two-Dimensional Gel Electrophoresis Used for Proteomic Analysis of Human Scleral Fibroblasts

Purpose: This study used two-dimensional gel electrophoresis (2-DE) to analyze protein profiles for normal human scleral fibroblasts in order to provide a baseline for future study of proteomics of the sclera in experimental conditions. In addition, differences in the presence and amount of proteins from fibroblasts isolated from the anterior or posterior sclera were analyzed. Methods: The fibroblasts from anterior and posterior sclera of two healthy donors were cultured separately. Proteins were extracted from the cell lines, run on 2-DE, and stained by Commassie blue R-250. The gel images were analyzed to detect differences in expression levels (at least a fivefold difference in intensity) and location of the protein spots between the anterior and posterior sclera. These protein spots were trimmed from the gels, digested with trypsin, identified by MALDI mass spectrometry, and functionally categorized with human cDNA and protein databases from NCBI. Results: The number of spots detected was 455 and 453 protein spots from the anterior and posterior scleral fibroblasts, respectively. The patterns of gel maps were very similar between the anterior and posterior sclera in each donor and between the donors in either the anterior or posterior sclera. Nine proteins showed a stronger expression in the anterior sclera compared with the posterior sclera. These proteins together with the two proteins that appeared only in the anterior sclera were mainly associated with anabolic metabolism in cells. Eight proteins showed a stronger expression in the posterior sclera, and seven of them were mainly associated with catabolic metabolism in cells. Among all 19 protein spots identified as being differentially expressed between fibroblasts originally isolated from the anterior or posterior sclera, 14 proteins had a pI (3.86–7.95) and molecular weight (23–66 kDa) consistent with those found in human from the database of NCBI and from SwissProt Entry Name. Conclusions: The distribution and levels of expression in proteins are very similar for both the anterior and posterior sclera in vitro, with only approximately 4% of the proteins demonstrating a differential level of expression (at least fivefold) between the two segments of the sclera.

Effects of muscarinic receptor modulators on ocular biometry of guinea pigs.

This study investigated whether pilocarpine and cyclopentolate induce changes in ocular biometry of guinea pigs, in order to understand if guinea pigs have a similar response to these two agents as humans do.

Interocular evaluation of axial length and retinal thickness in people with myopic anisometropia.

Purpose: To investigate the changes in axial length and retinal thickness and their relationships with myopia in highly myopic anisometropia. Methods: A total of 87 Chinese subjects (25.28±11.98 years, mean±SD) were divided into two groups: anisometropia (n=38) and nonanisometropia (n=49). All eyes were measured for axial length, refractive status, and macular thickness (optical coherence tomography). Ocular biometric results were compared between eyes of subgroups. Linear correlation between refractive error and other biometric results was performed. Results: In the anisometropic group, the inner ring macula and part of the outer ring macula (nasal and inferior quadrants) in the higher myopic eyes were significantly thinner than in the fellow eyes (P⩽0.007), but the foveal thickness (minimum and average) was similar (P≥0.050) between the two eyes. However, the minimum and average foveal thicknesses were found to be significantly thicker in the highly myopic eyes than those in the emmetropic to moderate myopic eyes (P⩽0.016) in the nonanisometropic group. Among the eyes ranging from emmetropia to high myopia, the refractive error was negatively correlated to the axial length of the eye (P<0.001) and the thinning of inner ring macula is consistent with the increase in both myopia and axial length. There was a negative correlation in refractive error and axial length but no correlation in parafoveal thickness between eyes of the same subjects (P<0.001) in the anisometropic group. Conclusions: In people with myopic anisometropia, the higher myopic eye has a longer axial length but a thinner parafoveal region than its fellow eye. The axial growth in the development of high myopia seems to be centrally regulated; however, the changes in parafoveal thickness are likely manipulated by local mechanisms within the eye.