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Retina  |   March 2012
A Novel Experimental Mouse Model of Retinal Detachment: Complete Functional and Histologic Recovery of the Retina
Author Affiliations & Notes
  • Rui Zeng
    From the Eye Hospital, School of Ophthalmology and Optometry, Wenzhou Medical College, Wenzhou, China.
  • Ying Zhang
    From the Eye Hospital, School of Ophthalmology and Optometry, Wenzhou Medical College, Wenzhou, China.
  • Fanjun Shi
    From the Eye Hospital, School of Ophthalmology and Optometry, Wenzhou Medical College, Wenzhou, China.
  • Fansheng Kong
    From the Eye Hospital, School of Ophthalmology and Optometry, Wenzhou Medical College, Wenzhou, China.
  • Corresponding author: Fansheng Kong, Wenzhou Medical College, Xue Yuan Xi lu 270, Wenzhou, People's Republic of China; fanshengkong2003@yahoo.com
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 1685-1695. doi:10.1167/iovs.11-8241
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      Rui Zeng, Ying Zhang, Fanjun Shi, Fansheng Kong; A Novel Experimental Mouse Model of Retinal Detachment: Complete Functional and Histologic Recovery of the Retina. Invest. Ophthalmol. Vis. Sci. 2012;53(3):1685-1695. doi: 10.1167/iovs.11-8241.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To establish an experimental mouse model of retinal detachment (RD) created by corneal puncture (CP).

Methods.: Mouse corneas were punctured with a 30.5-gauge beveled needle, and the anterior chamber was penetrated. Histologic and functional changes of the retina were examined by light microscopy and electroretinography (ERG). Certain retinal cellular responses were examined by immunofluorescence microscopy. Internucleosomal DNA fragmentation in the retina was determined by terminal deoxynucleotidyl transferase–mediated uridine 5′-triphosphate-biotin nick-end labeling (TUNEL).

Results.: CP caused transient leakage of aqueous humor along the needle shaft and immediate formation of multiple retinal blebs, which shrank and flattened within 24 hours. Bleb formation was associated with detachment of the neuroretina from the retinal pigment epithelium (RPE). After CP, the RPE cells underwent extensive transformation during retinal detachment/reattachment, but they resumed normal morphology on retinal reattachment around 10 to 13 days after CP. Relative to pre-CP ERG amplitudes, the punctured eyes showed decreases of 45% and 24% in scotopic and 7% and 12% in photopic b- and a-wave amplitudes, respectively, within 10 to 20 minutes after CP. The ERG amplitudes recovered fully by 12 hours after CP. No infiltrated cells were observed in the subretinal space, and no proliferating or TUNEL-positive cells were observed in the retina of the punctured eyes.

Conclusions.: Puncturing the mouse cornea can create transient RD, and the functional and histologic changes in the retina can subsequently recover. This experimental mouse model of RD mimics human traction and serous RD.

Retinal detachment (RD) is a major cause of vision loss and blindness. Surgical procedures to reduce the mechanical force that pulls the neuroretina away from the RPE and to create effective chorioretinal adhesion are commonly used to treat RD patients. With the improvement and development of microsurgery equipment and techniques, surgical treatment can achieve retinal reattachment in 95% of patients; however, vision recovery is not optimal, with only some of the patients showing significant improvement in postoperative vision. 1,2 To improve vision recovery, studies have sought to understand the biology of RD and to identify the factors involved in functional recovery, leading to more effective therapies. In this context, animal RD models have been valuable for the study of the pathologic progression of RD and for testing new methods and therapies. 
Many studies have used the mouse as a model to study retinal detachment/reattachment. Although mouse eyes are significantly different from human eyes (they are smaller, have fewer cones, and lack a macula), they have many advantages in the study of retinal diseases. Mice are easy to breed and handle, making them a convenient tool for the study of human eye diseases. Recently, RD created in mice by subretinal injection has drawn the attention of retinal gene therapy researchers. During the past decade, many retina-related mutants have been found or engineered in the mouse, and mouse congenital retinal disease models have been used for preclinical study to test the efficacy of gene therapy for human inherited retinal diseases. 3 The most effective way of infecting RPE cells and photoreceptor cells is vector injection through the subretinal space. 4 6 One disadvantage of subretinal injection, in mice or humans, is the formation of an RD, which may damage the retinal cells and jeopardize the efficacy of the therapy delivered via subretinal injection. 7 Improved understanding of RD in the mouse can help identify the iatrogenic factors behind human RD. 
Most existing animal RD models are based on directly manipulating the retina, including manually peeling off the neuroretina from the RPE and subretinal injection of aqueous media, such as saline solution, balanced salt solution, or hyaluronate, to simulate the pathologic manifestations of human RD. 8 14 In this study, RD was induced in mice by puncturing through the cornea without directly manipulating the retina by either mechanical or liquid means. The RD induced by corneal puncture (CP) fully recovered at both the histologic and functional levels. 
Materials and Methods
Animals
C57BL/6J mice were housed and bred at the Wenzhou Medical College's animal facility under a 12-hour light/12-hour dark cycle. All animal experiments were conducted under IACUC-approved protocols in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the rules approved by national and local institutions. One hundred thirty 6- to 8-week-old C57BL/6J mice were used in the study. 
Creating Retinal Detachment via CP
The adult C57BL/6J mice were anesthetized by intraperitoneal injection of ketamine (72 mg/kg) and xylazine (4 mg/kg). Before and after anesthetization, 0.5% tropicamide/0.5% phenylephrine hydrochloride eye drops were topically applied to the cornea to dilate the pupil. After the pupil was dilated, a drop of 2.5% methylcellulose was topically applied to the cornea to serve as a magnifying lens to visualize the fundus through a microsurgery microscope. 
The corneal-punctured right eye was the experimental eye, and the left eye without CP was the control eye. Pupil dilation was applied to the experimental and control eyes simultaneously. 
The procedure used to create RD in the mouse was similar to parts of previously described protocols for rat and mouse subretinal injection. 5,14 16 Briefly, under direct observation aided by a microsurgery microscope (OPMI Pico; Carl Zeiss Meditec, Oberkochen, Germany), a full-thickness CP was made by gently piercing the cornea and entering the anterior chamber with a 30.5-gauge disposable needle, leading to rapid outflow of aqueous humor along the shaft of the needle. Care was taken to avoid injuring the iris and lens during entry into the anterior chamber. Immediately after corneal piercing, the fundus was lifted up and formed multiple retinal blebs around the retinal papillae. Occasionally, if retinal uplift was not apparent after the cornea was pierced, it was achieved by using the needle to gently lift the edge of the corneal opening made by the puncture to let more aqueous humor flow out. Small amounts of 5% atropine, neomycin, and dexamethasone ophthalmic ointments were applied to the eye after CP to prevent related inflammation, cornea–iris adhesion, and bacterial infection. 
Observation and Photography of Anterior Segment and Fundus of the Punctured Eye
The anterior segment and the fundus of live eyes or in eyecups made of punctured eyes were visualized under the microsurgery microscope. A clearer image of the anterior segment and the fundus of live eyes were obtained by administering a drop of methylcellulose to the cornea, forming a relatively stable magnifier. After the desired area was in focus under a microsurgery microscope, the zoom lens of a digital camera (Power Shot SD600; Canon Inc., Tokyo, Japan) was placed on the surface of one of the ocular lenses of the microsurgery microscope, and a photograph of the fundus was taken. 
Histology, Nuclei Staining, Immunofluorescence Staining, and TUNEL
Mouse eyes were enucleated and fixed with 10% formaldehyde in phosphate-buffered saline for histology or with 4% paraformaldehyde in phosphate-buffered saline for immunofluorescence staining and TUNEL. Eyecups were made from fixed eyes by gently removing the cornea and lens, with care taken to avoid pressuring the eye tissue beyond the corneal limbus. 
For histology, the fixed eyecups were dehydrated in a graded series of ethanols, embedded in paraffin, and sectioned at 5 μm. The sections were deparaffinized, stained with hematoxylin and eosin (HE), and examined and photographed with a photomicroscope (Axiophot; Carl Zeiss Meditec). 
For immunofluorescence staining and TUNEL assay, the fixed eyecups were transferred to 30% sucrose in 0.1 M phosphate-buffered solution for 2 hours and then transferred into optimal cutting temperature embedding compound (OCT; Fisher Scientific, Pittsburgh, PA). After the blocks were flash frozen in liquid nitrogen, 10- to 12-μm-thick sections were obtained with a cryostat. The sections were mounted on gelatin-coated slides. The retinal sections for immunofluorescence staining were blocked with 5% bovine serum albumin in 0.1 M phosphate buffer for 1 hour at room temperature, then incubated with mouse anti-rhodopsin antibody (1:300, mAB5356; Millipore, Billerica, MA), rabbit anti-GFAP antibody (1:500, ab7260; Abcam, Cambridge, UK), or rabbit anti-Ki-67 antibody (1:300; Chemicon-Millipore) overnight at 4°C. The sections were subsequently incubated for 2 hours at room temperature with goat anti-rabbit-FITC, goat anti-rabbit-Cy3, or goat anti-mouse-Cy3 antibody, depending on the primary antibody. The retinal sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI, 1 mg/mL in PBS; Sigma-Aldrich) to visualize cell nuclei. 
Figure 1.
 
Change in the anterior chamber after CP. (A) Anterior chamber of a normal mouse eye. (B) After CP, the anterior chamber was diminished and the lens moved toward the cornea. (C) Twelve hours after CP, the anterior chamber re-formed and the lens returned to its regular position. AC, anterior chamber.
Figure 1.
 
Change in the anterior chamber after CP. (A) Anterior chamber of a normal mouse eye. (B) After CP, the anterior chamber was diminished and the lens moved toward the cornea. (C) Twelve hours after CP, the anterior chamber re-formed and the lens returned to its regular position. AC, anterior chamber.
TUNEL examination was performed with a cell viability kit (In Situ Cell Death Detection Kit; Roche Diagnostics, Mannheim, Germany) based on the manufacturer's instructions. Positive controls were incubated with DNase I (1 μg/mL 50 mM Tris-HCl [pH 7.5] and 1 mg/mL BSA) for 10 minutes at room temperature before labeling. All the fluorescence-labeled retinal sections were examined using an inverted microscope (CK40; Olympus, Tokyo, Japan) and photographed with a real-time digital camera (Spot RT; Diagnostic Instruments, Sterling Heights, MI). 
ERG Recording and Analysis
After overnight dark adaptation, the animals were anesthetized with a mixture of ketamine (72 mg/kg) and xylazine (4 mg/kg). The pupils were dilated with a single drop of 0.5% tropicamide/0.5% phenylephrine hydrochloride eye drops. Before the electrodes were attached, a drop of 0.5% proparacaine hydrochloride was applied for corneal anesthesia. All procedures were performed under dim red light (>650 nm). CP before ERG recording was performed with the aide of a magnifying lens. ERGs from both eyes were recorded simultaneously. A pair of silver loop electrodes was placed on the surface of the right and left corneas with a drop of 2.5% methylcellulose to enhance attachment, a pair of reference electrodes was inserted into the right and left cheeks, and a ground electrode was inserted into the tail of the animal, to record the ERGs. The animals were tested in a Ganzfeld illumination dome connected to an electrophysiological diagnostic system (RETI Port21; Roland Consult, Wiesbaden, Germany) with a thermal platform that was maintained at 37°C. Full-field scotopic ERGs of both eyes were elicited simultaneously with 10-second flashes of blue light of 3.0-cd · s/m2 intensity. Electrical responses detected at the corneal contact electrodes were amplified (1–500 Hz). Light was spectrally filtered with a 500-nm interference filter. After the scotopic ERGs were recorded, both eyes were light adapted for 10 minutes under a light source of 30 cd · s/m2 intensity, and photopic ERGs were elicited simultaneously with 10-second flashes of white light at 1.96 cd · s/m2 intensity. The amplitude of the a-wave was measured from the prestimulus baseline to the trough of the a-wave, and the amplitude of the b-wave was measured from the trough of the a-wave to the crest of the b-wave. In each mouse, the ratio of the a- or b-wave of the right eye to that of the left eye recorded before CP was set at 100%. The ratio of the punctured right eye to the nonpunctured left eye obtained at various time points after CP was then calculated relative to the ratio obtained before CP to show the change in ERG amplitude of the punctured eyes: change ratio = [(RE amplitude/LE amplitude) of post-CP]/[(RE amp/LE amp) of pre-CP]. 
For the calculated ratio of ERG amplitudes, data in each group (n = 10–13) were expressed as the mean ± SE, and the significance of the difference in ERG amplitudes between each time point after CP and before CP was determined by t-test. 
Quantification of Retinal Thickness
The thickness of the outer nuclear layer (ONL), inner nuclear layer (INL), and inner plexiform layer (IPL) at a middle region between the pars plana and the optic nerve head in retinal sections was analyzed. The middle region of a retinal section showing retinal twist or retinal fold was excluded from retinal thickness measurements. The measurements of five eyes from each time point before and after CP were averaged. Data are presented as the mean ± SE. A two-sample t-test was used to compare the thickness before and after CP. P < 0.05 indicated statistical significance. 
Results
Status of Anterior Segment and Fundus Appearance after CP
After applying a drop of methylcellulose to the surface of the cornea, we used a needle to puncture the cornea. The needle broke through the anterior chamber, causing aqueous humor outflow along its shaft, disappearance of the anterior chamber, pupil constriction, and forward lens migration (Fig. 1A compared with 1B). At the same time, the retina marked by blood vessels was lifted up around the optic nerve head and formed multiple blebs (Fig. 2A compared with 2C). In some mouse eyes, retinal uplift was not obvious, but a gentle popping at the edge of the corneal hole with the needle made the uplift detectable. The anterior chamber recovered, and the lens returned to its regular position by 12 hours after CP (Fig. 1C). When the needle pierced the pars plana or sclera and entered the vitreous cavity, no retinal uplift was observed (Fig. 2B; Supplementary Movie S1). 
Figure 2.
 
(A) Fundus photograph of a non–corneal-punctured normal mouse eye. (B) Fundus photograph of a mouse eye that was punctured by a needle through the pars plana region. (C) Fundus photograph of a mouse eye that was punctured through the cornea. The fundus was lifted after CP.
Figure 2.
 
(A) Fundus photograph of a non–corneal-punctured normal mouse eye. (B) Fundus photograph of a mouse eye that was punctured by a needle through the pars plana region. (C) Fundus photograph of a mouse eye that was punctured through the cornea. The fundus was lifted after CP.
We observed the transformation of the appearance of the live fundus at various time points after CP. Also, knowing that the mouse lens is nearly spherical and that the image visualized through the lens may not be accurate, we made eyecups from formaldehyde-fixed mouse eyes at various time points after CP and directly visualized the appearance of the fundus. The image of the live eyes showed that the uplifted retina (Fig. 3B) caused by CP shrank 0.5 hour after CP and gradually flattened, forming an irregular, yellowish patch on the fundus (Fig. 3C). The retina returned to an almost normal fundus appearance at 10 days after CP (compare Fig. 3A with 3D). The eyecups showed that the irregular multiple blebs that corresponded to retinal uplift in live eyes were formed after CP (Fig. 3F), but the sclera maintained its spherical shape (data not shown). By 3 hours after CP, the retinal blebs became smaller and flatter (Fig. 3G) and returned to an almost normal appearance at approximately 10 days after CP (Figs. 3D, 3H). There were no breaks or holes in the retinas after CP. 
Figure 3.
 
Changes in the fundus after CP. Representative fundus photographs taken from both live mouse eyes (AD) and formaldehyde-fixed mouse eyecups (EH) to show the fundus of mouse eyes without CP (A, E) and the fundus of mouse eyes immediately (B, F), 3 (C, G), and 10 (D, H) days after CP.
Figure 3.
 
Changes in the fundus after CP. Representative fundus photographs taken from both live mouse eyes (AD) and formaldehyde-fixed mouse eyecups (EH) to show the fundus of mouse eyes without CP (A, E) and the fundus of mouse eyes immediately (B, F), 3 (C, G), and 10 (D, H) days after CP.
Change in Retinal Histology and RPE Cell Morphology during Progression of Retinal Detachment and Reattachment
To trace changes in the retina during the progression of retinal detachment/reattachment, we examined eyes without CP and eyes enucleated at 0 (immediately), 0.5, 1, 3, 5, 7, 10, 13, 15, and 20 days after CP by light microscopy. The cross sections of the eyecup showed, immediately after CP, that part of the neuroretina had detached from the RPE to form blebs and infolds. The subretinal space underneath the detached neuroretina was filled with HE-stained material, similar in color to the outer segment (OS) of the photoreceptor cells (Figs. 4C, 4D). At 1 day after CP, the retina blebs or infolded retina became flat and smooth, and the RPE cells were enlarged (Figs. 4E, 4F). Gradually, with progressive enlargement of the RPE cells and diminishing of the subretinal space, the material contained in the detached subretinal space was absorbed, and the detached neuroretina retreated and reattached to the RPE. By 10 to 13 days after CP, most of the detached neuroretina had flattened and reattached to the RPE (Figs. 4G, 4H). 
Figure 4.
 
Histologic changes of the retina after CP. The HE-stained sections crossing the optic nerve head area show an eye without CP (control, normal eye) (A, B), and the eyes immediately (C, D), 3 (E, F), and 10 (G, H) days after CP. The rectangles in (A), (C), (E), and (G) are imaged at higher magnification in (B), (D), (F), and (H), respectively. Retinal detachment is defined as the separation between the neuroretina and the RPE. Retinal reattachment is defined as reattachment of the neuroretina to the RPE without any separation between. Separation between the neuroretina and the RPE happened immediately after CP, and gradually diminished with reattachment of the neuroretina back to the RPE. During this process, RPE cells undergo major changes, becoming extremely expanded and enlarged, containing giant vacuoles, before gradually returning to a regular sheetlike monolayer. Scale bars: (A, C, E) 400 μm; (B, D, F) 200 μm; (G) 300 μm; (H) 150 μm.
Figure 4.
 
Histologic changes of the retina after CP. The HE-stained sections crossing the optic nerve head area show an eye without CP (control, normal eye) (A, B), and the eyes immediately (C, D), 3 (E, F), and 10 (G, H) days after CP. The rectangles in (A), (C), (E), and (G) are imaged at higher magnification in (B), (D), (F), and (H), respectively. Retinal detachment is defined as the separation between the neuroretina and the RPE. Retinal reattachment is defined as reattachment of the neuroretina to the RPE without any separation between. Separation between the neuroretina and the RPE happened immediately after CP, and gradually diminished with reattachment of the neuroretina back to the RPE. During this process, RPE cells undergo major changes, becoming extremely expanded and enlarged, containing giant vacuoles, before gradually returning to a regular sheetlike monolayer. Scale bars: (A, C, E) 400 μm; (B, D, F) 200 μm; (G) 300 μm; (H) 150 μm.
During the whole progression of retinal detachment/reattachment, the thickness of the ONL, INL, and IPL relative to the ILM–OLM thickness remained the same as that before CP (Fig. 5). 
Figure 5.
 
The thickness ratio of the ONL, INL, and IPL relative to the thickness from the inner limiting to the outer limiting membrane (ILM-OLM) before and after CP. The thickness ratio of ONL, INL, and IPL relative to the thickness of ILM-OLM did not change significantly after CP. The thickness average of five eye sections was plotted; bars, SE.
Figure 5.
 
The thickness ratio of the ONL, INL, and IPL relative to the thickness from the inner limiting to the outer limiting membrane (ILM-OLM) before and after CP. The thickness ratio of ONL, INL, and IPL relative to the thickness of ILM-OLM did not change significantly after CP. The thickness average of five eye sections was plotted; bars, SE.
RPE cells lining the detached subretinal space showed extensive morphologic transformation during retinal detachment/reattachment. Immediately after detachment of the neuroretina from the RPE, the RPE maintained its sheetlike monolayer structure, but the apical side of each RPE cell became convex (Fig. 6B). Approximately 1 day after CP, the shape of the RPE cells expanded from flat cuboid to columnar (Fig. 6C). At 3 days after CP, the enlargement of the RPE cells reached its maximum. The size of an enlarged RPE cell could be 10 to 20 times the size of a normal RPE cell. The enlarged RPE cells irregularly abutted or overlapped to form a pseudomultilayer. A giant intracellular vacuole was formed in each RPE cell that occupied most of the enlarged RPE cells (Figs. 6D, 6E). The nuclei of the RPE cells also moved from the basal to the apical side, and the densely congregated pigment clump, which appears in normal RPE cells, became looser and dissociated. Many individual pigment granules were evenly distributed in the cytoplasm of the RPE cells. Approximately 10 to 13 days after CP, all the enlarged RPE cells recovered to almost regular cell size to form a sheetlike monolayer again, yet a few of the RPE cells still contained the intracellular vacuole (Fig. 6A compared with 6F, 6G). 
Figure 6.
 
Morphologic changes of RPE cells during the progression of retinal detachment/reattachment. HE-stained retinal sections of normal mouse eye (A), and the eyes enucleated immediately (B), 1 (C), 3 (D, E), 5 (F), and 10 (G) days after CP. Arrows: RPE cells; Arrowheads: nuclei of RPE cells; (*) detached subretinal space. IS, inner segment; OS, outer segment; RPEL, retinal pigment epithelium layer; CM, choroid membrane. Scale bars, 40 μm.
Figure 6.
 
Morphologic changes of RPE cells during the progression of retinal detachment/reattachment. HE-stained retinal sections of normal mouse eye (A), and the eyes enucleated immediately (B), 1 (C), 3 (D, E), 5 (F), and 10 (G) days after CP. Arrows: RPE cells; Arrowheads: nuclei of RPE cells; (*) detached subretinal space. IS, inner segment; OS, outer segment; RPEL, retinal pigment epithelium layer; CM, choroid membrane. Scale bars, 40 μm.
Electroretinographic Analysis
To evaluate whether CP affects retinal function, we examined the retinal ERGs. The punctured eye showed ∼45% and 27% of dark-adapted b- and a-wave amplitude reduction immediately after CP, and the reduction of ERG amplitude recovered to a normal level approximately 12 hours after CP (Figs. 7A, 7C), whereas photopic ERG recording showed approximately 7% and 12% of light-adapted b- and a-wave reduction immediately after CP and recovered to a normal level approximately 12 hours after CP (Fig. 7B). The ERG amplitudes remained at a normal level relative to contralateral nonpunctured eyes at 12 hours after CP. 
Figure 7.
 
Time course of relative scotopic and photopic ERG a- and b-wave amplitudes of mouse eyes after CP. For each mouse, the right eye underwent CP, the left eye was the control. Simultaneous ERG measurements for both right and left eyes were taken 3 days before CP and at 0 (immediately after CP), 0.5, 1, 2, 3, 5, and 60 days after CP. (A) Average relative scotopic a- and b-wave amplitudes of corneal-punctured eyes. (B) Average relative photopic a- and b-wave amplitudes of corneal-punctured eyes. (C) Representative dark-adapted ERG waveforms of a mouse eye. Both scotopic a- and b-wave reduced immediately after cornea puncture (P < 0.05) and had almost full ERG recovery at 12 hours (0.5 day) after CP. The ERG measurements represent an average of 10 to 13 eyes. Error bars, ±SE. Scale bar, (C) 200 μV.
Figure 7.
 
Time course of relative scotopic and photopic ERG a- and b-wave amplitudes of mouse eyes after CP. For each mouse, the right eye underwent CP, the left eye was the control. Simultaneous ERG measurements for both right and left eyes were taken 3 days before CP and at 0 (immediately after CP), 0.5, 1, 2, 3, 5, and 60 days after CP. (A) Average relative scotopic a- and b-wave amplitudes of corneal-punctured eyes. (B) Average relative photopic a- and b-wave amplitudes of corneal-punctured eyes. (C) Representative dark-adapted ERG waveforms of a mouse eye. Both scotopic a- and b-wave reduced immediately after cornea puncture (P < 0.05) and had almost full ERG recovery at 12 hours (0.5 day) after CP. The ERG measurements represent an average of 10 to 13 eyes. Error bars, ±SE. Scale bar, (C) 200 μV.
DAPI Staining and Immunoreactivity for Rhodopsin in the Detached Subretinal Space
To investigate the potential inflammatory response during retinal detachment/reattachment, we used DAPI to locate nuclei of any inflammatory cells that may have infiltrated the detached subretinal space. The retinal sections of eyes enucleated at 0, 1, 3, 5, and 10 days after CP were examined, and no DAPI-stained nuclei were observed in the detached subretinal space or the area between the nuclei of the RPE cells and the ONL (Figs. 8B, 8F, 8J, 8N, 8R). 
Figure 8.
 
Immunofluorescence micrographs showing the immunolabeling (red) pattern for the antibody to rhodopsin and DAPI staining (blue) for nuclei on frozen retinal sections. (A, E, I, M, Q) Bright-field images of retinal sections from normal eye and eyes enucleated at 0 (immediately), 3, 5, and 10 days after CP. (B, F, J, N, R) DAPI staining of (A), (E), (I), (M), and (Q). (C, G, K, O, S) Merged images of DAPI staining and anti-rhodopsin antibody labeling of (A), (E), (I), (M), and (Q). (D, H, L, P, T) Magnified image of anti-rhodopsin antibody labeling of (C), (G), (K), (O), and (S). The detached subretinal space is filled with anti-rhodopsin labeling and has no DAPI-stained nuclei, indicating no cells migrated into the detached subretinal space after retinal detachment. (*) Detached subretinal space. Arrowheads: suspected detached subretinal space. IS, inner segment; CM, choroid membrane. Scale bars: (AC, IK, MO, QS) 50 μm; (EG) 100 μm; (D, H, L, P, T) 20 μm.
Figure 8.
 
Immunofluorescence micrographs showing the immunolabeling (red) pattern for the antibody to rhodopsin and DAPI staining (blue) for nuclei on frozen retinal sections. (A, E, I, M, Q) Bright-field images of retinal sections from normal eye and eyes enucleated at 0 (immediately), 3, 5, and 10 days after CP. (B, F, J, N, R) DAPI staining of (A), (E), (I), (M), and (Q). (C, G, K, O, S) Merged images of DAPI staining and anti-rhodopsin antibody labeling of (A), (E), (I), (M), and (Q). (D, H, L, P, T) Magnified image of anti-rhodopsin antibody labeling of (C), (G), (K), (O), and (S). The detached subretinal space is filled with anti-rhodopsin labeling and has no DAPI-stained nuclei, indicating no cells migrated into the detached subretinal space after retinal detachment. (*) Detached subretinal space. Arrowheads: suspected detached subretinal space. IS, inner segment; CM, choroid membrane. Scale bars: (AC, IK, MO, QS) 50 μm; (EG) 100 μm; (D, H, L, P, T) 20 μm.
To see whether the HE-stained material in the subretinal space may include debris from broken outer segments of photoreceptor cells, we examined the immunoreactivity of rhodopsin on retinal sections after CP. Both outer segments of photoreceptor cells and the subretinal space underneath the detached neuroretina showed rhodopsin labeling. During the progression of retinal detachment/reattachment, the rhodopsin labeling in the subretinal space reduced progressively in accordance with the enlargement of the RPE cells and diminishing of the subretinal space (Figs. 8G, 8H, 8K, 8L, 8O, 8P, 8S, 8T). 
Detection of Müller Cell Reactivity, Proliferation, and Apoptosis of Retinal Cells during Retinal Detachment and Reattachment
Strong Müller cell reactivity was observed in other animal experimental models of RD. 17 19 We tested the immunoreactivity of glial fibrillary acidic protein (GFAP) in the retinal sections of eyes without CP and eyes enucleated 3, 7, and 10 days after CP. Although the retina sections showed transformation of the subretinal space and RPE cells in accordance with the progression of retinal detachment/reattachment (Figs. 9B, 9D, 9F, 9H), immunoreactivity for GFAP was consistently detected only in the endfoot region of Müller cells and retinal astrocytes in the ganglion cell layer (GCL) of the retina (Figs. 9A, 9C, 9E, 9G). 
Figure 9.
 
Immunolabeling pattern of GFAP on frozen retinal sections. Normal retinal section (A) and retina sections at 3 (C), 7 (E), and 10 (G) days after CP were labeled with anti-GFAP antibody (red) and counterstained with DAPI (blue) to show the nuclei. (B, D, F, H) Bright-field images of (A), (C), (E), and (G). GFAP labeling is mainly limited in the endfoot region of Müller cells and retinal astrocytes in the GCL of the retina before and after CP. IS, inner segment; CM, choroid membrane. Scale bars, 50 μm.
Figure 9.
 
Immunolabeling pattern of GFAP on frozen retinal sections. Normal retinal section (A) and retina sections at 3 (C), 7 (E), and 10 (G) days after CP were labeled with anti-GFAP antibody (red) and counterstained with DAPI (blue) to show the nuclei. (B, D, F, H) Bright-field images of (A), (C), (E), and (G). GFAP labeling is mainly limited in the endfoot region of Müller cells and retinal astrocytes in the GCL of the retina before and after CP. IS, inner segment; CM, choroid membrane. Scale bars, 50 μm.
Proliferation was another major cell activity induced by RD in many other animal experimental models of RD 20,21 and also appears in many human retinal diseases. 22,23 We tested the immunoreactivity of Ki-67 protein, a nuclear protein used as a proliferation marker, in the retinal sections of eyes enucleated before and after CP. A retinal section of embryonic mouse eye (15 dpc; days post coitus) was used as a positive control. Ki-67 protein labeling appeared in the nuclei of epithelial cells of the palpebral conjunctiva (PC), corneal cells, and the retinal cells that were close to the ciliary body (CB) on the retinal sections of the embryonic mouse eye (Figs. 10A, 10B). The retinal sections of adult mouse eyes enucleated at 0, 3, 7, and 10 days after CP did not show any nuclear staining for Ki-67 (Figs. 10E–J). 
Figure 10.
 
Ki-67 immunolabeling pattern on frozen retinal sections. (A) Embryonic mouse eye section (15 dpc) incubated with both Ki-67 antibody and FITC-conjugated secondary antibody as the positive control. Ki-67 antibody labeling occurs in the nuclei of epithelial cells of PC, cornea cells, and the retinal cells that are close to the CB on the eye section. (B) Merged image of DAPI staining and the FITC image of Ki-67 antibody labeling of (A). DAPI staining overlaps with Ki-67 staining in the nuclei. (C) Embryonic mouse eye section (15 dpc) incubated with only FITC-conjugated secondary antibody as the negative control. No nuclei are labeled. (D) Merged image of DAPI staining and FITC image of (C). (E, G, I, K) Retinal sections of adult mouse eyes enucleated at 0, 3, 7, and 10 days after CP. (F, H, J, F) Merged image of DAPI staining and FITC image of Ki-67 antibody labeling of (F), (H), (J), (F). There is no immunoreactivity for Ki-67 in the nuclei of adult mouse retina before and after CP. C, cornea; R, retina; IS, inner segment. Scale bar, 50 μm.
Figure 10.
 
Ki-67 immunolabeling pattern on frozen retinal sections. (A) Embryonic mouse eye section (15 dpc) incubated with both Ki-67 antibody and FITC-conjugated secondary antibody as the positive control. Ki-67 antibody labeling occurs in the nuclei of epithelial cells of PC, cornea cells, and the retinal cells that are close to the CB on the eye section. (B) Merged image of DAPI staining and the FITC image of Ki-67 antibody labeling of (A). DAPI staining overlaps with Ki-67 staining in the nuclei. (C) Embryonic mouse eye section (15 dpc) incubated with only FITC-conjugated secondary antibody as the negative control. No nuclei are labeled. (D) Merged image of DAPI staining and FITC image of (C). (E, G, I, K) Retinal sections of adult mouse eyes enucleated at 0, 3, 7, and 10 days after CP. (F, H, J, F) Merged image of DAPI staining and FITC image of Ki-67 antibody labeling of (F), (H), (J), (F). There is no immunoreactivity for Ki-67 in the nuclei of adult mouse retina before and after CP. C, cornea; R, retina; IS, inner segment. Scale bar, 50 μm.
Retinal cell apoptosis was also examined in cross sections of eyes from various time points after CP by TUNEL assay. Only the positive control section was TUNEL positive (Fig. 11A). No TUNEL-positive cells were observed in the neuroretina and RPE of the eyes at various time points after CP (Figs. 11B–G). 
Figure 11.
 
TUNEL positivity in the retina of corneal-punctured eyes at various time points. (A) Representative fluorescence image of positive control, which was tested on a cross section of normal mouse eye. (B, C, E, F, G) Representative fluorescence images of cross sections of normal eye and the eyes enucleated at 0, 3, 7, and 10 days after CP. (D) Combination of FITC, DAPI, and Cy3 channel (autofluorescence) fluorescence images of (C), showing nuclear staining (blue) and tissue background autofluorescence (overlap of green and red). (H) Bright-field image of (G). (H, arrows) show the vacuoles in RPE. No TUNEL-positive cells were observed in the retinal cross sections before and after CP. Scale bars, 100 μm.
Figure 11.
 
TUNEL positivity in the retina of corneal-punctured eyes at various time points. (A) Representative fluorescence image of positive control, which was tested on a cross section of normal mouse eye. (B, C, E, F, G) Representative fluorescence images of cross sections of normal eye and the eyes enucleated at 0, 3, 7, and 10 days after CP. (D) Combination of FITC, DAPI, and Cy3 channel (autofluorescence) fluorescence images of (C), showing nuclear staining (blue) and tissue background autofluorescence (overlap of green and red). (H) Bright-field image of (G). (H, arrows) show the vacuoles in RPE. No TUNEL-positive cells were observed in the retinal cross sections before and after CP. Scale bars, 100 μm.
Discussion
With the availability of many spontaneous and engineered mouse models of inherited retinal diseases, 24 30 the preclinical study of retinal gene therapy in the mouse is gaining more attention. Transcorneal subretinal injection is one of the emerging methods of delivering therapeutic transgene vectors targeting RPE and photoreceptor cells. This technique has been increasingly popular because it is easy to learn, allows high throughput, does not require surgical preparation, and has a high success rate. 15 More important, it has fewer potential complications such as hemorrhage and retinal trauma that can affect the efficacy of the therapy. Transcorneal subretinal injection of the mouse eye starts with making a pilot hole in the cornea. Then, a blunt needle connected to a syringe is inserted through the hole, bypassing the lens and passing through the vitreous cavity into the subretinal space. The fluorescein-containing fluid in the syringe is then slowly injected into the subretinal space to induce retinal blebs. 9,15,16,31,32 The authors considered the formation of retinal blebs after injection, which can be viewed through the mouse pupil under a dissecting microscope, as a marker of success of subretinal injection. In our studies, the fundus was lifted up to form retinal blebs immediately after the needle pierced the cornea and entered the anterior chamber of the mouse eye (Fig. 2). Retinal blebs could be successfully achieved in almost all the corneal-punctured mouse eyes. Although retinal blebs occasionally formed after a CP were not as obvious as others, gentle picking on the edge of the corneal hole with the needle used for the CP could quickly further expand and lift up the minor retinal blebs. 
Considering that uplift of the fundus induced by CP may indeed be an RD created without direct artificial manipulation of the retina, we performed histologic and functional studies of punctured mouse corneas. The results revealed that CP can induce RD between the neuroretina and the RPE in C57BL/6J mice. The RD was not accompanied with retinal holes or breaks. We assume that the reason for inducing RD by CP in mouse eyes is that the rapid outflow of aqueous humor along the shaft of the needle leads the lens, which is larger than that in other animals, to move forward and cause a sudden drop in intraocular pressure, sucking the retina and sclera inward. The sudden inward-pulling force breaks the connection between the neuroretina and the RPE to form an RD. The RD can be created by CP with even smaller or larger needles than 30.5-gauge size sharp-pointed needles (data not shown) used in this study, but not by inserting a needle through the sclera into the vitreous cavity. We also tested CP in the eyes of other animals and found that it can cause an RD in the eye of ICR (Institute of Cancer Research) mice as well, but not in the eyes of Sprague-Dawley (SD) rats and guinea pigs (data not shown). Creating RD by CP of the mouse eye provides us a method of developing a novel experimental mouse model of retinal detachment. 
In our mouse RD model, we found a rapid reduction of ERG amplitudes soon after CP. Surprisingly, the ERG amplitude recovered almost completely within 12 hours after CP and was maintained (Fig. 7). Our findings differ from those reported by Nour et al., 14 who showed that the ERG amplitudes of dark adapted a- and b-wave were approximately 36% and 44%, respectively, of the control mouse eyes at 1 day after subretinal injection, and both were approximately 61% of the control mouse eyes at 14 days after subretinal injection. 14 In the two studies, different methods were used to calculate the ERG recovery ratio: Nour et al. used the ratio of ERG amplitudes of subretinal injected eyes relative to those of mock-injected eyes, and we used the ratio of ERG amplitudes of corneal-punctured eyes relative to that measured before CP with the contralateral nonpunctured eye used as the control, to eliminate potential interfering factors such as environment, food, anesthesia, and shock caused by the surgical procedure. Despite the difference between the ways of calculating the ERG recovery ratio, the quicker ERG recovery in our RD model prompts us to suggest that detached retina can have a quick, fully functional recovery. Such a recovery is possible if the microenvironment of the subretinal space in an RD is in a self-recoverable state and the RPE cells and photoreceptor cells involved are not impaired by mechanical manipulation or toxins from injected fluid or the vitreous body leaking into the subretinal space through the broken retina. 
Morphologic changes in RPE cells during retinal detachment/reattachment have been described in many RD studies. Despite the various animals used, the major apparent morphologic changes in RPE cells were remodeling of processes on the apical surface of RPE cells 33 and proliferation. 34,35 RPE cells are thought to transdifferentiate into myofibroblasts and other cell types, such as glial cells, during retinal detachment, causing proliferative vitreoretinopathy. 36,37 From 1 to 8 weeks after an RD created by a break in the peripheral retina, RPE cells in the posterior pole region in wild-type mouse eyes became multilayered proliferating cells. 38 In this study, the gross morphology of RPE cells underwent dramatic changes during the progression of retinal detachment/reattachment (Figs. 4, 6), and histologic changes in RPE cells indicate that they actively functioned during this process. We assume that the cells execute their fluid transfer and phagocytosis functions 39,40 to remove fluid and broken outer segments of photoreceptor cells and other debris from the subretinal space during the progression of retinal detachment/reattachment. Also, although RPE cells formed a pseudomultilayer during retinal detachment/reattachment in this study, we doubt that RPE cells proliferate in this RD model, since no anti-Ki-67 antibody-labeled nuclei of RPE were observed throughout the study and the affected RPE layer eventually became a sheetlike monolayer of cells, similar to that in normal mouse eyes. We show in this study only the gross morphologic change in RPE cells during progression of retinal detachment/reattachment. Further investigation will reveal the details of the cell activity involved in this RD model. 
Previous clinical and experimental studies reported that the recovery of cone function after temporary RD lags behind retinal reattachment. 41 43 Nour et al. 14 reported that after subretinal injection in mouse eyes, the detached retina reattached within 24 hours, whereas ERG recovery took weeks. In this study, the images of live fundus and fundus in eyecups showed that the retinal blebs could shrink and flatten within 24 hours, but it took days for the appearance of the fundus to return to normal. Some of the RPE cells in the detached area still contained vacuoles 10 days after CP (Fig. 3). Thus, in this RD model, histologic recovery lagged behind ERG recovery. 
We noted that most of the corneal-punctured eyes showed a gap between the apical surface of the RPE and the neuroretina, which was less frequently observed in the sections of normal control eyes (Figs. 4, 6). Since the gap was not stained and contained no debris, and since some part of the contour of one edge of the gap matched the opposite edge, we believe the gap was an artificial separation caused by histologic processing such as dehydration or sectioning. The fact that the newly reattached retina was more fragile to histologic processing than normal retina makes us think that the recovery of interaction strength between the neuroretina and the RPE may be a valuable criterion to predict recovery of RD. Based on the data gained in this study, we conclude that the interaction strength between the neuroretina and the RPE can recover to an almost normal level about 2 weeks after RD if the RD is caused solely by disruption of the interaction between the neuroretina and the RPE; but if other deleterious factors beyond RD itself are involved, total recovery of the interaction strength between the neuroretina and the RPE may take longer, or may not be achieved, even when the fundus looks normal. 
Many experimental animal models of RD showed activation and proliferation of RPE cells and Müller cells in damaged retina, 34,38,44 46 macrophage infiltration into the subretinal space, 47 and apoptosis of photoreceptor cells during retinal detachment/reattachment. 14,48 51 We found that, throughout the progression of retinal detachment/reattachment, Müller cell reactivity, which is indicated by anti-GFAP antibody labeling, in damaged retina appeared similar to that in nondamaged (or normal) retina (Fig. 9), the immunoreactivity of proliferation marker Ki-67 was not observed in either the neuroretina or the RPE of adult mouse RD retina (Fig. 10), and no cells appeared in the subretinal space (Fig. 8). Moreover, we did not find TUNEL-positive cells in either the neuroretina or the RPE (Fig. 11). Considering that the RD models showing active immunoreactivity of GFAP and Ki-67 protein, cell proliferation and infiltration, and apoptosis in damaged retina were created by either peeling off the neuroretina from the RPE or subretinal injection of fluid, we assume that both photoreceptor cells and RPE cells are able to survive a limited RD in which the lesion does not extend beyond the disruption of the interaction between the neuroretina and the RPE. We think that mechanical manipulation of the retina, toxicity from the subretinal injected fluid, or vitreous body leakage into the subretinal space through the retinal break can cause additional or even fatal damage to photoreceptor cells that have already been affected by the detachment. 
In the eye clinic, although retinal reattachment varies depending on the extent, type, and duration of RD, two kinds of detachment-related retinal diseases have similarity to this model: tractional RD with no retinal break and central serous chorioretinitis, in which a fluid layer remains between the neuroretina and the RPE with no retinal breaks. Patients with this disease can spontaneously recover visual function in days or weeks after the fluid is absorbed. We believe our RD model with complete functional and histologic recovery will provide us an alternative means of understanding the pathology and pathophysiology of retinal detachment/reattachment and identify deleterious factors coming from RD-related treatment and therapy so that unnecessary iatrogenic damage to the retina can be avoided. 
We report an experimental mouse RD model in which the retina is not directly manipulated by either mechanical or liquid means to induce RD, has no breaks, and can have complete functional and histologic recovery. This model can be a useful tool for identifying influential factors for recovery. Since the model has no breaks or holes in the retina while containing debris and fluid between the separated neuroretina and RPE, it may also be useful in the study of subretinal exudate-related retinal disease such as central serous chorioretinitis. 
Supplementary Materials
Text sm1, MOV - Text sm1, MOV 
Footnotes
 Supported in part by Major Projects of the National Science and Technology of China Grant 2009ZX09503, Key Projects of National High-Tech R&D Program (863) of China Grant 2007AA021004, and a retinal gene therapy study grant from the Eye Hospital, School of Ophthalmology and Optometry, Wenzhou Medical College, Wenzhou, China.
Footnotes
 Disclosure: R. Zeng, None; Y. Zhang, None; F. Shi, None; F. Kong, None
The authors thank Xiaogang Chen for technical assistance and Ying Xia, Lili Tu, Philip Kaplan, Mark A Hallett, and IOVS volunteer editor Timothy W. Corson for proofreading and editing the manuscript. 
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Figure 1.
 
Change in the anterior chamber after CP. (A) Anterior chamber of a normal mouse eye. (B) After CP, the anterior chamber was diminished and the lens moved toward the cornea. (C) Twelve hours after CP, the anterior chamber re-formed and the lens returned to its regular position. AC, anterior chamber.
Figure 1.
 
Change in the anterior chamber after CP. (A) Anterior chamber of a normal mouse eye. (B) After CP, the anterior chamber was diminished and the lens moved toward the cornea. (C) Twelve hours after CP, the anterior chamber re-formed and the lens returned to its regular position. AC, anterior chamber.
Figure 2.
 
(A) Fundus photograph of a non–corneal-punctured normal mouse eye. (B) Fundus photograph of a mouse eye that was punctured by a needle through the pars plana region. (C) Fundus photograph of a mouse eye that was punctured through the cornea. The fundus was lifted after CP.
Figure 2.
 
(A) Fundus photograph of a non–corneal-punctured normal mouse eye. (B) Fundus photograph of a mouse eye that was punctured by a needle through the pars plana region. (C) Fundus photograph of a mouse eye that was punctured through the cornea. The fundus was lifted after CP.
Figure 3.
 
Changes in the fundus after CP. Representative fundus photographs taken from both live mouse eyes (AD) and formaldehyde-fixed mouse eyecups (EH) to show the fundus of mouse eyes without CP (A, E) and the fundus of mouse eyes immediately (B, F), 3 (C, G), and 10 (D, H) days after CP.
Figure 3.
 
Changes in the fundus after CP. Representative fundus photographs taken from both live mouse eyes (AD) and formaldehyde-fixed mouse eyecups (EH) to show the fundus of mouse eyes without CP (A, E) and the fundus of mouse eyes immediately (B, F), 3 (C, G), and 10 (D, H) days after CP.
Figure 4.
 
Histologic changes of the retina after CP. The HE-stained sections crossing the optic nerve head area show an eye without CP (control, normal eye) (A, B), and the eyes immediately (C, D), 3 (E, F), and 10 (G, H) days after CP. The rectangles in (A), (C), (E), and (G) are imaged at higher magnification in (B), (D), (F), and (H), respectively. Retinal detachment is defined as the separation between the neuroretina and the RPE. Retinal reattachment is defined as reattachment of the neuroretina to the RPE without any separation between. Separation between the neuroretina and the RPE happened immediately after CP, and gradually diminished with reattachment of the neuroretina back to the RPE. During this process, RPE cells undergo major changes, becoming extremely expanded and enlarged, containing giant vacuoles, before gradually returning to a regular sheetlike monolayer. Scale bars: (A, C, E) 400 μm; (B, D, F) 200 μm; (G) 300 μm; (H) 150 μm.
Figure 4.
 
Histologic changes of the retina after CP. The HE-stained sections crossing the optic nerve head area show an eye without CP (control, normal eye) (A, B), and the eyes immediately (C, D), 3 (E, F), and 10 (G, H) days after CP. The rectangles in (A), (C), (E), and (G) are imaged at higher magnification in (B), (D), (F), and (H), respectively. Retinal detachment is defined as the separation between the neuroretina and the RPE. Retinal reattachment is defined as reattachment of the neuroretina to the RPE without any separation between. Separation between the neuroretina and the RPE happened immediately after CP, and gradually diminished with reattachment of the neuroretina back to the RPE. During this process, RPE cells undergo major changes, becoming extremely expanded and enlarged, containing giant vacuoles, before gradually returning to a regular sheetlike monolayer. Scale bars: (A, C, E) 400 μm; (B, D, F) 200 μm; (G) 300 μm; (H) 150 μm.
Figure 5.
 
The thickness ratio of the ONL, INL, and IPL relative to the thickness from the inner limiting to the outer limiting membrane (ILM-OLM) before and after CP. The thickness ratio of ONL, INL, and IPL relative to the thickness of ILM-OLM did not change significantly after CP. The thickness average of five eye sections was plotted; bars, SE.
Figure 5.
 
The thickness ratio of the ONL, INL, and IPL relative to the thickness from the inner limiting to the outer limiting membrane (ILM-OLM) before and after CP. The thickness ratio of ONL, INL, and IPL relative to the thickness of ILM-OLM did not change significantly after CP. The thickness average of five eye sections was plotted; bars, SE.
Figure 6.
 
Morphologic changes of RPE cells during the progression of retinal detachment/reattachment. HE-stained retinal sections of normal mouse eye (A), and the eyes enucleated immediately (B), 1 (C), 3 (D, E), 5 (F), and 10 (G) days after CP. Arrows: RPE cells; Arrowheads: nuclei of RPE cells; (*) detached subretinal space. IS, inner segment; OS, outer segment; RPEL, retinal pigment epithelium layer; CM, choroid membrane. Scale bars, 40 μm.
Figure 6.
 
Morphologic changes of RPE cells during the progression of retinal detachment/reattachment. HE-stained retinal sections of normal mouse eye (A), and the eyes enucleated immediately (B), 1 (C), 3 (D, E), 5 (F), and 10 (G) days after CP. Arrows: RPE cells; Arrowheads: nuclei of RPE cells; (*) detached subretinal space. IS, inner segment; OS, outer segment; RPEL, retinal pigment epithelium layer; CM, choroid membrane. Scale bars, 40 μm.
Figure 7.
 
Time course of relative scotopic and photopic ERG a- and b-wave amplitudes of mouse eyes after CP. For each mouse, the right eye underwent CP, the left eye was the control. Simultaneous ERG measurements for both right and left eyes were taken 3 days before CP and at 0 (immediately after CP), 0.5, 1, 2, 3, 5, and 60 days after CP. (A) Average relative scotopic a- and b-wave amplitudes of corneal-punctured eyes. (B) Average relative photopic a- and b-wave amplitudes of corneal-punctured eyes. (C) Representative dark-adapted ERG waveforms of a mouse eye. Both scotopic a- and b-wave reduced immediately after cornea puncture (P < 0.05) and had almost full ERG recovery at 12 hours (0.5 day) after CP. The ERG measurements represent an average of 10 to 13 eyes. Error bars, ±SE. Scale bar, (C) 200 μV.
Figure 7.
 
Time course of relative scotopic and photopic ERG a- and b-wave amplitudes of mouse eyes after CP. For each mouse, the right eye underwent CP, the left eye was the control. Simultaneous ERG measurements for both right and left eyes were taken 3 days before CP and at 0 (immediately after CP), 0.5, 1, 2, 3, 5, and 60 days after CP. (A) Average relative scotopic a- and b-wave amplitudes of corneal-punctured eyes. (B) Average relative photopic a- and b-wave amplitudes of corneal-punctured eyes. (C) Representative dark-adapted ERG waveforms of a mouse eye. Both scotopic a- and b-wave reduced immediately after cornea puncture (P < 0.05) and had almost full ERG recovery at 12 hours (0.5 day) after CP. The ERG measurements represent an average of 10 to 13 eyes. Error bars, ±SE. Scale bar, (C) 200 μV.
Figure 8.
 
Immunofluorescence micrographs showing the immunolabeling (red) pattern for the antibody to rhodopsin and DAPI staining (blue) for nuclei on frozen retinal sections. (A, E, I, M, Q) Bright-field images of retinal sections from normal eye and eyes enucleated at 0 (immediately), 3, 5, and 10 days after CP. (B, F, J, N, R) DAPI staining of (A), (E), (I), (M), and (Q). (C, G, K, O, S) Merged images of DAPI staining and anti-rhodopsin antibody labeling of (A), (E), (I), (M), and (Q). (D, H, L, P, T) Magnified image of anti-rhodopsin antibody labeling of (C), (G), (K), (O), and (S). The detached subretinal space is filled with anti-rhodopsin labeling and has no DAPI-stained nuclei, indicating no cells migrated into the detached subretinal space after retinal detachment. (*) Detached subretinal space. Arrowheads: suspected detached subretinal space. IS, inner segment; CM, choroid membrane. Scale bars: (AC, IK, MO, QS) 50 μm; (EG) 100 μm; (D, H, L, P, T) 20 μm.
Figure 8.
 
Immunofluorescence micrographs showing the immunolabeling (red) pattern for the antibody to rhodopsin and DAPI staining (blue) for nuclei on frozen retinal sections. (A, E, I, M, Q) Bright-field images of retinal sections from normal eye and eyes enucleated at 0 (immediately), 3, 5, and 10 days after CP. (B, F, J, N, R) DAPI staining of (A), (E), (I), (M), and (Q). (C, G, K, O, S) Merged images of DAPI staining and anti-rhodopsin antibody labeling of (A), (E), (I), (M), and (Q). (D, H, L, P, T) Magnified image of anti-rhodopsin antibody labeling of (C), (G), (K), (O), and (S). The detached subretinal space is filled with anti-rhodopsin labeling and has no DAPI-stained nuclei, indicating no cells migrated into the detached subretinal space after retinal detachment. (*) Detached subretinal space. Arrowheads: suspected detached subretinal space. IS, inner segment; CM, choroid membrane. Scale bars: (AC, IK, MO, QS) 50 μm; (EG) 100 μm; (D, H, L, P, T) 20 μm.
Figure 9.
 
Immunolabeling pattern of GFAP on frozen retinal sections. Normal retinal section (A) and retina sections at 3 (C), 7 (E), and 10 (G) days after CP were labeled with anti-GFAP antibody (red) and counterstained with DAPI (blue) to show the nuclei. (B, D, F, H) Bright-field images of (A), (C), (E), and (G). GFAP labeling is mainly limited in the endfoot region of Müller cells and retinal astrocytes in the GCL of the retina before and after CP. IS, inner segment; CM, choroid membrane. Scale bars, 50 μm.
Figure 9.
 
Immunolabeling pattern of GFAP on frozen retinal sections. Normal retinal section (A) and retina sections at 3 (C), 7 (E), and 10 (G) days after CP were labeled with anti-GFAP antibody (red) and counterstained with DAPI (blue) to show the nuclei. (B, D, F, H) Bright-field images of (A), (C), (E), and (G). GFAP labeling is mainly limited in the endfoot region of Müller cells and retinal astrocytes in the GCL of the retina before and after CP. IS, inner segment; CM, choroid membrane. Scale bars, 50 μm.
Figure 10.
 
Ki-67 immunolabeling pattern on frozen retinal sections. (A) Embryonic mouse eye section (15 dpc) incubated with both Ki-67 antibody and FITC-conjugated secondary antibody as the positive control. Ki-67 antibody labeling occurs in the nuclei of epithelial cells of PC, cornea cells, and the retinal cells that are close to the CB on the eye section. (B) Merged image of DAPI staining and the FITC image of Ki-67 antibody labeling of (A). DAPI staining overlaps with Ki-67 staining in the nuclei. (C) Embryonic mouse eye section (15 dpc) incubated with only FITC-conjugated secondary antibody as the negative control. No nuclei are labeled. (D) Merged image of DAPI staining and FITC image of (C). (E, G, I, K) Retinal sections of adult mouse eyes enucleated at 0, 3, 7, and 10 days after CP. (F, H, J, F) Merged image of DAPI staining and FITC image of Ki-67 antibody labeling of (F), (H), (J), (F). There is no immunoreactivity for Ki-67 in the nuclei of adult mouse retina before and after CP. C, cornea; R, retina; IS, inner segment. Scale bar, 50 μm.
Figure 10.
 
Ki-67 immunolabeling pattern on frozen retinal sections. (A) Embryonic mouse eye section (15 dpc) incubated with both Ki-67 antibody and FITC-conjugated secondary antibody as the positive control. Ki-67 antibody labeling occurs in the nuclei of epithelial cells of PC, cornea cells, and the retinal cells that are close to the CB on the eye section. (B) Merged image of DAPI staining and the FITC image of Ki-67 antibody labeling of (A). DAPI staining overlaps with Ki-67 staining in the nuclei. (C) Embryonic mouse eye section (15 dpc) incubated with only FITC-conjugated secondary antibody as the negative control. No nuclei are labeled. (D) Merged image of DAPI staining and FITC image of (C). (E, G, I, K) Retinal sections of adult mouse eyes enucleated at 0, 3, 7, and 10 days after CP. (F, H, J, F) Merged image of DAPI staining and FITC image of Ki-67 antibody labeling of (F), (H), (J), (F). There is no immunoreactivity for Ki-67 in the nuclei of adult mouse retina before and after CP. C, cornea; R, retina; IS, inner segment. Scale bar, 50 μm.
Figure 11.
 
TUNEL positivity in the retina of corneal-punctured eyes at various time points. (A) Representative fluorescence image of positive control, which was tested on a cross section of normal mouse eye. (B, C, E, F, G) Representative fluorescence images of cross sections of normal eye and the eyes enucleated at 0, 3, 7, and 10 days after CP. (D) Combination of FITC, DAPI, and Cy3 channel (autofluorescence) fluorescence images of (C), showing nuclear staining (blue) and tissue background autofluorescence (overlap of green and red). (H) Bright-field image of (G). (H, arrows) show the vacuoles in RPE. No TUNEL-positive cells were observed in the retinal cross sections before and after CP. Scale bars, 100 μm.
Figure 11.
 
TUNEL positivity in the retina of corneal-punctured eyes at various time points. (A) Representative fluorescence image of positive control, which was tested on a cross section of normal mouse eye. (B, C, E, F, G) Representative fluorescence images of cross sections of normal eye and the eyes enucleated at 0, 3, 7, and 10 days after CP. (D) Combination of FITC, DAPI, and Cy3 channel (autofluorescence) fluorescence images of (C), showing nuclear staining (blue) and tissue background autofluorescence (overlap of green and red). (H) Bright-field image of (G). (H, arrows) show the vacuoles in RPE. No TUNEL-positive cells were observed in the retinal cross sections before and after CP. Scale bars, 100 μm.
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