We describe a simple and efficient method for isolating cells of the retinal pigment epithelium (RPE) cells from the eyes of young pigmented guinea pigs. This procedure allows for follow-up molecular biology studies on the isolated RPE, including gene expression analyses.
This protocol describes the isolation of cells of the retinal pigment epithelium (RPE) from the eyes of young pigmented guinea pigs for potential application in molecular biology studies, including gene expression analyses. In the context of eye growth regulation and myopia, the RPE likely plays a role as a cellular relay for growth modulatory signals, as it is located between the retina and the two walls of the eye, such as the choroid and sclera. While protocols for isolating the RPE have been developed for both chicks and mice, these protocols have proven not to be directly translatable to the guinea pig, which has become an important and widely used mammalian myopia model. In this study, molecular biology tools were used to examine the expression of specific genes to confirm that the samples were free of contamination from the adjacent tissues. The value of this protocol has already been demonstrated in an RNA-Seq study of RPE from young pigmented guinea pigs exposed to myopia-inducing optical defocus. Beyond eye growth regulation, this protocol has other potential applications in studies of retinal diseases, including myopic maculopathy, one of the leading causes of blindness in myopes, in which the RPE has been implicated. The main advantage of this technique is that it is relatively simple and once perfected, yields high-quality RPE samples suitable for molecular biology studies, including RNA analysis.
The RPE comprises a unique monolayer of pigmented cells located between the neural retina and the vascular choroid, and the RPE has well-recognized roles in the development and maintenance of normal retinal function, including phototransduction1,2. More recently, the RPE has been assigned an additional key role in eye growth regulation3 and, thus, the development of myopia4. This assignment is based on the RPE's critical location, interposed between the retina and choroid and the now broad acceptance that eye growth and, thus, refractive errors are regulated locally5. The RPE is believed to play a key role as a signal relay, linking the retina, the assumed source of growth modulatory signals, to the choroid and sclera, the two targets of the relayed signals6,7,8.
The increase in axial length that characterizes most myopia cannot be considered benign, with pathophysiological changes involving the retina, choroid, and/or sclera representing unavoidable and now well-recognized consequences of excessive ocular elongation7,9. In this context, the RPE is perhaps the most vulnerable, since, being a nonmitotic tissue, it is only able to accommodate the expanding vitreous chamber by the stretching and thinning of individual cells. While its role in myopia-related pathologies, such as myopic macular degeneration, is yet to be fully understood, the RPE has been implicated in the pathogenesis of a number of other retinal diseases, including geographic atrophy, one of the leading causes of blindness, which is associated with documented abnormalities in the retina, RPE, and choroid10,11,12.
The successful isolation of RPE cells, free from contamination from adjacent ocular tissues, potentially opens up many research opportunities to gain new insights into the mechanisms underlying a variety of eye/retinal diseases. However, the isolation of the RPE has proven challenging, with many published studies making use of combined retina/RPE or RPE/choroid samples for this reason13,14,15. Studies involving the successful isolation of the RPE of a quality suitable for molecular biology studies has been limited to chick and mouse eyes16,17. For example, the simultaneous RPE isolation and RNA stabilization (SRIRS) method described by Wang et al.18. To isolate RPE cells in mice does not appear to work well in guinea pig eyes. The protocol described here represents a refinement of an approach that was initially prototyped with tree shrew eyes by one of the authors (M.F.) and has proven to yield high-quality RPE samples, appropriate for RNA and other molecular biology analyses, from the eyes of young pigmented guinea pigs19.
All animal care and treatments used in this study conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The experimental protocols were approved by the Animal Care and Use Committee of the University of California, Berkeley.
1. Enucleation of the guinea pig eye
2. Isolation of the ocular posterior eye cup and RPE/choroid/sclera complex
3. Isolation of the RPE from the choroid
4. RPE-RNA extraction
The analysis of the RPE samples collected using the above protocol showed well-preserved RNA (RIN >8.0, Figure 2B), with 240.2 ng ± 35.1 ng per eye (n = 8, NanoDrop, Figure 2B). To further evaluate the quality of the isolated RPE samples, particularly the absence of choroidal and scleral contaminants, we examined the expression of representative genes for each of the latter tissues in the RPE samples19. The RPE samples demonstrated a significantly higher expression of Rpe65 (an RPE-specific gene) compared with the Rpe65 expression levels in the choroid and sclera (Table 1 and Figure 2C). In contrast, the RPE samples showed minimal expression of Col1a1, the selected choroid-sclera-specific gene (Figure 2D).
Figure 1: Procedure for collecting the RPE sheets. (A) A 2-week-old guinea pig eye with the first incision. (B) The anterior segment (cornea, iris, and lens), vitreous,and retina are then separated from the posterior eye cup (RPE, choroid, and sclera). (C) A jet stream of PBS, delivered via a 30 G needle, is used detach the RPE (D) as a sheet (black arrows) from the choroid. The procedure from the first incision to the RPE collection takes 5-7 min. Abbreviation: RPE = retinal pigment epithelium. Please click here to view a larger version of this figure.
Figure 2: Procedure for assessing the quality of the isolated RPE samples. (A) Representative image of an RPE sample in a 1.5 mL microcentrifuge tube after being spun down. (B) Representative bioanalyzer output for RNA extracted from the collected RPE samples. (C,D) The gene expression levels of Rpe65, an RPE-specific gene, and Col1a1, which is not or minimally expressed in the RPE, measured by RT-qPCR in the RPE, choroid, and scleral samples from n = 3 untreated animals; both data sets were normalized to β-actin. ** P < 0.01; *** P < 0.001. This figure has been modified from Goto et al.19. Abbreviations: RPE = retinal pigment epithelium; β-actin = beta-actin; Col1a1 = collagen type-I alpha-1 chain; Rpe65 = retinoid isomerohydrolase; RT-qPCR = reverse-transcription quantitative polymerase chain reaction. Please click here to view a larger version of this figure.
Gene | Forward Primer (5' to 3') | Reverse Primer (5' to 3') | ||||
Col1a1 | GCCTCAGGCAAGACAGTCATT | GCTAACGGTAAAGCCGAATTCC | ||||
Rpe65 | GCCCTTCTGCACAAGTTTGAC | CAGTGCGGATGAACCTTCTGT | ||||
β-actin | GGCCGAGCGGGAAATT | CCAGGGCAACATAGCATAGCTT |
Table 1: Nucleotide sequences of the primers used for PCR amplification in the sample quality analysis. Abbreviations: β-actin = Beta-actin; Col1a1 = collagen type I alpha 1 chain; Rpe65 = retinoid isomerohydrolase.
In this article, we describe a method for isolating RPE, appropriate for RPE gene expression analyses, from the eyes of young, pigmented guinea pigs. The merits of this protocol are that it yields high-quality RPE samples that are relatively free from contamination, with RNA suitably preserved for RNA-specific analyses and, yet, is relatively simple and efficient. While in the example provided here, the RPE samples were collected from the eyes of a young (2 weeks old) guinea pig, the protocol has also been used successfully to collect RPE samples from older (young adult) animals.
For researchers with minimal prior experience with ocular surgery or dissection, protocol step 2.1 and step 2.2 can be challenging. The critical detail in step 2.1 is the location of the initial incision in the sclera, which should be accurately placed 1.0 mm behind the limbus so that the iris and lens are removed together with the cornea when detaching the anterior segment. If, instead, the iris remains attached to the posterior ocular segment, it is challenging to find the zonule of Zinn, which is key to successfully detaching the retina in the next step. As noted in the protocol, for the successful detachment of the retina, it is also critical that the retina not be grasped directly with the forceps to avoid its fragmentation. The guinea pig retina appears more fragile than the mouse retina, likely because of its avascular nature20. Ideally, the isolated posterior eye cup, comprising the RPE, choroid, and sclera, should be immersed in tissue storage reagent within 5 min of eye enucleation to ensure the adequate preservation of RNA in the collected RPE sample.
The SRIRS method, which was developed for the specific purpose of collecting high-quality samples of RPE from the eyes of mice, appears to have achieved that goal for mouse eyes; it is reported to be both efficient and effective18. This technique has also been successfully used to collect RPE from healthy human donor eyes21. However, based on the authors' experience, this SRIRS method is not suitable for collecting RPE from the eyes of the guinea pig, tree shrew, and opossum, although the underlying reasons for this are not clear. By reporting the technique described here to isolate and collect RPE from the eyes of young guinea pigs, we hope to address an important unmet need in the myopia research field.
In terms of the limitations of the protocol described, the main one is the need for a period of hands-on training to ensure the efficient collection of samples, as the time to completion is key, in addition to the purity of the collected RPE samples. Researchers who have no experience with ocular micro-dissection or surgery will be most in need of training. Although several RPE isolation methods have been reported22,23, the method as described here is not suitable for RPE cell cultures or protein analyses due to the use of an RNA stabilizing reagent.
The RPE has long been recognized to play critical roles not only in maintaining the health and function of the retina but also in related diseases. The fact that the RPE is now also recognized to play a key role in ocular growth regulation and myopia development has considerably broadened the scope of research questions, for which the ability to collect high-quality RPE samples from either the eyes of guinea pigs, as described here, or other mammals used as animal models may be key to providing new insights. Such studies may also offer new insights into the pathological complications of myopia, including myopic maculopathy, for which the prevalence figures can be expected to rise in parallel with myopia prevalence figures per se.
The authors have nothing to disclose.
This study is supported by the Japan Society for the Promotion of Science Overseas Research Fellowships (S.G.), a Loris and David Rich Postdoctoral Scholar (S.G.), and a grant from the National Eye Institute of the National Institutes of Health (R01EY012392; C.F.W.).
1 mL Syringe with Slip Tip | Bd Vacutainer Labware Medical | 22-253-260 | |
2-Mercaptoethanol | Invitrogen | 21985-023 | |
6 Well Tissue Culture Plate with Lid, Flat Bottom, Sterile | pectrum Chemical Mfg. Corp | 970-95008 | |
12 Well Tissue Culture Plate with Lid, Individual, Sterile | Thomas Scientific LLC | 1198D72 | |
Agilent 2100 Bioanalyzer | automated electrophoresis to check RNA quality | ||
Balanced Salt Solutions | Gibco | 10010031 | |
Bonn Micro Forceps, Straight Smooth, 0.3 mm Tip, 7 cm | Fine Science Tools, Inc. | 11083-07 | |
Dumont forceps no. 5 | ROBOZ | RS-5045 | |
Hypodermic disposable needles | Exelint International, Co. | 26419 | |
Hypodermic disposable needles | Exelint International, Co. | 26437 | |
MiniSpin Microcentrifuges | Eppendorf | 540108 | Max. Speed: 8,000 g |
RNAlater Stabilization Solution | Invitrogen | AM7020 | tissue storage reagent |
RNeasy Mini kits | Qiagen | 74104 | RNA isolation kit |
Student Vannas Spring Scissors | Fine Science Tools, Inc. | 91500-09 |