Abstract
AIM
To investigate the effect of intravitreal injection of DL-alpha-aminoadipic acid (DL-α-AAA) on ocular refractive state and retinal dopamine, transforming growth factor-β2 (TGFβ2), vasoactive intestinal polypeptide (VIP) in guinea pig form-deprived myopia.
METHODS
Four-week-old pigmented guinea pigs were randomly assigned to 4 groups: normal control, deprivation, deprivation plus DL-α-AAA, deprivation plus saline. Form deprivation was induced with the self-made translucent eye shields, and lasted for 14 days. 8µg DL-α-AAA was injected into the vitreous chamber of deprived eyes. The corneal radius of curvature, refraction and axial length were measured. Retinal dopamine content was evaluated by the high-performance liquid chromatography with electrochemical detection, and TGFβ2 and VIP protein were detected by Western blotting.
RESULTS
Fourteen days of eye occlusion caused the axial length to elongate and become myopic in the form-deprived eyes, with the decrease of retinal dopamine and the increase of TGFβ2 and vasoactive intestinal polypeptide (VIP) protein. Intravitreal injection of DL-α-AAA could inhibit the myopic shift from (-3.65±1.06)D to (-1.48±0.63)D, P<0.01 due to goggles occluding and cause the decrease of retinal TGFβ2 protein in the deprived eyes. However, intravitreal injection of DL-α-AAA had no significant effect on retinal dopamine and VIP protein in deprived eyes. Retinal TGFβ2 protein correlated highly with the ocular refraction (y=-3.34+0.31/x, F=74.75, P<0.001) and axial length (y=8.39-0.02/x, F=48.32, P<0.001) in different treatment groups.
CONCLUSION
Intravitreal injection of DL-α-AAA is effectively able to suppress the development of form deprivation myopia, which may be associated with retinal TGFβ2 protein in guinea pigs.
Keywords: DL-alpha-aminoadipic acid, form-deprivation myopia, TGFβ2, Müller cell, retina
INTRODUCTION
Alpha-aminoadipic acid (α-AAA) is a six-carbon chemical analog of the excitatory amino acid, L-glutamic acid. It is well known that D-, L- and DL-isomers have gliotoxic effects in the retina both in vitro [1] and in vivo [2]. With regard to these isomers, D-α-AAA and DL-α-AAA individually exhibit selective damage to retinal Müller cells; however, L-isomer is found to cause additional neuronal injury. In our previous study, it has been shown that the intravitreal DL-α-AAA specifically acts on retinal Müller cells, and effectively ameliorates the development of form-deprived myopia in guinea pig [3]. Subsequently, we revealed that retinal Müller cells in the guinea pig myopic eye can synthesize dopamine, transforming growth factor-β2 (TGFβ2), vasoactive intestinal polypeptide (VIP) [4],[5]. But it is unclear whether the DL-α-AAA inhibition on myopia is associated with these retinal factors. In the present study, we investigate the association between DL-α-AAA inhibitory effect on myopia and retinal dopamine, TGFβ2 and VIP.
MATERIALS AND METHODS
Materials
Healthy triad color guinea pigs (clean grade, n=48, aged 4 weeks, weighed 180-220g) were obtained from the Animal Center of Xiangya Medical College, and were randomly divided into four groups: normal control, deprivation, deprivation plus DL-α-AAA, deprivation plus saline. All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No.86-23, revised 1986). Approval for the project was obtained from the Animal Ethics Committees of Xiangya Medical College.
Guinea pigs were intraperitoneally injected with 30mg/kg sodium pentobarbital. Then, the self-made translucent eye shields were sutured to the tissue surrounding the right eye socket on the right eye. It was observed that occluders did not compromise the cornea, and form-deprived eyes could freely blink behind occluders. Animals were then reared under 12-12 hours light/darkness cycle.
Fourteen days after occlusion, the occluders were removed. Its corneal radius of curvature was measured with a keratometer (Topcon, OM-4, Japan), and refraction was measured with a streak retinoscope. Type-A ultrasonic examination (Cinescan A/B, Quantel Medical, French) was conducted to determine the axial length. Each animal was measured three times and the mean value was used for the following analysis.
Methods
DL-α-AAA (Sigma, St. Louis, MO, USA) was freshly prepared by dissolving into physiological saline solution. At the first day and the seventh day of occlusion, guinea pigs in the deprivation plus DL-α-AAA group were intravitreally injected with 5µL DL-α-AAA solution contained 8µg DL-α-AAA using a 25µL Hamilton syringe. As a control, physiological saline solution (vehicle) was injected in the same way. In the current study, the dosage of DL-α-AAA was chosen on the basis of previous work in guinea pigs [3].
Retinal dopamine content was detected by the high-performance liquid chromatography (HPLC) [4] and was plotted as nanogram dopamine per milligram retina.
Retinal samples for western blotting detection were frozen in liquid nitrogen. The blot was probed with a polyclonal antibody against rabbit TGFβ2 and VIP (Santa Cruz, USA), working concentration 1:500. Target strap is performed grey value analysis by Bandscan 5.0 image analysis software, GAPDH as the internal control; the relative expression of target protein is calculated.
Statistical Analysis
All data was expressed as mean±SD, and analyzed with SPSS11.5 software. One-way ANOVA was used to analyze ocular refraction, axial length, corneal radius of curvature, and the content of retinal DA, TGFβ2, and VIP. The correlation in axial length, refraction and retinal TGFβ2 was analyzed by the regression of an inverse function.
Results
Effect of DL-α-AAA on Ocular Refractive State
Fourteen days after occlusion, the deprived eyes became myopic with a significant increase in its axial length, which showed statistically significant difference when compared with its fellow control eyes and age-matched normal control eyes (P<0.01). Intravitreal injection of DL-α-AAA significantly reduced the degree of myopia (from -3.65±1.06 D to -1.48±0.63 D, P<0.01) and retarded the increase of axial length (from 8.41±0.09mm to 8.30±0.06mm, P<0.01) in the deprived eyes. However, the myopia development was not suppressed completely by 8µg DL-α-AAA treatment, because its refraction showed statistically significant difference when compared with its fellow control eyes (P<0.01) and normal control eyes (P<0.01). In contrast, intravitreal injection of saline (vehicle) caused no statistically significant effect on the refraction in the deprived eyes. No statistically significant difference was observed in corneal radius of curvature in different treatment groups (Table 1).
Table 1. Effect of DL-α-AAA on refraction, axial length and corneal radius of curvature.
| Groups | Corneal curvature(mm) | Refraction (D) | Axial length (mm) |
| Normal control | |||
| Right | 3.57±0.03 | +0.80±0.25 | 8.16±0.05 |
| Left | 3.58±0.02 | +0.83±0.25 | 8.15±0.04 |
| Deprivation | |||
| Right | 3.59±0.02 | -3.65±1.06b,d,f | 8.41±0.09b,d,f |
| Left | 3.57±0.03 | +0.86±0.20 | 8.17±0.06 |
| Deprivation plus DL-α-AAA | |||
| Right | 3.58±0.04 | -1.48±0.63b,d | 8.30±0.06b,d |
| Left | 3.58±0.01 | +0.86±0.22 | 8.15±0.05 |
| Deprivation plus saline | |||
| Right | 3.57±0.04 | -3.55±1.12b,d,f | 8.40±0.08b,d,f |
| Left | 3.59±0.03 | +0.84±0.25 | 8.16±0.06 |
Data are expressed as the means±SD; bP<0.01 vs the left eye; dP<0.01 vs the normal control group; fP<0.01 vs the deprivation plus DL-α-AAA group.
(n=12)
Effect of DL-α-AAA on Retinal Dopamine, TGFβ2 and VIP
Fourteen days of form-deprivation induced a significant decrease in retinal dopamine content of the deprived eyes when compared with fellow control eyes (P<0.01) and age-matched normal control eyes (P<0.01). However, 14 days of form-deprivation induced a significant increase in retinal TGFβ2 and VIP protein (P<0.01). Intravitreal injection of DL-α-AAA significantly caused the decrease of retinal TGFβ2 protein in the deprived eyes, when compared with those in the deprivation group (P<0.01). There was no statistically significant difference in retinal dopamine content and VIP protein of the deprived eyes between the deprivation group and the deprivation plus DL-α-AAA group. In contrast, intravitreal injection of saline (vehicle) caused no statistically significant effect on retinal dopamine content, TGFβ2 and VIP protein in the deprived eyes (Table 2, Figure 1).
Table 2. Effect of DL-α-AAA on retinal dopamine, TGFβ2 and VIP.
| Groups | Dopamine(ng) | TGFβ2 | VIP |
| Normal control | 1.52±0.57 | 0.09±0.03 | 0.14±0.05 |
| Deprivation | 0.65±0.15b | 0.69±0.13b,d | 0.47±0.09b |
| Deprivation plus DL-α-AAA | 0.70±0.17b | 0.43±0.10b,d | 0.45±0.11b |
| Deprivation plus saline | 0.67±0.18b | 0.71±0.15 b,d | 0.49±0.15b |
Data are expressed as the means ± SEM; bP<0.01 vs the normal control; dP<0.01 vs the deprivation plus DL-α-AAA group.
(n=12)
Figure 1. Western blotting technique detection of retinal TGFβ2 and VIP protein in the guinea pig.
1,2: normal control; 3,4: deprivation; 5,6: deprivation plus DL-α-AAA; 7,8: deprivation plus saline.
The ocular refraction correlated highly with retinal TGFβ2 protein in different treatment groups (y=-3.34+0.31/x, R2=0.62, DF=46, F=74.75, P<0.001). The axial length and retinal TGFβ2 protein were also correlated in different treatment groups (y=8.39-0.02/x, R2=0.51, DF=46, F=48.32, P<0.001) (Figure 2).
Figure 2. A: Relation between the ocular refraction and retinal TGFβ2 protein in different treatment groups (y=-3.34+0.31/x, F=74.75, P<0.001); B: Relation between the axial length and retinal TGFβ2 protein in different treatment groups (y=8.39-0.02/x, F=48.32, P<0.001).
DISCUSSION
Myopia is a visual disorder affecting about one half of the world's population. It is also a socio-economic-health problem of considerable proportions. Its prevalence shows a rising tendency, especially in Asian countries such as China [6],[7], Japan [8],[9] and Singapore [10],[11]. However, there is no generally accepted and approved method or drug for preventing myopia.
Müller cells are the predominant class of glial cells in the vertebrate retina. They span the entire width of retina from the inner to the outer limitant membrance, and its processes surround most of retinal neurons. Together with retinal neurons, they constitute a “neuron-glia” regulatory network that is involved in the retinal functional activities, including supporting and nourishing neurons, maintaining the stability of extracellular ion concentration, and participating in glutamate cycle and synaptic signal transduction, etc [12]. It has been shown that Müller cells may participate in the formation of myopia in the guinea pig, and it is a significant source for signaling factors of TGFβ2 in the retina [3],[5].
Seko et al [13] has reported that the content of TGFβ2 is increased in the posterior pole of retina-retinal pigment epithelium-choroid complex and sclera of the deprived eyes in chicken, suggesting that TGFβ2 participate in the development of myopia. However, Honda et al [14] has reported that TGFβ2 protein are reduced in retina of the deprived eyes in chicken. In addition, Jobling et al [15] has found that form deprivation in the early stage has no significant effect on TGFβ2 content in retina, and TGFβ2 content begins to decrease on day five in the tree shrew. In our study, form deprivation induced a significant increase in retinal TGFβ2 protein of the deprived eyes in the guinea pig. Together these results suggest that there are the controversial results of retinal TGFβ2 in the form deprivation myopia, which may be associated with the species differences. So, the exact relation between retinal TGFβ2 and myopia remains to be verified by further studies.
DL-α-AAA has gliotoxic effects in the retina and exhibits selective damage to retinal müller cells [16]. In the present study, we found that intravitreal injection of DL-α-AAA significantly reduced the degree of myopia and retinal TGFβ2 protein in the deprived eyes, and retinal TGFβ2 protein correlated highly with the ocular refraction and axial length. These evidences indicate that retinal TGFβ2 protein may participate in the inhibitory effect of DL-α-AAA on myopia in guinea pig. Although retinal müller cells is also a significant source for retinal dopamine and VIP, intravitreal injection of DL-α-AAA caused no significant change of retinal dopamine and VIP in the guinea pig myopic eyes.
In summary, our data showed that intravitreal injection of DL-α-AAA could effectively retard the myopic development in the form-deprived guinea pig eyes, which may be associated with the change of retinal TGFβ2 protein.
Footnotes
Foundation item: National Natural Science Foundation of China (No. 30600694)
REFERENCES
- 1.Reichelt W, Stabel-Burow J, Pannicke T, Weichert H, Heinemann U. The glutathione level of retinal Müller glial cells is dependent on the high-affinity sodium-dependent uptake of glutamate. Neuroscience. 1997;77(4):1213–1224. doi: 10.1016/s0306-4522(96)00509-x. [DOI] [PubMed] [Google Scholar]
- 2.Wang X, Iannaccone A, Jablonski MM. Contribution of müller cells toward the regulation of photoreceptor outer segment assembly. Neuron Glia Biol. 2004;1(3):291–296. doi: 10.1017/S1740925X05000049. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Mao JF, Liu SZ, Qin WJ, Li FY, Tan Q, Wu XY. The modulation effect of retinal Müller cells on form deprivation myopia in guinea pig. Chin Ophthal Res. 2008;26(12):881–884. [Google Scholar]
- 4.Mao JF, Liu SZ, Qin WJ, Li FY, Wu XY, Tan Q. Levodopa Inhibits the Development of Form-Deprivation Myopia in Guinea Pigs. Optom Vis Sci. 2010;87(1):53–60. doi: 10.1097/OPX.0b013e3181c12b3d. [DOI] [PubMed] [Google Scholar]
- 5.Mao JF, Liu SZ, Qin WJ, Xiang Q. Modulation of TGFβ2 and dopamine by PKC in retinal Müller cells of guinea pig myopic eye. Int J Ophthalmol. 2011;4(4):357–360. doi: 10.3980/j.issn.2222-3959.2011.04.06. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.He MG, Lin Z, Huang J, Lu Y, Wu CF, Xu JJ. Population-based survey of refractive error in school-aged children in Liwan District, Guangzhou. Zhonghua Yan Ke Za Zhi. 2008;44(6):491–496. [PubMed] [Google Scholar]
- 7.Cheng CY, Hsu WM, Liu JH, Tsai SY, Chou P. Refractive errors in an elderly Chinese population in Taiwan: the Shihpai Eye study. Invest Ophthalmol Vis Sci. 2003;44(11):4630–4638. doi: 10.1167/iovs.03-0169. [DOI] [PubMed] [Google Scholar]
- 8.Sawada A, Tomidokoro A, Araie M, Iwase A, Yamamoto T. Refractive errors in an elderly Japanese population: the Tajimi study. Ophthalmology. 2008;115(2):363–370. doi: 10.1016/j.ophtha.2007.03.075. [DOI] [PubMed] [Google Scholar]
- 9.Matsumura H, Hirai H. Prevalence of myopia and refractive changes in students from 3 to 17 years of age. Surr Ophthalmol. 1999;44(suppl.1):s109–115. doi: 10.1016/s0039-6257(99)00094-6. [DOI] [PubMed] [Google Scholar]
- 10.Wu HM, Seet B, Yap EP, Saw SM, Lim TH, Chia KS. Does education explain ethic differences in myopia prevalence? A population-based study of young adult males in Singapore. Optom Vis Sci. 2001;78(4):234–239. doi: 10.1097/00006324-200104000-00012. [DOI] [PubMed] [Google Scholar]
- 11.Saw SM, Goh PP, Cheng A, Shankar A, Tan DT, Ellwein LB. Ethnicity-specific prevalences of refractive errors vary in Asian children in neighbouring Malaysia and Singapore. Br J Ophthalmol. 2006;90(10):1230–1235. doi: 10.1136/bjo.2006.093450. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Newman E, Reichenbach A. The Müller cell: a functional element of the retina. Trends Neurosci. 1996;19(8):307–312. doi: 10.1016/0166-2236(96)10040-0. [DOI] [PubMed] [Google Scholar]
- 13.Seko Y, Shimokawa H, Tokoro T. Expression of bFGF and TGF-beta 2 in experimental myopia in chicks. Invest Ophthalmol Vis Sci. 1995;36(6):1183–1187. [PubMed] [Google Scholar]
- 14.Honda S, Fujii S, Sekiya Y, Yamamoto M. Retinal control on the axial length mediated by transforming growth factor-beta in chick eye. Invest Ophthalmol Vis Sci. 1996;37(12):2519–2526. [PubMed] [Google Scholar]
- 15.Jobling AI, Wan R, Gentle A. Retinal and choroidal TGF-beta in the tree shrew model of myopia: isoform expression, activation and effects on function. Exp Eye Res. 2009;88(3):458–466. doi: 10.1016/j.exer.2008.10.022. [DOI] [PubMed] [Google Scholar]
- 16.Kato S, Ishita S, Sugawara K, Mawatari K. Cystine/glutamate antiporter expression in retinal Müller glial cells: implications for DL-alpha-aminoadipate toxicity. Neuroscience. 1993;57(2):473–482. doi: 10.1016/0306-4522(93)90080-y. [DOI] [PubMed] [Google Scholar]