November 2003
Volume 44, Issue 11
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Retinal Cell Biology  |   November 2003
Synaptic Plasticity in Mammalian Photoreceptors Prepared as Sheets for Retinal Transplantation
Author Affiliations
  • Mohamad A. Khodair
    From the Departments of Neurosciences and Ophthalmology, Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey, Newark, New Jersey.
  • Marco A. Zarbin
    From the Departments of Neurosciences and Ophthalmology, Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey, Newark, New Jersey.
  • Ellen Townes-Anderson
    From the Departments of Neurosciences and Ophthalmology, Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey, Newark, New Jersey.
Investigative Ophthalmology & Visual Science November 2003, Vol.44, 4976-4988. doi:10.1167/iovs.03-0036
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      Mohamad A. Khodair, Marco A. Zarbin, Ellen Townes-Anderson; Synaptic Plasticity in Mammalian Photoreceptors Prepared as Sheets for Retinal Transplantation. Invest. Ophthalmol. Vis. Sci. 2003;44(11):4976-4988. doi: 10.1167/iovs.03-0036.

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

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Abstract

purpose. To investigate systematically the early morphologic changes in axon terminals of adult mammalian rod and cone photoreceptors prepared as a sheet for subretinal transplantation.

methods. An in vitro system was designed to maintain adult porcine retinas for up to 48 hours. Photoreceptor sheets, prepared by vibratome sectioning, and full-thickness retinas were cultured at temperatures similar to those in pretransplantation storage (4°C) and after transplantation (37°C). Changes in the outer nuclear and outer plexiform layers were analyzed, using immunohistochemistry, laser scanning confocal microscopy, and image analysis.

results. Morphologic changes were observed in photoreceptor sheets as early as 10 minutes after incubation. The most significant change was the retraction of photoreceptor axons and terminals toward their cell bodies. Retraction was temperature dependent, being exacerbated at 37°C compared with 4°C, at its maximum by 24 hours of culture, and present in sheets obtained from both superior and inferior retina. The cause of this movement was not preparation techniques associated with vibratome sectioning or gelatin removal. Retraction was also present in full-thickness neural retina incubated at 37°C. Reduction in outer nuclear layer cell counts and thickness were also evident in these preparations, primarily in photoreceptor sheets.

conclusions. Adult photoreceptor sheets, a potential graft preparation for retinal transplantation, show a rapid retraction of axon terminals toward the cell bodies during culture. Although retraction may impede synaptic integration after transplantation, this intrinsic plasticity could be redirected to stimulate graft–host interaction.

For the past two decades, retinal cell transplantation has been investigated as a potential treatment for inherited retinal dystrophies such as retinitis pigmentosa and age-related macular degeneration. Although survival of graft tissue has been demonstrated, restoration of vision in humans has not been achieved. 1 2 3 4 5 6 7 In rodent models, some indications of improved visual function have been reported. 8 9 10 11 12 The cause of this functional improvement, however, is unclear. 
It is generally acknowledged that a robust improvement in vision will require trophic support of the degenerating retina and functional synaptic connectivity between the graft and the host. Presently, the evidence of synapse formation is sparse. 9 13 14 15 16 If donor photoreceptor cells or sheets are used for transplantation, the structural integrity as well as the synaptic plasticity of the photoreceptor terminals may play a pivotal role in determining the degree of graft–host synaptic interaction. Structural alterations in rod terminals in cultures of isolated photoreceptors 17 or after experimental retinal detachment 18 occur within hours to days. We propose therefore that synaptic changes occurring during graft preparation or shortly after transplantation may be critical to subsequent graft–host synaptic integration. 
To examine this hypothesis, an in vitro system was developed to monitor synaptic changes in adult porcine photoreceptors prepared for transplantation as a sheet of cells. Photoreceptor sheets have the advantages of maintaining correct orientation and lamination over a relatively large area and reducing contamination of the transplant with other retinal cells, compared with cell suspensions or microaggregates. 9 19 20 21 22 23 24 Adult tissue was chosen because mature photoreceptor sheets have shown comparable results to those of immature sheets when transplanted into the subretinal space 9 20 23 especially when transvitreal grafting procedures were used. 1 Moreover, the presynaptic plasticity displayed by adult photoreceptors during disease 18 25 26 27 28 29 30 31 32 33 34 and in culture 17 35 36 supports the notion that adult photoreceptors are capable of synapse formation after transplantation. Finally, the porcine retina was used because the anatomy, size, and vasculature of the porcine eye are close to those of the human, 37 38 39 and the porcine retina is well endowed with cones that form an area centralis. 40  
Photoreceptor sheets were studied for up to 48 hours at two temperatures that mimic pretransplantation storage conditions (4°C) and posttransplantation conditions (37°C). For identifying the changes in rod and cone cell terminals, antibodies to the synaptic vesicle integral membrane proteins synaptic vesicle protein-2 (SV2) and synaptophysin were used in conjunction with either the nuclear stain propidium iodide, which highlights the nuclear layers, or a rod-specific opsin antibody, which identifies rod photoreceptor cell membranes. Structural plasticity was analyzed with laser scanning confocal microscopy and imaging software. Parts of this work have been reported in abstract form (Khodair MA, et al. IOVS 2000;41:ARVO Abstract 4538; Khodair MA, et al. IOVS 2001;42:ARVO Abstract 4192). 
Materials and Methods
Animals
Twenty-one young adult Yorkshire pigs, males and females 3 to 5 months of age and weighing 25 to 55 kg (Animal Biotech Industries, Danbora, PA) served as donors of retinal tissue. Animals were maintained on a 12-hour light–dark cycle and fed porcine chow ad libitum. Animals were killed at 9 AM by anesthetizing with 2.2 mL telazol (7 mg/kg; 100 mg/mL), 0.7 mL xylazine (2.2 mg/kg; 100 mg/mL), and 1.2 mL atropine (0.02 mg/kg; 0.54 mg/mL) administered intramuscularly, followed by death by intravenous overdose of pentobarbital sodium (1.0 mL/4.5 kg). Experimental procedures adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the University of Medicine and Dentistry of New Jersey-New Jersey Medical School Institutional Animal Care and Use Committee. 
Preparation of Photoreceptor Sheets
Photoreceptor sheets were prepared by a method modified from Huang et al. 21 Briefly, the anterior segment and vitreous body were removed as one unit, minimizing traction on the retina. Retina, with attached retinal pigment epithelium (RPE), choroid, and sclera, was obtained from the central and midperipheral regions of both the superior and inferior halves of the eyecup as 7-mm buttons, using a trephine. Tissue was kept at 4°C at all times in Dulbecco’s modified minimum essential medium (DMEM, M-0643; Sigma-Aldrich, St. Louis, MO), supplemented with 10% (vol/vol) fetal calf serum, 10 μg/mL porcine insulin, 5.5 mM d-glucose, 1.0 mM pyruvate, 0.1 mM taurine, 2.0 mM ascorbic acid, 100 U/mL penicillin, 100 μg/mL streptomycin, and 250 ng/mL amphotericin B (pH 7.4), aerated with humidified 5% CO2-95% O2. A sterile 32.5% gelatin block was affixed to the specimen table of a vibratome sectioning system, equipped with cooling and semiautomatic advance systems (Series 2000; Ted Pella Inc., Redding, CA) and submerged in calcium-free, magnesium-free 1× Dulbecco’s phosphate-buffered salt solution (D-PBS; Cellgro; Mediatech Inc., Herndon, VA). Double-edged stainless steel razor blades (American Safety Razor Co., Staunton, VA) were gas sterilized, soaked in xylene, flushed with 70% ethanol, and mounted on the vibratome. The neural retina was detached gently from the underlying tissues and mounted onto the gelatin block, with the photoreceptor outer segments (OS) oriented downward. Gelatin was prepared from lyophilized gelatin (Sigma-Aldrich, G1890; Type A from porcine skin, 300 bloom) reconstituted in 1× D-PBS. A thin film of 2% molten gelatin (37°C) was introduced between the retina and the underlying gelatin block, and a dome of 8% molten gelatin (37°C) was poured on top of the retina and allowed to harden to create stability during vibratome sectioning. Cutting of the retinal sections proceeded in increments of 10 to 20 μm. To preserve the integrity of the photoreceptor terminals, vibratome sectioning was terminated at a level where one to two cells of the inner nuclear layer (INL) remained. This level was determined based on knowledge of the differential thickness of the retinal layers provided by a histologic analysis of porcine retina (manuscript in preparation), and the distribution of the retinal vasculature. 39 The vibratome was then advanced to undercut the remaining retina with an underlying 100-μm-thick gelatin layer. The vibratome-sectioned retina and underlying gelatin layer were covered by another 100-μm-thick gelatin layer. The gelatin-embedded photoreceptor sheet was incubated for 30 to 60 seconds at 37°C, to allow the gelatin layers to coalesce, and then placed on ice to resolidify. The final product was a photoreceptor sheet embedded in a gelatin sandwich, with a total thickness of approximately 250 to 300 μm. Although an effort was made to maintain all specimens at 4°C until the time of incubation, fluctuations in temperature were inevitable during such processes as gelatin embedding, vibratome sectioning, and formation of the gelatin sandwich. 
Culture of Photoreceptor Sheets and Full-Thickness Retinas
Photoreceptor sheets and full-thickness retinas were maintained in culture under the same conditions. For full-thickness preparations, retinas were embedded in gelatin sandwiches according to the procedure just described, but without sectioning the retina. The time elapsed between enucleation and the completion of sectioning ranged from 4 to 8 hours. The range in preparation time resulted from the fact that not all specimens could be processed simultaneously. For final incubation, which was 8 hours after enucleation for all specimens, preparations were placed in fortified DMEM (pH 7.4) (described above) in six-well dishes (Fisher Scientific, Morris Plains, NJ) with OS oriented toward the floor of the well and overlaid by 8 μm polycarbonate membrane inserts (Fisher Scientific). Specimens were incubated at 4°C (to simulate storage temperature) or 37°C (to simulate body temperature after subretinal transplantation) in humidified 5% CO2-95% O2. At the end of incubation, specimens were fixed in 4% paraformaldehyde in 0.125 M PBS (pH 7.4). For comparison of photoreceptor sheets versus full-thickness retinas, one eye of an animal was used to obtain sheets and the other eye to obtain full-thickness retinal preparations. Control specimens were full-thickness retinas obtained from the midperipheral regions of each half of the globe and fixed immediately after detachment from the underlying RPE. 
Specimens were fixed overnight at room temperature, rinsed in PBS, re-embedded in gelatin, and returned to fixative for at least an additional 24 hours at 4°C. Fixed specimens were rinsed in PBS, sectioned at 100 μm on an automated tissue cutter (Sorvall, Newtown, CT), and stored in PBS (pH 7.4) at 4°C. 
Immunohistochemistry
To evaluate photoreceptor terminals, the mouse monoclonal antibody anti-synaptic vesicle protein 2 (anti-SV2; the generous gift of Kathleen Buckley, Harvard Medical School, Boston, MA) and the rabbit polyclonal antibody anti-synaptophysin (Dako Corp., Carpinteria, CA) were used. Both the anti-SV2 and anti-synaptophysin antibodies are specific markers for presynaptic terminals across a wide variety of species, including the pig. 34 The nuclear stain, propidium iodide (Molecular Probes, Eugene, OR), was used as a marker for the nuclear layers (Fig. 1A) . A mouse monoclonal antibody specific for rod opsin, 4D2 (kindly provided by Robert Molday, University of British Columbia, Vancouver, BC, Canada) was used to identify photoreceptor membranes (Fig. 1B) . Goat anti-mouse and goat anti-rabbit conjugated to FITC and goat anti-mouse conjugated to tetramethylrhodamine isothiocyanate (TRITC) (Roche Diagnostics Corp., Indianapolis, IN) were used as secondary antibodies. Sections mounted onto glass slides were immunolabeled as described by Nachman-Clewner and Townes-Anderson. 17 In some experiments, retinas were stained with propidium iodide diluted 1:1000 in PBS, after immunolabeling. For double-label experiments, primary antibodies were diluted together in appropriate buffer and added to specimens simultaneously, as were the secondary antibodies. Omission of the primary antibodies constituted the control immunolabeling. Specimens were mounted in antifade medium containing 90% glycerol, 10% PBS, and 2.5% (wt/vol) 1,4 diazobicyclo[2,2,2] octane (DABCO, Sigma-Aldrich). 
Morphometric Analysis
Optical sections, 1 or 2 μm thick, of photoreceptor sheets and full-thickness retinas were obtained using a laser scanning confocal microscope equipped with an argon/krypton laser and 63× 1.4 numerical aperture (N/A) oil-immersion and 40× 1.2 N/A water-immersion objective lenses (LSM-410; Carl Zeiss Meditec, Oberkochen, Germany). The same objective lens was used throughout the analysis in any single set of experiments. A 488-nm excitation filter with a 515- to 540-nm narrow band-pass emission filter and a 568-nm excitation filter with a 590-nm long band-pass emission filter were used for FITC (green) and TRITC/propidium iodide (red) imaging, respectively. Brightness and contrast were set to obtain unsaturated images. These parameters were maintained throughout a single experiment. Thus, with normalized settings, changes in staining between specimens could be detected. Adjustments in brightness and contrast were performed later solely for presentation purposes. 
Digitized images were analyzed using NIH Image 1.62 software (available by ftp at zippy.nimh.nih.gov/ or at http://rsb.info.nih.gov/nih-image; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD). To quantify outer nuclear layer (ONL) thickness, measurements were taken of the distance from the base of the inner segment (IS) to the base of the innermost cell body of the ONL. The cell count of the ONL was determined by counting along a vertically oriented straight line, perpendicular to the outer plexiform layer (OPL; Fig. 1C ). To obtain the area of SV2 and synaptophysin staining in the ONL, we opened images in the fluorescein channel only, and a standard threshold was assigned to eliminate background fluorescence. The area of labeling was in squared pixels within a rectangular area that included the entire thickness of the ONL (Fig. 1C , dotted lines). Measurements were scaled at 6 and 4 pixels/μm for specimens examined with the 63× and 40× objective lenses, respectively. Nine sections were examined from each specimen for each time point in each condition. The cumulative data from each specimen were compared with the data from other specimens. Statistical analysis was performed with an unpaired t-test or a one-way analysis of variance (ANOVA) with Tukey’s post hoc test for all pair-wise multiple comparisons, where appropriate. Significance was accepted at P < 0.05. 
Results
Morphologic Changes in Cultured Photoreceptor Sheets
In control retina, fixed immediately after detachment, the OS and IS of photoreceptors were straight and oriented toward the RPE (Fig. 1A) . The ONL, identified by propidium iodide staining, was composed of five to seven layers of cells with nuclei uniformly spaced from one another. There were no signs of pyknotic changes. SV2 labeling was confined to the plexiform layers. In the distal half of the OPL, which contains the photoreceptor terminals, there was a bilaminar organization (Fig. 1D) . The smaller-sized rod spherules with their characteristic globular appearance were generally located sclerad to the larger triangular-shaped cone pedicles. This finding is in agreement with Peng et al., 34 who described the same arrangement of rod and cone terminals in the porcine retina using an antibody against a cone-specific marker, the cone transducin γ-subunit. Dark puncta present in these profiles most probably represent synaptic invaginations. Photoreceptor axons were mostly devoid of SV2 labeling. 
To examine early morphologic changes under a temperature similar to that encountered during pretransplantation storage, photoreceptor sheets were fixed after 10 minutes (Fig. 2A) or 24 hours (Fig. 2B) in culture at 4°C. In general, under such conditions, photoreceptor sheets maintained their structural integrity compared with control cells. This was evident by preservation of the OS and IS organization and orientation, maintenance of ONL thickness with only a small but significant reduction after 24 hours in culture (Fig. 3A) , preservation of the normal ONL cell stratification and order (Fig. 3B) , and uniformity of the SV2 terminal labeling, which remained largely confined to the OPL. Some increased labeling, however, was detected in the ONL, especially in specimens maintained in culture for 24 hours (Figs. 2B 3C) . Therefore, it appears that photoreceptor sheets prepared for transplantation and maintained at 4°C generally retained their structural integrity. 
Photoreceptor sheets from both superior and inferior halves of the eyecup were compared to determine whether regional differences in rod-cone densities, reported in previous studies 40 and confirmed by the results of a histologic study of the porcine retina conducted in our laboratories (manuscript in preparation), would influence the overall structural integrity of the photoreceptor sheet and/or result in variations in photoreceptor terminal morphology. There were no significant differences in ONL thickness or number of cell layers between superior and inferior retina (Figs. 3A 3B) . However, specimens obtained from the inferior retina showed significantly larger areas of SV2 labeling in the ONL than did superior specimens after 10 minutes in culture (Fig. 3C , P < 0.001). This discrepancy disappeared after 24 hours in culture, when no significant differences in SV2 labeling were observed between the hemiretinas (Fig. 3C)
To examine early morphologic changes at a temperature similar to that after subretinal transplantation, photoreceptor sheets were maintained in culture for the same time intervals but at 37°C (Figs. 2C 2D) . The gelatin sandwich around these specimens had melted by the time of fixation. By 24 hours at this temperature, the sheets were less organized, and the OS had become shorter, distorted, and disoriented (Fig. 2D) . A significant reduction in ONL thickness was observed as early as 10 minutes into the culture period (Fig. 3D , P < 0.001). ONL layers also exhibited disorganization early on but a significant reduction in the number of cell layers only after 24 hours (Fig. 3E , P < 0.05). This reduction could not entirely account for the reduction in ONL thickness. These findings were similar in both the superior and inferior halves of the retina. 
SV2 labeling started to spread from the OPL into the ONL after 10 minutes (Fig. 2C) . After 24 hours, labeling had spread as far as the base of the IS (Fig. 2D) . Concomitant dropout of label was seen in some areas of the OPL (see Fig. 2F , inset). Absence of label in the OPL was most frequent in areas displaying abundant synaptic vesicle stain in the ONL. Both rod spherules and cone pedicles showed deformities in their shape and disappearance of localized dark puncta, the sites of synaptic invagination. Cone pedicles, however, appeared to be more resistant to change than rod spherules, especially at early stages. The spread of SV2 labeling into the ONL was most extensive in specimens kept in culture at 37°C for 24 hours and resulted in an approximately ninefold increase in the SV2-labeled area of the ONL in both superior and inferior retinas when compared to the control samples (Fig. 3F , P < 0.001). Corresponding specimens maintained in culture at 4°C for 24 hours showed only three- and fivefold increases in ONL labeling for superior and inferior retinas, respectively (Fig. 3C , P < 0.001). 
The presence of SV2 in the ONL as well as the occasional absence of label in areas of the OPL closely resembled the pattern of synaptic vesicle protein labeling seen after experimental retinal detachment. 18 25 This labeling pattern represents retraction of photoreceptor axons and terminals toward their cell bodies after retinal detachment 18 25 and appeared to indicate axonal retraction in our specimens as well. 
Morphologic Changes Reexamined with Alternate Immunohistochemical Markers
To confirm the structural changes in photoreceptor sheets at 37°C, photoreceptor sheets were prepared from the superior hemiretinas and double labeled using anti-synaptophysin and anti-rod opsin (4D2) antibodies (Figs. 2E 2F) . Opsin, primarily localized in the rod OS, is found throughout the photoreceptor membrane after detachment of the retina from the RPE. 41 Thus, opsin was used to demarcate the rod photoreceptor cell membrane. Culture time was also extended to 48 hours. Sheets were maintained in culture for less than 45 minutes, 24 hours, or 48 hours. Full-thickness retinas from the midperipheral areas, fixed immediately after detachment from the RPE, served as the control (Fig. 1B)
Consistent with data obtained from the SV2–propidium iodide double-labeling experiments, the ONL began to thin almost immediately and was only 60% of control thickness at 48 hours (Fig. 4A , P < 0.05). The ONL cell layers were also reduced, but only after 24 hours in culture (Fig. 4A , P < 0.05). The reduction in cell layers, as noted in the SV2-propidium iodide–stained specimens, was too small to explain the reduction in ONL thickness. The area of synaptophysin labeling in the ONL increased approximately ninefold by 45 minutes in culture and then tripled after 24 hours but did not change thereafter (Fig. 4B , P < 0.001). Using anti-opsin, rod OS were brightly stained. However, some higher than control staining in the IS was evident by 45 minutes in culture. By 24 to 48 hours, opsin staining was widespread, delineating the entire photoreceptor cell membrane, and colocalization of opsin and synaptophysin in photoreceptor terminals was detected in the ONL (Fig. 2F) . Thus, labeling with anti-synaptophysin confirmed the dramatic spread of vesicle membrane staining with anti-SV2, and anti-opsin confirmed that rod cells were affected. 
Photoreceptor Sheets versus Full-Thickness Retinas
To determine whether vibratome sectioning influences photoreceptor axonal and terminal retraction, indicated by synaptic vesicle labeling in the ONL, full-thickness retinas were compared with photoreceptor sheets. In one experiment, specimens were maintained at 4°C or 37°C for less than 45 minutes and 24 hours and labeled with SV2 and propidium iodide. In general, full-thickness retinas showed less disruption of their morphology than photoreceptor sheets. Substantial spread of labeling occurred, however, in both full-thickness retinas and photoreceptor sheets (Figs. 5A 5B)
For photoreceptor sheets and full-thickness retinal specimens kept at 4°C, there was a small but significant increase in ONL SV2 labeling in superior and inferior hemiretinas at 45 minutes compared with controls (Figs. 6A 6B , P < 0.05). By 24 hours, a larger increase in the area of labeling was seen, however. The photoreceptor sheets had significantly larger areas of labeling than the full-thickness retinal preparations (Figs. 6A 6B , P < 0.001). Vibratome sectioning may allow some fluctuations in temperature, resulting in changes in SV2 staining not seen in full-thickness retina. At 37°C, photoreceptor sheets and full-thickness retinas from both the superior and inferior hemiretinas had an approximately three- to fourfold increase in the ONL area labeled with SV2 by 45 minutes in culture (Figs. 6C 6D , P < 0.001). By 24 hours, the area of labeling in both preparations reached a 14- to 24-fold increase (Figs. 6C 6D , P < 0.001). The fact that both vibratome-sectioned photoreceptor sheets and full-thickness retinas exhibited the same amount of labeling in the ONL at 37°C (Figs. 6C 6D) indicates that vibratome sectioning alone is not the cause of this phenomenon. 
For photoreceptor sheets, specimens obtained from the inferior hemiretina had somewhat higher areas of SV2 labeling in the ONL than those obtained from the superior retina (P < 0.001) after 45 minutes in culture at both 4°C (Figs. 6A 6B) and 37°C (Figs. 6C 6D) . This difference in labeling disappeared after 24 hours in culture. These findings corroborate the results of the previous experiments and indicate that photoreceptor terminals of the inferior retina initially show a higher rate and/or larger magnitude of retraction than those obtained from the superior retina. 
Morphometric analysis of ONL thickness and cell layers showed significant changes, primarily in photoreceptor sheets. Data from superior and inferior (not shown) retinas showed similar results. At 37°C, there was a 14% to 33% reduction in ONL thickness after 45 minutes and 24 hours, respectively (Fig. 7B) . A significant reduction in thickness was also seen at 4°C but only after 24 hours in culture in photoreceptor sheets (Fig. 7A , P < 0.05). In full-thickness specimens, no significant reduction of ONL thickness was observed. For ONL cell layers, there was a small reduction in both preparations only after 24 hours in culture at 37°C (Fig. 7B , P < 0.05). Thus, full-thickness retina was more resistant to changes in ONL thickness than were photoreceptor sheets. 
In a second experiment, gelatin-embedded photoreceptor sheets and full-thickness retinas were prepared from the superior hemiretina and labeled with anti-synaptophysin and anti-opsin antibodies. Specimens were maintained in culture for less than 45 minutes, 24 hours, and 48 hours at 37°C only. Consistent with the data obtained from the previous experiment, both photoreceptor sheets and full-thickness retinas showed extensive spread of synaptophysin labeling into the ONL, which presumably represents photoreceptor axonal and synaptic terminal retraction, beginning during the first 45 minutes of culture (Fig. 8A) . Least-squares regression analysis was used to analyze the rate of change in synaptic terminal labeling. Half-maximum retraction was reached after only 1.75 ± 0.01 and 2.6 ± 0.53 hours in vibratome-sectioned photoreceptor sheets and full-thickness retinal sheets, respectively. The rate of retraction then plateaued after the 24-hour time point. 
As before, there was a significant reduction in ONL thickness only in photoreceptor sheets (Fig. 8B , P < 0.05). Regression analysis showed that reduction of ONL thickness occurred, for the most part, within the first 45 minutes in culture. There was a small but statistically significant decrease in the number of ONL cell layers by 24 hours in culture in photoreceptor sheets and intact retina (Fig. 8C , P < 0.05). According to regression analysis, the highest rate of reduction in ONL layers occurred during the first 45 minutes of culture. Thus, whether in sheets or intact retina, once incubation at 37°C began, changes occurred rapidly. 
Effect of Gelatin on Morphologic Changes
Because increased temperature increased morphologic change, it is possible that melting, and thus removing, gelatin at 37°C allows movement in the outer retina. To examine whether the absence or presence of the gelatin support influenced the extent of retraction of photoreceptor terminals and ONL thickness, photoreceptor sheets were first incubated in medium at 37°C for 30 minutes to allow the gelatin to melt. Culture medium was then replaced, and retinas were incubated at 4°C for less than 30 minutes or 24 hours. Controls were photoreceptor sheets maintained in culture at either 4°C or 37°C throughout the experiment. Culture medium was replaced for the control and experimental specimens. All specimens were also compared with full-thickness retinas fixed immediately after detachment from the RPE. 
When compared with specimens that were maintained at 4°C in culture (i.e., where the gelatin was not removed, Fig. 5C ), specimens kept at 4°C after removal of the gelatin showed no substantial increase in the area of the ONL labeled with SV2 even after 24 hours in culture (Fig. 5D) . In morphometric analysis, both specimens showed significantly less ONL SV2 labeling than specimens maintained at 37°C for 24 hours in culture (Fig. 9A , P < 0.001). All specimens, however, showed significantly higher ONL SV2 labeling than control full-thickness retinas (P < 0.001). Results from the superior retina (Fig. 9A) were similar to those from the inferior retinal specimens (data not shown). In summary, the pattern of photoreceptor axonal and terminal retraction was similar in photoreceptor sheets with or without gelatin support maintained at 4°C in culture, and both showed a significantly different pattern than that of specimens maintained in culture at 37°C. These results indicate that gelatin has no effect on retraction, but temperature plays a key role in the spread of labeling. 
For ONL thickness, sheets from which gelatin had been removed before culture at 4°C showed a significant reduction, like those maintained in culture at 37°C (Fig. 9B) . In contrast, there was no significant difference between the control full-thickness retina, fixed immediately after detachment, and photoreceptor sheets with gelatin support maintained in culture at 4°C for 30 minutes. Only after 24 hours in culture did these sheets show a reduction in ONL thickness (P < 0.001). Similar results were obtained from the inferior hemiretina (data not shown). It appears therefore that gelatin maintained at 4°C can provide some support to the photoreceptor sheet preparations. 
For ONL cell layering, the only significant reduction in the number of ONL layers was seen in photoreceptor sheets maintained in culture at 37°C in both superior (Fig. 9C) and inferior (data not shown) hemiretinas (P < 0.05). These results suggest that cell loss was minimized at low temperatures in culture, irrespective of gelatin support. 
Discussion
Structural Plasticity of Photoreceptor Axons
Recent studies have demonstrated that the processes of adult retinal neurons respond to insult or injury by extensive remodeling. Adult photoreceptor axonal and terminal retraction, 17 42 as well as neurite outgrowth, presynaptic varicosity development, and synapse formation, 35 36 have been observed in vitro. Retraction of photoreceptor terminals and subsequent neurite extension have also been seen in studies of experimental retinal detachment and reattachment 18 25 (Lewis GP, et al. IOVS 2002;43:ARVO E-Abstract 4540). In addition, reactive sprouting and/or synaptogenesis by photoreceptors has been observed in several animal models of retinal degeneration, including the light-damaged mouse retina, 27 29 the rd and rds mouse, 26 28 29 34 43 and the rhodopsin Pro347Leu transgenic pig, 34 and in human donor retinas from eyes with retinitis pigmentosa. 30 32 44 Thus, it is evident that adult photoreceptor terminals are capable of structural plasticity. 
In this study, we focused on changes in terminals of donor photoreceptors prepared as sheets for transplantation. The hallmark of our findings is the substantial and consistent spread of the synaptic vesicle protein markers SV2 and synaptophysin from the OPL deep into the ONL concomitant with a loss of label in the OPL. Erickson et al. 25 and Lewis et al. 18 suggested that this phenomenon, also observed in their models of experimental retinal detachment, represents retraction of photoreceptor axons and terminals toward their cell bodies. An alternative explanation of the redistribution of synaptic vesicle labeling in the present study is defective transport of the synaptic vesicles to the photoreceptor terminals, without actual retraction of these terminals. However, accumulation of synaptic vesicle protein labeling within photoreceptor IS, which would occur if transport was blocked, was not observed in any of the specimens. Moreover, some of the SV2- and synaptophysin-labeled profiles in the ONL bore a striking resemblance in size and shape to adult photoreceptor terminals. Thus, retraction of terminals seems to be responsible for the SV2 and synaptophysin label in the ONL in the porcine retina. 
This axonal activity may reduce the possibility of graft–host neuronal connectivity after transplantation. Retraction may represent a step in a process of structural and biochemical alterations that renders photoreceptors incapable of reestablishing synaptic contacts with second-order neurons. Alternatively, retraction of photoreceptor axons and terminals may be viewed as a protective mechanism that the photoreceptor undergoes in response to injury or insult, a process of cytoarchitectural remodeling that allows cells to preserve their resources and enhance their recovery once conditions again become favorable. Similar reasoning can be applied to the shortened OS after injury, in which case shortening may be an attempt to conserve the cell’s energy for survival. 45 In either case, however, preventing or reversing retraction may be an essential step in establishing a functional neural network after transplantation. 
Temporal Course of Morphologic Changes
Analysis of the rate of morphologic change in the ONL and photoreceptor terminals demonstrated that most of the observed changes occurred during the first 24 hours of incubation. This was true for both photoreceptor sheets and full-thickness retinas. This finding recalls that of Erickson et al. 25 and Lewis et al., 18 who demonstrated a rapid response of retinal cells to experimental retinal detachment, including disorganization of the OPL, retraction of photoreceptor terminals, extension of rod bipolar and horizontal cell processes into the ONL, and photoreceptor cell death. Nachman-Clewner and Townes-Anderson 17 and Nachman-Clewner et al. 42 have also demonstrated that axonal retraction in cultured photoreceptors occurs within hours after dissociation of the retina. Early changes after detachment are not limited to structural changes but involve metabolic changes as well. Sherry and Townes-Anderson 46 have demonstrated that alterations in the glutamatergic system of photoreceptors appear as early as 5 minutes after detachment. A decrease in RNA synthesis 47 was also reported to occur within the first 24 hours after detachment. The present findings emphasize the importance of studying the early changes that photoreceptors undergo, because they may influence synaptogenesis and graft–host integration. 
Possible Mechanisms Underlying Retraction
What initiates retraction of adult photoreceptor terminals? Our results indicate that in sheet preparations, increased temperature, but not loss of gelatin, is associated with increased retraction. Temperature dependence in turn indicates that structural change requires a metabolically active cell. The trigger for retraction may result from direct perturbation of the photoreceptor terminals, loss of Müller glial cells, and/or removal or injury of the photoreceptor postsynaptic targets, the bipolar and horizontal neurons (i.e., detargeting) during and after vibratome sectioning. These possibilities seem plausible in light of the well-recognized role of both target-derived and Müller glial cell-derived trophic and metabolic support of photoreceptors, 48 49 as well as the crucial role of Müller cells in providing structural stability for retinal neurons. 49 None of these possibilities seems likely to be the major contributor to retraction, however, as retraction of equal magnitude was observed in full-thickness retinal preparations in which photoreceptor terminals were not disturbed by vibratome sectioning, and Müller cells as well as second-order neurons were spared. It remains possible, however, that even the spared glial and second-order neuronal cells in the full-thickness preparations are not completely functional or may even enhance retraction due to the reactive changes these cells undergo in response to detachment and/or injury. 45 49 Conversely, retraction is unlikely to be triggered by the release of diffusible factor(s), substance(s), or signal(s) from the inner retina, because it also occurred in vibratome sectioned photoreceptor sheets in which the inner retina had been removed. 
Common to both photoreceptor sheet and full-thickness retinal preparations, however, are the mechanical trauma caused by cutting the retina using the trephine and detachment of photoreceptors from the RPE, and the loss of trophic, metabolic, and functional support normally provided by RPE. A massive wave of depolarization, a phenomenon known as spreading depression involving both neurons and glia, accompanies mechanical insult and may cause retraction. 50 51 Spreading depression in the retina causes extensive redistribution of ions and amino acid neurotransmitters between the intra- and extracellular compartments 46 51 52 and, presumably, activation of voltage-gated channels. The presynaptic localization of voltage-gated L-type Ca2+ channels at the photoreceptor ribbon synapse has been demonstrated in rods cells 42 53 where they regulate neurotransmitter release. 54 For developing and mature neurons, depolarization-induced neurite retraction is mediated by a rise in intracellular Ca2+ concentration, whereas blocking depolarization-induced Ca2+ influx through voltage-gated L-type Ca2+ channels prevents retraction and inhibits neurite outgrowth. 55 56 57 In adult rod photoreceptors specifically, Nachman-Clewner et al. 42 have demonstrated that terminal retraction in culture is Ca2+-dependent and that blockage of Ca2+ influx through voltage-gated L-type Ca2+ channels using a dihydropyridine antagonist prevents retraction. Therefore, a detachment of photoreceptors from RPE during retinal preparation that is accompanied by membrane depolarization and Ca2+ influx may initiate the photoreceptor axonal and terminal retraction observed in the porcine specimens. 
Reduction in the ONL and Possible Underlying Mechanisms
There was a small but significant reduction in the number of cell layers in the ONL of photoreceptor sheets and full-thickness retinas after 24 to 48 hours in culture at 37°C. Several investigators have reported photoreceptor cell death after experimental 58 59 and human 60 retinal detachment. Cell death presumably occurs at least partly as a result of ischemia. Recent studies have shown that oxygen supplementation minimizes the degree of photoreceptor cell death in experimental retinal detachment. 59 The high O2 tension in our cultures may have minimized, although not prevented, cell death. 
There was also a significant reduction in ONL thickness in photoreceptor sheets. This result cannot be explained solely by cell death, because the reduction in the number of cell layers was too small and occurred too slowly to account for the reduction in ONL thickness. Moreover, cell death represented by reduction in ONL cell layering occurred to a similar extent in full-thickness preparations that do not exhibit a significant reduction in ONL thickness. Other mechanisms may explain the reduction in ONL thickness. First, in photoreceptor sheets, Müller cells are lost as a result of vibratome sectioning. Second, horizontal expansion of the sheet preparations may occur, especially at 37°C, when gelatin support is absent. Finally, discrepancies in ONL thickness of photoreceptor sheets and full-thickness retinal preparations may result from hypertrophy of reactive Müller cells in the latter but not in the former preparation. 45 61  
Morphologic Variations in Rod and Cone Photoreceptors
Although specific markers to differentiate rod and cone populations were not used, most photoreceptor terminals that remain in the OPL after 45 minutes of incubation, based on shape, size, and position, were probably cone pedicles. Some differences in the rate of retraction were observed in superior and inferior retinas. Retraction occurred in the inferior retina at a higher rate during early stages in culture. Later, there was an increase in ONL labeling of the superior retina. Because there is a twofold difference in cone density in the superior retina compared with the inferior retina 49 (Khodair MA, et al. IOVS 2000;41:ARVO Abstract 4538), one interpretation is that rod cells show retraction early, but with time, cones, which are more numerous in the superior retina, eventually show axonal retraction as well. Studies using differential rod and cone labeling are needed to confirm this explanation, especially seeing that Lewis et al. 18 have reported that retraction occurs exclusively in rod cells in a feline model of experimental retinal detachment. 
In conclusion, an in vitro system has been developed for studying morphologic changes in photoreceptor cells prepared as sheets for transplantation at temperatures that simulate those encountered during preparation, storage, and transplantation. It is hoped that this system will shed some light on factors that influence the outcome of transplantation, a potential remedy for a variety of retinal degenerative diseases. Moreover, axonal changes that may be an impediment to graft–host synaptic integration have been shown to occur very soon after photoreceptor sheet preparation. Pharmacological manipulations to prevent these changes may enhance the success of transplantations and overcome the current obstacle posed by the paucity of graft–host synaptic contacts. 
 
Figure 1.
 
(A, B) Porcine full-thickness retina fixed immediately after detachment from RPE. Retinas prepared in this manner served as controls. (A) Retina immunolabeled with SV2 (green), stained with propidium iodide (red) and viewed in a 2-μm optical section with laser scanning confocal microscopy. OS (arrows) and IS (arrowheads) appear intact with the OS uniformly organized and properly oriented. (B) An alternative staining, retina immunolabeled with synaptophysin (green) and rod-specific opsin (4D2; red) and viewed in a 1-μm optical section. SV2 and synaptophysin uniformly label both plexiform layers. Propidium iodide stained the nuclear layers, whereas 4D2 was confined to rod OS. (B) Cone IS exhibited autofluorescence that was distinct from 4D2 labeling of rod OS. (C) Morphometric analysis, illustrated in a photoreceptor sheet. To quantify the spread of synaptic labeling, the area of SV2 labeling within the ONL was measured within a set rectangular frame (dashed lines). ONL thickness was quantified by measuring the distance from the base of the IS to the base of the innermost cell body of the ONL (double-headed arrow). A count of the ONL cell layers was made along the same perpendicular line. (D) A higher magnification of (A) depicting the characteristic large, triangular-shaped cone pedicles (arrows) and smaller globular rod spherules (arrowheads). Note the bilaminar arrangement of the photoreceptor terminals with the spherules located sclerad to the cone terminals. On occasion, dark puncta were present in photoreceptor terminals. They represent synaptic invaginations (arrow Image not available and arrowhead Image not available ). OS, outer segments; IS, inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer, INL, inner nuclear layer, IPL, inner plexiform layer; GCL, ganglion cell layer; and NFL, nerve fiber layer. Scale bar: (A) 50 μm; (B) 25 μm; (D) 10 μm.
Figure 1.
 
(A, B) Porcine full-thickness retina fixed immediately after detachment from RPE. Retinas prepared in this manner served as controls. (A) Retina immunolabeled with SV2 (green), stained with propidium iodide (red) and viewed in a 2-μm optical section with laser scanning confocal microscopy. OS (arrows) and IS (arrowheads) appear intact with the OS uniformly organized and properly oriented. (B) An alternative staining, retina immunolabeled with synaptophysin (green) and rod-specific opsin (4D2; red) and viewed in a 1-μm optical section. SV2 and synaptophysin uniformly label both plexiform layers. Propidium iodide stained the nuclear layers, whereas 4D2 was confined to rod OS. (B) Cone IS exhibited autofluorescence that was distinct from 4D2 labeling of rod OS. (C) Morphometric analysis, illustrated in a photoreceptor sheet. To quantify the spread of synaptic labeling, the area of SV2 labeling within the ONL was measured within a set rectangular frame (dashed lines). ONL thickness was quantified by measuring the distance from the base of the IS to the base of the innermost cell body of the ONL (double-headed arrow). A count of the ONL cell layers was made along the same perpendicular line. (D) A higher magnification of (A) depicting the characteristic large, triangular-shaped cone pedicles (arrows) and smaller globular rod spherules (arrowheads). Note the bilaminar arrangement of the photoreceptor terminals with the spherules located sclerad to the cone terminals. On occasion, dark puncta were present in photoreceptor terminals. They represent synaptic invaginations (arrow Image not available and arrowhead Image not available ). OS, outer segments; IS, inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer, INL, inner nuclear layer, IPL, inner plexiform layer; GCL, ganglion cell layer; and NFL, nerve fiber layer. Scale bar: (A) 50 μm; (B) 25 μm; (D) 10 μm.
Figure 2.
 
(AD) Effects of temperature on photoreceptor sheets. Photoreceptor sheets were fixed within 10 minutes (A, C) or after 24 hours (B, D) of being placed in culture, immunolabeled with SV2 (green), stained with propidium iodide (red), and viewed in 2-μm optical sections by laser scanning confocal microscopy. Monochromes are in the green channel only, showing SV2 labeling. (A) After vibratome sectioning, only one to two cell layers of the INL remained along the inner surface of the photoreceptor sheet (arrows). When maintained for 10 minutes at 4°C, the photoreceptor terminals appeared intact, and the SV2 labeling was confined to the outer plexiform layer. (B) After 24 hours in culture at 4°C, photoreceptor terminals remained largely intact, and the overall structure of the photoreceptor sheet was well preserved, although a small amount of SV2 labeling was present in the ONL (arrowheads). (C) In photoreceptor sheets maintained at 37°C for 10 minutes, SV2 labeling was also mostly confined to the OPL, but some areas of SV2 labeling were present in the ONL (arrowheads). (D) Photoreceptor sheets maintained at 37°C for 24 hours showed morphologic changes. SV2 labeling was present deep within the ONL (arrowheads). There was also general disorganization of the ONL and reduction in its thickness. Both the OS and IS appeared distorted. (E, F) To confirm results obtained with SV2–propidium iodide double-labeling, specimens were fixed at various time points after incubation, immunolabeled for synaptophysin (synaptic vesicle protein) and 4D2 (rod opsin), using FITC-conjugated (green) and TRITC-conjugated (red) secondary antibodies, respectively, and viewed in 1-μm optical sections by laser scanning confocal microscopy. Monochromes are in the green channel only, showing synaptophysin labeling. Photoreceptor sheets were maintained in culture for up to 48 hours at 37°C. (E). Photoreceptor sheets maintained in culture for less than 45 minutes at 37°C maintained their structural integrity. Similar to SV2-labeled specimens, small areas of synaptophysin labeling were present in the ONL (arrowheads). (F) By 48 hours, disorganization of the OPL and ONL was evident. Synaptophysin labeling was no longer confined to the OPL but spread over a wide area of the ONL (arrowheads). OS and IS were distorted; 4D2 labeling was present in all layers of the sheet, and occasionally 4D2 and synaptophysin labeling colocalized (arrows). Inset: a higher magnification showing areas of drop out of label from the OPL (arrowheads) as labeling spread into the ONL, as well as colocalization of synaptophysin and 4D2 at a single photoreceptor synaptic terminal that retracted and was present in proximity to the cell soma (arrow). Scale bar: (D) 50 μm; (F, and inset) 25 μm.
Figure 2.
 
(AD) Effects of temperature on photoreceptor sheets. Photoreceptor sheets were fixed within 10 minutes (A, C) or after 24 hours (B, D) of being placed in culture, immunolabeled with SV2 (green), stained with propidium iodide (red), and viewed in 2-μm optical sections by laser scanning confocal microscopy. Monochromes are in the green channel only, showing SV2 labeling. (A) After vibratome sectioning, only one to two cell layers of the INL remained along the inner surface of the photoreceptor sheet (arrows). When maintained for 10 minutes at 4°C, the photoreceptor terminals appeared intact, and the SV2 labeling was confined to the outer plexiform layer. (B) After 24 hours in culture at 4°C, photoreceptor terminals remained largely intact, and the overall structure of the photoreceptor sheet was well preserved, although a small amount of SV2 labeling was present in the ONL (arrowheads). (C) In photoreceptor sheets maintained at 37°C for 10 minutes, SV2 labeling was also mostly confined to the OPL, but some areas of SV2 labeling were present in the ONL (arrowheads). (D) Photoreceptor sheets maintained at 37°C for 24 hours showed morphologic changes. SV2 labeling was present deep within the ONL (arrowheads). There was also general disorganization of the ONL and reduction in its thickness. Both the OS and IS appeared distorted. (E, F) To confirm results obtained with SV2–propidium iodide double-labeling, specimens were fixed at various time points after incubation, immunolabeled for synaptophysin (synaptic vesicle protein) and 4D2 (rod opsin), using FITC-conjugated (green) and TRITC-conjugated (red) secondary antibodies, respectively, and viewed in 1-μm optical sections by laser scanning confocal microscopy. Monochromes are in the green channel only, showing synaptophysin labeling. Photoreceptor sheets were maintained in culture for up to 48 hours at 37°C. (E). Photoreceptor sheets maintained in culture for less than 45 minutes at 37°C maintained their structural integrity. Similar to SV2-labeled specimens, small areas of synaptophysin labeling were present in the ONL (arrowheads). (F) By 48 hours, disorganization of the OPL and ONL was evident. Synaptophysin labeling was no longer confined to the OPL but spread over a wide area of the ONL (arrowheads). OS and IS were distorted; 4D2 labeling was present in all layers of the sheet, and occasionally 4D2 and synaptophysin labeling colocalized (arrows). Inset: a higher magnification showing areas of drop out of label from the OPL (arrowheads) as labeling spread into the ONL, as well as colocalization of synaptophysin and 4D2 at a single photoreceptor synaptic terminal that retracted and was present in proximity to the cell soma (arrow). Scale bar: (D) 50 μm; (F, and inset) 25 μm.
Figure 3.
 
Quantitative analysis of photoreceptor sheets maintained in culture at 4°C and 37°C. Controls were intact full-thickness retinas fixed immediately after detachment from RPE. (A, D) ONL thickness. (A) At 4°C, only after 24 hours in culture was there a significant reduction in ONL thickness compared with control specimens (*P < 0.001). (D) In contrast, at 37°C a significant reduction in ONL thickness was seen by 10 minutes, and at 24 hours in culture, both superior and inferior photoreceptor sheets were compared with their respective controls (*P < 0.001). These sheets were also significantly reduced in thickness compared with their 4°C counterparts at 10 minutes and 24 hours (P < 0.05). (B, E) ONL cell layering. (B) At 4°C no significant reduction in the number of ONL cell layers was seen in photoreceptor sheets obtained from either hemiretina for up to 24 hours in culture. (E) At 37°C, a small but significant reduction in the number of ONL cell layers was seen in both superior and inferior photoreceptor sheets only after 24 hours in culture (*P < 0.05). (C, F) ONL SV2 labeling. (C) Small but significant increases in SV2 labeling were present in the ONL of photoreceptor sheets obtained from both superior (*P < 0.01) and inferior (**P < 0.001) hemiretinas after 10 minutes in culture at 4°C compared with the control. (F) At 37°C there was a significant increase in the area of labeling by 10 minutes in culture in photoreceptor sheets obtained from both the superior and inferior hemiretinas compared with the control (*P < 0.05). At both temperatures at 10 minutes, photoreceptor sheets from the inferior hemiretina showed significantly larger areas of SV2 labeling than those from the superior hemiretina (P < 0.001). By 24 hours, this discrepancy had disappeared, with a significantly larger area of the ONL showing SV2 labeling in photoreceptor sheets obtained from either hemiretina compared with control cultures (**P < 0.001) or with specimens maintained in culture for only 10 minutes (P < 0.05). The area of SV2 labeling was greatest in sheets maintained in culture for 24 hours at 37°C. These specimens showed an approximate ninefold increase in labeling over the control (**P < 0.001) and more than double that in specimens maintained at 4°C for 24 hours (P < 0.001). These results underscore the significant role of temperature in these structural changes. Data are reported as mean ± SEM; n = 10 eyes of five animals.
Figure 3.
 
Quantitative analysis of photoreceptor sheets maintained in culture at 4°C and 37°C. Controls were intact full-thickness retinas fixed immediately after detachment from RPE. (A, D) ONL thickness. (A) At 4°C, only after 24 hours in culture was there a significant reduction in ONL thickness compared with control specimens (*P < 0.001). (D) In contrast, at 37°C a significant reduction in ONL thickness was seen by 10 minutes, and at 24 hours in culture, both superior and inferior photoreceptor sheets were compared with their respective controls (*P < 0.001). These sheets were also significantly reduced in thickness compared with their 4°C counterparts at 10 minutes and 24 hours (P < 0.05). (B, E) ONL cell layering. (B) At 4°C no significant reduction in the number of ONL cell layers was seen in photoreceptor sheets obtained from either hemiretina for up to 24 hours in culture. (E) At 37°C, a small but significant reduction in the number of ONL cell layers was seen in both superior and inferior photoreceptor sheets only after 24 hours in culture (*P < 0.05). (C, F) ONL SV2 labeling. (C) Small but significant increases in SV2 labeling were present in the ONL of photoreceptor sheets obtained from both superior (*P < 0.01) and inferior (**P < 0.001) hemiretinas after 10 minutes in culture at 4°C compared with the control. (F) At 37°C there was a significant increase in the area of labeling by 10 minutes in culture in photoreceptor sheets obtained from both the superior and inferior hemiretinas compared with the control (*P < 0.05). At both temperatures at 10 minutes, photoreceptor sheets from the inferior hemiretina showed significantly larger areas of SV2 labeling than those from the superior hemiretina (P < 0.001). By 24 hours, this discrepancy had disappeared, with a significantly larger area of the ONL showing SV2 labeling in photoreceptor sheets obtained from either hemiretina compared with control cultures (**P < 0.001) or with specimens maintained in culture for only 10 minutes (P < 0.05). The area of SV2 labeling was greatest in sheets maintained in culture for 24 hours at 37°C. These specimens showed an approximate ninefold increase in labeling over the control (**P < 0.001) and more than double that in specimens maintained at 4°C for 24 hours (P < 0.001). These results underscore the significant role of temperature in these structural changes. Data are reported as mean ± SEM; n = 10 eyes of five animals.
Figure 4.
 
Quantitative analysis using alternate immunohistochemical markers, synaptophysin, and rod opsin (4D2). (A) ONL thickness and cell layers. ONL thickness decreased significantly at all time points compared with the control (*P < 0.05). A small but significant reduction in the number of cell layers was also seen in these specimens after 24 and 48 hours in culture (*P < 0.05). (B) Synaptophysin labeling. A significant increase in the area of ONL labeling was observed in photoreceptor sheets by 45 minutes (*P < 0.001). By 24 hours, the synaptophysin-labeled area tripled (*P < 0.001) but did not change significantly thereafter. The pattern of these changes closely resembled that in photoreceptor sheets labeled with SV2-propidium iodide and maintained under the same culture conditions. Mean ± SEM. n = 4 eyes of four animals.
Figure 4.
 
Quantitative analysis using alternate immunohistochemical markers, synaptophysin, and rod opsin (4D2). (A) ONL thickness and cell layers. ONL thickness decreased significantly at all time points compared with the control (*P < 0.05). A small but significant reduction in the number of cell layers was also seen in these specimens after 24 and 48 hours in culture (*P < 0.05). (B) Synaptophysin labeling. A significant increase in the area of ONL labeling was observed in photoreceptor sheets by 45 minutes (*P < 0.001). By 24 hours, the synaptophysin-labeled area tripled (*P < 0.001) but did not change significantly thereafter. The pattern of these changes closely resembled that in photoreceptor sheets labeled with SV2-propidium iodide and maintained under the same culture conditions. Mean ± SEM. n = 4 eyes of four animals.
Figure 5.
 
(A, B) Full-thickness retinal preparations versus photoreceptor sheets. (A) Full-thickness retina and (B) a photoreceptor sheet fixed after 24 hours in culture at 37°C and immunolabeled for SV2 using a FITC-conjugated secondary antibody (green). Propidium iodide (red) was used as a marker for the nuclear layers. SV2 staining was present deep within the ONL of both preparations (arrowheads). (C, D) Effect of gelatin on photoreceptor sheets using anti-SV2 and propidium iodide staining. (C) Photoreceptor sheet maintained in culture for 24 hours at 4°C after gelatin removal at 37°C for 30 minutes. In contrast to photoreceptor sheets maintained at 37°C, SV2 labeling was largely confined to the OPL. (D) Photoreceptor sheet maintained in culture for 24 hours at 4°C without gelatin removal. As seen before (Fig. 2B) , most staining remained in the OPL. Note tangential cut of specimen showing several rows of photoreceptor terminals. Scale bar, 50 μm.
Figure 5.
 
(A, B) Full-thickness retinal preparations versus photoreceptor sheets. (A) Full-thickness retina and (B) a photoreceptor sheet fixed after 24 hours in culture at 37°C and immunolabeled for SV2 using a FITC-conjugated secondary antibody (green). Propidium iodide (red) was used as a marker for the nuclear layers. SV2 staining was present deep within the ONL of both preparations (arrowheads). (C, D) Effect of gelatin on photoreceptor sheets using anti-SV2 and propidium iodide staining. (C) Photoreceptor sheet maintained in culture for 24 hours at 4°C after gelatin removal at 37°C for 30 minutes. In contrast to photoreceptor sheets maintained at 37°C, SV2 labeling was largely confined to the OPL. (D) Photoreceptor sheet maintained in culture for 24 hours at 4°C without gelatin removal. As seen before (Fig. 2B) , most staining remained in the OPL. Note tangential cut of specimen showing several rows of photoreceptor terminals. Scale bar, 50 μm.
Figure 6.
 
Quantitative analysis of SV2 labeling in full-thickness retinal preparations versus photoreceptor sheets. (A, B) Culture at storage temperature of 4°C. The area of the ONL labeled with SV2 was compared in preparations harvested from the superior (A) and the inferior (B) half of the eyecup. Controls were intact retinas fixed immediately after harvesting. After less than 45 minutes in culture, SV2 labeling in the ONL showed a small but significant increase compared with controls in both preparations obtained from both hemiretinas (*P < 0.05). The greatest increase in labeling occurred in specimens maintained in culture for 24 hours (**P < 0.001). Differences in labeling between photoreceptor sheets and full-thickness retinal preparations were only significant after a 24-hour incubation (P < 0.001), with photoreceptor sheets showing larger areas of labeling. (C, D) Culture at 37°C. By less than 45 minutes, preparations from superior (C) and inferior (D) hemiretinas, showed a significant increase in SV2 labeling compared with their respective controls (*P < 0.05). By 24 hours at 37°C the area of SV2 labeling had increased severalfold compared with control cultures (**P < 0.001) or with specimens maintained in culture for less than 45 minutes under the same conditions (P < 0.001). No differences in labeling between photoreceptor sheets and full-thickness retinal preparations were observed at any time point. Data are reported as mean ± SEM; n = 6 eyes of three animals.
Figure 6.
 
Quantitative analysis of SV2 labeling in full-thickness retinal preparations versus photoreceptor sheets. (A, B) Culture at storage temperature of 4°C. The area of the ONL labeled with SV2 was compared in preparations harvested from the superior (A) and the inferior (B) half of the eyecup. Controls were intact retinas fixed immediately after harvesting. After less than 45 minutes in culture, SV2 labeling in the ONL showed a small but significant increase compared with controls in both preparations obtained from both hemiretinas (*P < 0.05). The greatest increase in labeling occurred in specimens maintained in culture for 24 hours (**P < 0.001). Differences in labeling between photoreceptor sheets and full-thickness retinal preparations were only significant after a 24-hour incubation (P < 0.001), with photoreceptor sheets showing larger areas of labeling. (C, D) Culture at 37°C. By less than 45 minutes, preparations from superior (C) and inferior (D) hemiretinas, showed a significant increase in SV2 labeling compared with their respective controls (*P < 0.05). By 24 hours at 37°C the area of SV2 labeling had increased severalfold compared with control cultures (**P < 0.001) or with specimens maintained in culture for less than 45 minutes under the same conditions (P < 0.001). No differences in labeling between photoreceptor sheets and full-thickness retinal preparations were observed at any time point. Data are reported as mean ± SEM; n = 6 eyes of three animals.
Figure 7.
 
ONL thickness and cell layers in photoreceptor sheets versus full-thickness retinas from the superior half of the eyecup. (A) At 4°C, a small but significant reduction in ONL thickness (left) occurred in photoreceptor sheets, only after 24 hours in culture compared with the controls (*P < 0.05) but was insignificant compared with full-thickness preparations. No significant changes in ONL cell layering (right) were seen in either preparation when compared with controls or each other. (B) At 37°C, a significant reduction in ONL thickness (left) was observed in the photoreceptor sheets (*P < 0.05) at less than 45 minutes but not in full-thickness retinal preparations when compared with the control. After 24 hours in culture, the ONL thickness of photoreceptor sheets was further reduced, but that of full-thickness preparations remained nearly unchanged. Differences in thickness between photoreceptor sheets and full-thickness retinal preparations were significant only after 24 hours in culture (P < 0.001). Reduction in the number of ONL cell layers (right) was significant only after 24 hours in all specimens examined (*P < 0.05). No significant differences were detected between photoreceptor sheets and full-thickness retinal preparations. Data are reported as mean ± SEM; n = 6 eyes of three animals.
Figure 7.
 
ONL thickness and cell layers in photoreceptor sheets versus full-thickness retinas from the superior half of the eyecup. (A) At 4°C, a small but significant reduction in ONL thickness (left) occurred in photoreceptor sheets, only after 24 hours in culture compared with the controls (*P < 0.05) but was insignificant compared with full-thickness preparations. No significant changes in ONL cell layering (right) were seen in either preparation when compared with controls or each other. (B) At 37°C, a significant reduction in ONL thickness (left) was observed in the photoreceptor sheets (*P < 0.05) at less than 45 minutes but not in full-thickness retinal preparations when compared with the control. After 24 hours in culture, the ONL thickness of photoreceptor sheets was further reduced, but that of full-thickness preparations remained nearly unchanged. Differences in thickness between photoreceptor sheets and full-thickness retinal preparations were significant only after 24 hours in culture (P < 0.001). Reduction in the number of ONL cell layers (right) was significant only after 24 hours in all specimens examined (*P < 0.05). No significant differences were detected between photoreceptor sheets and full-thickness retinal preparations. Data are reported as mean ± SEM; n = 6 eyes of three animals.
Figure 8.
 
Line plots of morphologic changes in photoreceptor sheets and full-thickness retinal preparations maintained in culture at 37°C for 48 hours. (A) Synaptophysin ONL labeling. A significant increase in the ONL area labeled with synaptophysin compared with controls oc-
 
curred rapidly within the first 45 minutes in culture in both photoreceptor sheets and full-thickness preparations (P < 0.001). The area of labeling continued to increase during the first 24 hours up to four times that observed after 45 minutes in culture (P < 0.001), but then remained unchanged. (B) ONL thickness. A significant reduction in photoreceptor sheet ONL thickness was seen at all three time points examined when compared with the control (P < 0.05). The highest rate of reduction occurred during the first 45 minutes in culture; subsequent small reductions were statistically insignificant when compared with the 45-minute time point. For full-thickness retinas, a small reduction in ONL thickness occurred over time but was not significantly different from controls. (C) ONL cell layering. A small but significant decrease in the number of ONL cell layers occurred in both preparations only after 24 and 48 hours in culture (P < 0.05). Data reported as mean ± SEM; n = 8 eyes of four animals.
Figure 8.
 
Line plots of morphologic changes in photoreceptor sheets and full-thickness retinal preparations maintained in culture at 37°C for 48 hours. (A) Synaptophysin ONL labeling. A significant increase in the ONL area labeled with synaptophysin compared with controls oc-
 
curred rapidly within the first 45 minutes in culture in both photoreceptor sheets and full-thickness preparations (P < 0.001). The area of labeling continued to increase during the first 24 hours up to four times that observed after 45 minutes in culture (P < 0.001), but then remained unchanged. (B) ONL thickness. A significant reduction in photoreceptor sheet ONL thickness was seen at all three time points examined when compared with the control (P < 0.05). The highest rate of reduction occurred during the first 45 minutes in culture; subsequent small reductions were statistically insignificant when compared with the 45-minute time point. For full-thickness retinas, a small reduction in ONL thickness occurred over time but was not significantly different from controls. (C) ONL cell layering. A small but significant decrease in the number of ONL cell layers occurred in both preparations only after 24 and 48 hours in culture (P < 0.05). Data reported as mean ± SEM; n = 8 eyes of four animals.
Figure 9.
 
Effect of gelatin on photoreceptor sheets. Gelatin-embedded photoreceptor sheets from the superior eyecup were incubated at 37°C for 30 minutes to allow the gelatin to melt. The culture medium was then replaced, and sheets were maintained in culture at 4°C for about 15 minutes to 24 hours. Controls were photoreceptor sheets maintained at 4°C without gelatin removal, sheets maintained at 37°C, and intact full-thickness retinas fixed immediately after removal from the eye. (A) SV2 labeling. Loss of gelatin (37°C to 4°C group) did not significantly increase SV2 labeling in the ONL of photoreceptor sheets maintained in culture at 4°C even after 24 hours, compared with sheets in which gelatin was not removed (4°C group). Both groups showed significantly less SV2 labeling (*P < 0.001) compared with sheets maintained at 37°C for 24 hours (37°C group). SV2 labeling was significantly higher in all specimens after 24 hours in culture, compared with intact retina fixed immediately after removal from the eye (controls, P < 0.001). (B) ONL thickness. A significant reduction in ONL thickness was observed in the groups from which gelatin support had been removed (37°C to 4°C, 37°C groups, *P < 0.001) after 15 to 30 minutes and 24 hours. Thickness was preserved in photoreceptor sheets embedded in gelatin and maintained at 4°C for 15 to 30 minutes. After 24 hours, there was a significant reduction in ONL thickness compared with the control (*P < 0.001). The least reduction overall was seen in specimens maintained in culture at 4°C with gelatin support. (C) ONL cell layering. The only significant reduction in ONL cell layering compared with controls was seen in photoreceptor sheets maintained at 37°C (*P < 0.05). Data are reported as mean ± SEM; n = 5 eyes of five animals.
Figure 9.
 
Effect of gelatin on photoreceptor sheets. Gelatin-embedded photoreceptor sheets from the superior eyecup were incubated at 37°C for 30 minutes to allow the gelatin to melt. The culture medium was then replaced, and sheets were maintained in culture at 4°C for about 15 minutes to 24 hours. Controls were photoreceptor sheets maintained at 4°C without gelatin removal, sheets maintained at 37°C, and intact full-thickness retinas fixed immediately after removal from the eye. (A) SV2 labeling. Loss of gelatin (37°C to 4°C group) did not significantly increase SV2 labeling in the ONL of photoreceptor sheets maintained in culture at 4°C even after 24 hours, compared with sheets in which gelatin was not removed (4°C group). Both groups showed significantly less SV2 labeling (*P < 0.001) compared with sheets maintained at 37°C for 24 hours (37°C group). SV2 labeling was significantly higher in all specimens after 24 hours in culture, compared with intact retina fixed immediately after removal from the eye (controls, P < 0.001). (B) ONL thickness. A significant reduction in ONL thickness was observed in the groups from which gelatin support had been removed (37°C to 4°C, 37°C groups, *P < 0.001) after 15 to 30 minutes and 24 hours. Thickness was preserved in photoreceptor sheets embedded in gelatin and maintained at 4°C for 15 to 30 minutes. After 24 hours, there was a significant reduction in ONL thickness compared with the control (*P < 0.001). The least reduction overall was seen in specimens maintained in culture at 4°C with gelatin support. (C) ONL cell layering. The only significant reduction in ONL cell layering compared with controls was seen in photoreceptor sheets maintained at 37°C (*P < 0.05). Data are reported as mean ± SEM; n = 5 eyes of five animals.
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Figure 1.
 
(A, B) Porcine full-thickness retina fixed immediately after detachment from RPE. Retinas prepared in this manner served as controls. (A) Retina immunolabeled with SV2 (green), stained with propidium iodide (red) and viewed in a 2-μm optical section with laser scanning confocal microscopy. OS (arrows) and IS (arrowheads) appear intact with the OS uniformly organized and properly oriented. (B) An alternative staining, retina immunolabeled with synaptophysin (green) and rod-specific opsin (4D2; red) and viewed in a 1-μm optical section. SV2 and synaptophysin uniformly label both plexiform layers. Propidium iodide stained the nuclear layers, whereas 4D2 was confined to rod OS. (B) Cone IS exhibited autofluorescence that was distinct from 4D2 labeling of rod OS. (C) Morphometric analysis, illustrated in a photoreceptor sheet. To quantify the spread of synaptic labeling, the area of SV2 labeling within the ONL was measured within a set rectangular frame (dashed lines). ONL thickness was quantified by measuring the distance from the base of the IS to the base of the innermost cell body of the ONL (double-headed arrow). A count of the ONL cell layers was made along the same perpendicular line. (D) A higher magnification of (A) depicting the characteristic large, triangular-shaped cone pedicles (arrows) and smaller globular rod spherules (arrowheads). Note the bilaminar arrangement of the photoreceptor terminals with the spherules located sclerad to the cone terminals. On occasion, dark puncta were present in photoreceptor terminals. They represent synaptic invaginations (arrow Image not available and arrowhead Image not available ). OS, outer segments; IS, inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer, INL, inner nuclear layer, IPL, inner plexiform layer; GCL, ganglion cell layer; and NFL, nerve fiber layer. Scale bar: (A) 50 μm; (B) 25 μm; (D) 10 μm.
Figure 1.
 
(A, B) Porcine full-thickness retina fixed immediately after detachment from RPE. Retinas prepared in this manner served as controls. (A) Retina immunolabeled with SV2 (green), stained with propidium iodide (red) and viewed in a 2-μm optical section with laser scanning confocal microscopy. OS (arrows) and IS (arrowheads) appear intact with the OS uniformly organized and properly oriented. (B) An alternative staining, retina immunolabeled with synaptophysin (green) and rod-specific opsin (4D2; red) and viewed in a 1-μm optical section. SV2 and synaptophysin uniformly label both plexiform layers. Propidium iodide stained the nuclear layers, whereas 4D2 was confined to rod OS. (B) Cone IS exhibited autofluorescence that was distinct from 4D2 labeling of rod OS. (C) Morphometric analysis, illustrated in a photoreceptor sheet. To quantify the spread of synaptic labeling, the area of SV2 labeling within the ONL was measured within a set rectangular frame (dashed lines). ONL thickness was quantified by measuring the distance from the base of the IS to the base of the innermost cell body of the ONL (double-headed arrow). A count of the ONL cell layers was made along the same perpendicular line. (D) A higher magnification of (A) depicting the characteristic large, triangular-shaped cone pedicles (arrows) and smaller globular rod spherules (arrowheads). Note the bilaminar arrangement of the photoreceptor terminals with the spherules located sclerad to the cone terminals. On occasion, dark puncta were present in photoreceptor terminals. They represent synaptic invaginations (arrow Image not available and arrowhead Image not available ). OS, outer segments; IS, inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer, INL, inner nuclear layer, IPL, inner plexiform layer; GCL, ganglion cell layer; and NFL, nerve fiber layer. Scale bar: (A) 50 μm; (B) 25 μm; (D) 10 μm.
Figure 2.
 
(AD) Effects of temperature on photoreceptor sheets. Photoreceptor sheets were fixed within 10 minutes (A, C) or after 24 hours (B, D) of being placed in culture, immunolabeled with SV2 (green), stained with propidium iodide (red), and viewed in 2-μm optical sections by laser scanning confocal microscopy. Monochromes are in the green channel only, showing SV2 labeling. (A) After vibratome sectioning, only one to two cell layers of the INL remained along the inner surface of the photoreceptor sheet (arrows). When maintained for 10 minutes at 4°C, the photoreceptor terminals appeared intact, and the SV2 labeling was confined to the outer plexiform layer. (B) After 24 hours in culture at 4°C, photoreceptor terminals remained largely intact, and the overall structure of the photoreceptor sheet was well preserved, although a small amount of SV2 labeling was present in the ONL (arrowheads). (C) In photoreceptor sheets maintained at 37°C for 10 minutes, SV2 labeling was also mostly confined to the OPL, but some areas of SV2 labeling were present in the ONL (arrowheads). (D) Photoreceptor sheets maintained at 37°C for 24 hours showed morphologic changes. SV2 labeling was present deep within the ONL (arrowheads). There was also general disorganization of the ONL and reduction in its thickness. Both the OS and IS appeared distorted. (E, F) To confirm results obtained with SV2–propidium iodide double-labeling, specimens were fixed at various time points after incubation, immunolabeled for synaptophysin (synaptic vesicle protein) and 4D2 (rod opsin), using FITC-conjugated (green) and TRITC-conjugated (red) secondary antibodies, respectively, and viewed in 1-μm optical sections by laser scanning confocal microscopy. Monochromes are in the green channel only, showing synaptophysin labeling. Photoreceptor sheets were maintained in culture for up to 48 hours at 37°C. (E). Photoreceptor sheets maintained in culture for less than 45 minutes at 37°C maintained their structural integrity. Similar to SV2-labeled specimens, small areas of synaptophysin labeling were present in the ONL (arrowheads). (F) By 48 hours, disorganization of the OPL and ONL was evident. Synaptophysin labeling was no longer confined to the OPL but spread over a wide area of the ONL (arrowheads). OS and IS were distorted; 4D2 labeling was present in all layers of the sheet, and occasionally 4D2 and synaptophysin labeling colocalized (arrows). Inset: a higher magnification showing areas of drop out of label from the OPL (arrowheads) as labeling spread into the ONL, as well as colocalization of synaptophysin and 4D2 at a single photoreceptor synaptic terminal that retracted and was present in proximity to the cell soma (arrow). Scale bar: (D) 50 μm; (F, and inset) 25 μm.
Figure 2.
 
(AD) Effects of temperature on photoreceptor sheets. Photoreceptor sheets were fixed within 10 minutes (A, C) or after 24 hours (B, D) of being placed in culture, immunolabeled with SV2 (green), stained with propidium iodide (red), and viewed in 2-μm optical sections by laser scanning confocal microscopy. Monochromes are in the green channel only, showing SV2 labeling. (A) After vibratome sectioning, only one to two cell layers of the INL remained along the inner surface of the photoreceptor sheet (arrows). When maintained for 10 minutes at 4°C, the photoreceptor terminals appeared intact, and the SV2 labeling was confined to the outer plexiform layer. (B) After 24 hours in culture at 4°C, photoreceptor terminals remained largely intact, and the overall structure of the photoreceptor sheet was well preserved, although a small amount of SV2 labeling was present in the ONL (arrowheads). (C) In photoreceptor sheets maintained at 37°C for 10 minutes, SV2 labeling was also mostly confined to the OPL, but some areas of SV2 labeling were present in the ONL (arrowheads). (D) Photoreceptor sheets maintained at 37°C for 24 hours showed morphologic changes. SV2 labeling was present deep within the ONL (arrowheads). There was also general disorganization of the ONL and reduction in its thickness. Both the OS and IS appeared distorted. (E, F) To confirm results obtained with SV2–propidium iodide double-labeling, specimens were fixed at various time points after incubation, immunolabeled for synaptophysin (synaptic vesicle protein) and 4D2 (rod opsin), using FITC-conjugated (green) and TRITC-conjugated (red) secondary antibodies, respectively, and viewed in 1-μm optical sections by laser scanning confocal microscopy. Monochromes are in the green channel only, showing synaptophysin labeling. Photoreceptor sheets were maintained in culture for up to 48 hours at 37°C. (E). Photoreceptor sheets maintained in culture for less than 45 minutes at 37°C maintained their structural integrity. Similar to SV2-labeled specimens, small areas of synaptophysin labeling were present in the ONL (arrowheads). (F) By 48 hours, disorganization of the OPL and ONL was evident. Synaptophysin labeling was no longer confined to the OPL but spread over a wide area of the ONL (arrowheads). OS and IS were distorted; 4D2 labeling was present in all layers of the sheet, and occasionally 4D2 and synaptophysin labeling colocalized (arrows). Inset: a higher magnification showing areas of drop out of label from the OPL (arrowheads) as labeling spread into the ONL, as well as colocalization of synaptophysin and 4D2 at a single photoreceptor synaptic terminal that retracted and was present in proximity to the cell soma (arrow). Scale bar: (D) 50 μm; (F, and inset) 25 μm.
Figure 3.
 
Quantitative analysis of photoreceptor sheets maintained in culture at 4°C and 37°C. Controls were intact full-thickness retinas fixed immediately after detachment from RPE. (A, D) ONL thickness. (A) At 4°C, only after 24 hours in culture was there a significant reduction in ONL thickness compared with control specimens (*P < 0.001). (D) In contrast, at 37°C a significant reduction in ONL thickness was seen by 10 minutes, and at 24 hours in culture, both superior and inferior photoreceptor sheets were compared with their respective controls (*P < 0.001). These sheets were also significantly reduced in thickness compared with their 4°C counterparts at 10 minutes and 24 hours (P < 0.05). (B, E) ONL cell layering. (B) At 4°C no significant reduction in the number of ONL cell layers was seen in photoreceptor sheets obtained from either hemiretina for up to 24 hours in culture. (E) At 37°C, a small but significant reduction in the number of ONL cell layers was seen in both superior and inferior photoreceptor sheets only after 24 hours in culture (*P < 0.05). (C, F) ONL SV2 labeling. (C) Small but significant increases in SV2 labeling were present in the ONL of photoreceptor sheets obtained from both superior (*P < 0.01) and inferior (**P < 0.001) hemiretinas after 10 minutes in culture at 4°C compared with the control. (F) At 37°C there was a significant increase in the area of labeling by 10 minutes in culture in photoreceptor sheets obtained from both the superior and inferior hemiretinas compared with the control (*P < 0.05). At both temperatures at 10 minutes, photoreceptor sheets from the inferior hemiretina showed significantly larger areas of SV2 labeling than those from the superior hemiretina (P < 0.001). By 24 hours, this discrepancy had disappeared, with a significantly larger area of the ONL showing SV2 labeling in photoreceptor sheets obtained from either hemiretina compared with control cultures (**P < 0.001) or with specimens maintained in culture for only 10 minutes (P < 0.05). The area of SV2 labeling was greatest in sheets maintained in culture for 24 hours at 37°C. These specimens showed an approximate ninefold increase in labeling over the control (**P < 0.001) and more than double that in specimens maintained at 4°C for 24 hours (P < 0.001). These results underscore the significant role of temperature in these structural changes. Data are reported as mean ± SEM; n = 10 eyes of five animals.
Figure 3.
 
Quantitative analysis of photoreceptor sheets maintained in culture at 4°C and 37°C. Controls were intact full-thickness retinas fixed immediately after detachment from RPE. (A, D) ONL thickness. (A) At 4°C, only after 24 hours in culture was there a significant reduction in ONL thickness compared with control specimens (*P < 0.001). (D) In contrast, at 37°C a significant reduction in ONL thickness was seen by 10 minutes, and at 24 hours in culture, both superior and inferior photoreceptor sheets were compared with their respective controls (*P < 0.001). These sheets were also significantly reduced in thickness compared with their 4°C counterparts at 10 minutes and 24 hours (P < 0.05). (B, E) ONL cell layering. (B) At 4°C no significant reduction in the number of ONL cell layers was seen in photoreceptor sheets obtained from either hemiretina for up to 24 hours in culture. (E) At 37°C, a small but significant reduction in the number of ONL cell layers was seen in both superior and inferior photoreceptor sheets only after 24 hours in culture (*P < 0.05). (C, F) ONL SV2 labeling. (C) Small but significant increases in SV2 labeling were present in the ONL of photoreceptor sheets obtained from both superior (*P < 0.01) and inferior (**P < 0.001) hemiretinas after 10 minutes in culture at 4°C compared with the control. (F) At 37°C there was a significant increase in the area of labeling by 10 minutes in culture in photoreceptor sheets obtained from both the superior and inferior hemiretinas compared with the control (*P < 0.05). At both temperatures at 10 minutes, photoreceptor sheets from the inferior hemiretina showed significantly larger areas of SV2 labeling than those from the superior hemiretina (P < 0.001). By 24 hours, this discrepancy had disappeared, with a significantly larger area of the ONL showing SV2 labeling in photoreceptor sheets obtained from either hemiretina compared with control cultures (**P < 0.001) or with specimens maintained in culture for only 10 minutes (P < 0.05). The area of SV2 labeling was greatest in sheets maintained in culture for 24 hours at 37°C. These specimens showed an approximate ninefold increase in labeling over the control (**P < 0.001) and more than double that in specimens maintained at 4°C for 24 hours (P < 0.001). These results underscore the significant role of temperature in these structural changes. Data are reported as mean ± SEM; n = 10 eyes of five animals.
Figure 4.
 
Quantitative analysis using alternate immunohistochemical markers, synaptophysin, and rod opsin (4D2). (A) ONL thickness and cell layers. ONL thickness decreased significantly at all time points compared with the control (*P < 0.05). A small but significant reduction in the number of cell layers was also seen in these specimens after 24 and 48 hours in culture (*P < 0.05). (B) Synaptophysin labeling. A significant increase in the area of ONL labeling was observed in photoreceptor sheets by 45 minutes (*P < 0.001). By 24 hours, the synaptophysin-labeled area tripled (*P < 0.001) but did not change significantly thereafter. The pattern of these changes closely resembled that in photoreceptor sheets labeled with SV2-propidium iodide and maintained under the same culture conditions. Mean ± SEM. n = 4 eyes of four animals.
Figure 4.
 
Quantitative analysis using alternate immunohistochemical markers, synaptophysin, and rod opsin (4D2). (A) ONL thickness and cell layers. ONL thickness decreased significantly at all time points compared with the control (*P < 0.05). A small but significant reduction in the number of cell layers was also seen in these specimens after 24 and 48 hours in culture (*P < 0.05). (B) Synaptophysin labeling. A significant increase in the area of ONL labeling was observed in photoreceptor sheets by 45 minutes (*P < 0.001). By 24 hours, the synaptophysin-labeled area tripled (*P < 0.001) but did not change significantly thereafter. The pattern of these changes closely resembled that in photoreceptor sheets labeled with SV2-propidium iodide and maintained under the same culture conditions. Mean ± SEM. n = 4 eyes of four animals.
Figure 5.
 
(A, B) Full-thickness retinal preparations versus photoreceptor sheets. (A) Full-thickness retina and (B) a photoreceptor sheet fixed after 24 hours in culture at 37°C and immunolabeled for SV2 using a FITC-conjugated secondary antibody (green). Propidium iodide (red) was used as a marker for the nuclear layers. SV2 staining was present deep within the ONL of both preparations (arrowheads). (C, D) Effect of gelatin on photoreceptor sheets using anti-SV2 and propidium iodide staining. (C) Photoreceptor sheet maintained in culture for 24 hours at 4°C after gelatin removal at 37°C for 30 minutes. In contrast to photoreceptor sheets maintained at 37°C, SV2 labeling was largely confined to the OPL. (D) Photoreceptor sheet maintained in culture for 24 hours at 4°C without gelatin removal. As seen before (Fig. 2B) , most staining remained in the OPL. Note tangential cut of specimen showing several rows of photoreceptor terminals. Scale bar, 50 μm.
Figure 5.
 
(A, B) Full-thickness retinal preparations versus photoreceptor sheets. (A) Full-thickness retina and (B) a photoreceptor sheet fixed after 24 hours in culture at 37°C and immunolabeled for SV2 using a FITC-conjugated secondary antibody (green). Propidium iodide (red) was used as a marker for the nuclear layers. SV2 staining was present deep within the ONL of both preparations (arrowheads). (C, D) Effect of gelatin on photoreceptor sheets using anti-SV2 and propidium iodide staining. (C) Photoreceptor sheet maintained in culture for 24 hours at 4°C after gelatin removal at 37°C for 30 minutes. In contrast to photoreceptor sheets maintained at 37°C, SV2 labeling was largely confined to the OPL. (D) Photoreceptor sheet maintained in culture for 24 hours at 4°C without gelatin removal. As seen before (Fig. 2B) , most staining remained in the OPL. Note tangential cut of specimen showing several rows of photoreceptor terminals. Scale bar, 50 μm.
Figure 6.
 
Quantitative analysis of SV2 labeling in full-thickness retinal preparations versus photoreceptor sheets. (A, B) Culture at storage temperature of 4°C. The area of the ONL labeled with SV2 was compared in preparations harvested from the superior (A) and the inferior (B) half of the eyecup. Controls were intact retinas fixed immediately after harvesting. After less than 45 minutes in culture, SV2 labeling in the ONL showed a small but significant increase compared with controls in both preparations obtained from both hemiretinas (*P < 0.05). The greatest increase in labeling occurred in specimens maintained in culture for 24 hours (**P < 0.001). Differences in labeling between photoreceptor sheets and full-thickness retinal preparations were only significant after a 24-hour incubation (P < 0.001), with photoreceptor sheets showing larger areas of labeling. (C, D) Culture at 37°C. By less than 45 minutes, preparations from superior (C) and inferior (D) hemiretinas, showed a significant increase in SV2 labeling compared with their respective controls (*P < 0.05). By 24 hours at 37°C the area of SV2 labeling had increased severalfold compared with control cultures (**P < 0.001) or with specimens maintained in culture for less than 45 minutes under the same conditions (P < 0.001). No differences in labeling between photoreceptor sheets and full-thickness retinal preparations were observed at any time point. Data are reported as mean ± SEM; n = 6 eyes of three animals.
Figure 6.
 
Quantitative analysis of SV2 labeling in full-thickness retinal preparations versus photoreceptor sheets. (A, B) Culture at storage temperature of 4°C. The area of the ONL labeled with SV2 was compared in preparations harvested from the superior (A) and the inferior (B) half of the eyecup. Controls were intact retinas fixed immediately after harvesting. After less than 45 minutes in culture, SV2 labeling in the ONL showed a small but significant increase compared with controls in both preparations obtained from both hemiretinas (*P < 0.05). The greatest increase in labeling occurred in specimens maintained in culture for 24 hours (**P < 0.001). Differences in labeling between photoreceptor sheets and full-thickness retinal preparations were only significant after a 24-hour incubation (P < 0.001), with photoreceptor sheets showing larger areas of labeling. (C, D) Culture at 37°C. By less than 45 minutes, preparations from superior (C) and inferior (D) hemiretinas, showed a significant increase in SV2 labeling compared with their respective controls (*P < 0.05). By 24 hours at 37°C the area of SV2 labeling had increased severalfold compared with control cultures (**P < 0.001) or with specimens maintained in culture for less than 45 minutes under the same conditions (P < 0.001). No differences in labeling between photoreceptor sheets and full-thickness retinal preparations were observed at any time point. Data are reported as mean ± SEM; n = 6 eyes of three animals.
Figure 7.
 
ONL thickness and cell layers in photoreceptor sheets versus full-thickness retinas from the superior half of the eyecup. (A) At 4°C, a small but significant reduction in ONL thickness (left) occurred in photoreceptor sheets, only after 24 hours in culture compared with the controls (*P < 0.05) but was insignificant compared with full-thickness preparations. No significant changes in ONL cell layering (right) were seen in either preparation when compared with controls or each other. (B) At 37°C, a significant reduction in ONL thickness (left) was observed in the photoreceptor sheets (*P < 0.05) at less than 45 minutes but not in full-thickness retinal preparations when compared with the control. After 24 hours in culture, the ONL thickness of photoreceptor sheets was further reduced, but that of full-thickness preparations remained nearly unchanged. Differences in thickness between photoreceptor sheets and full-thickness retinal preparations were significant only after 24 hours in culture (P < 0.001). Reduction in the number of ONL cell layers (right) was significant only after 24 hours in all specimens examined (*P < 0.05). No significant differences were detected between photoreceptor sheets and full-thickness retinal preparations. Data are reported as mean ± SEM; n = 6 eyes of three animals.
Figure 7.
 
ONL thickness and cell layers in photoreceptor sheets versus full-thickness retinas from the superior half of the eyecup. (A) At 4°C, a small but significant reduction in ONL thickness (left) occurred in photoreceptor sheets, only after 24 hours in culture compared with the controls (*P < 0.05) but was insignificant compared with full-thickness preparations. No significant changes in ONL cell layering (right) were seen in either preparation when compared with controls or each other. (B) At 37°C, a significant reduction in ONL thickness (left) was observed in the photoreceptor sheets (*P < 0.05) at less than 45 minutes but not in full-thickness retinal preparations when compared with the control. After 24 hours in culture, the ONL thickness of photoreceptor sheets was further reduced, but that of full-thickness preparations remained nearly unchanged. Differences in thickness between photoreceptor sheets and full-thickness retinal preparations were significant only after 24 hours in culture (P < 0.001). Reduction in the number of ONL cell layers (right) was significant only after 24 hours in all specimens examined (*P < 0.05). No significant differences were detected between photoreceptor sheets and full-thickness retinal preparations. Data are reported as mean ± SEM; n = 6 eyes of three animals.
Figure 8.
 
Line plots of morphologic changes in photoreceptor sheets and full-thickness retinal preparations maintained in culture at 37°C for 48 hours. (A) Synaptophysin ONL labeling. A significant increase in the ONL area labeled with synaptophysin compared with controls oc-
 
curred rapidly within the first 45 minutes in culture in both photoreceptor sheets and full-thickness preparations (P < 0.001). The area of labeling continued to increase during the first 24 hours up to four times that observed after 45 minutes in culture (P < 0.001), but then remained unchanged. (B) ONL thickness. A significant reduction in photoreceptor sheet ONL thickness was seen at all three time points examined when compared with the control (P < 0.05). The highest rate of reduction occurred during the first 45 minutes in culture; subsequent small reductions were statistically insignificant when compared with the 45-minute time point. For full-thickness retinas, a small reduction in ONL thickness occurred over time but was not significantly different from controls. (C) ONL cell layering. A small but significant decrease in the number of ONL cell layers occurred in both preparations only after 24 and 48 hours in culture (P < 0.05). Data reported as mean ± SEM; n = 8 eyes of four animals.
Figure 8.
 
Line plots of morphologic changes in photoreceptor sheets and full-thickness retinal preparations maintained in culture at 37°C for 48 hours. (A) Synaptophysin ONL labeling. A significant increase in the ONL area labeled with synaptophysin compared with controls oc-
 
curred rapidly within the first 45 minutes in culture in both photoreceptor sheets and full-thickness preparations (P < 0.001). The area of labeling continued to increase during the first 24 hours up to four times that observed after 45 minutes in culture (P < 0.001), but then remained unchanged. (B) ONL thickness. A significant reduction in photoreceptor sheet ONL thickness was seen at all three time points examined when compared with the control (P < 0.05). The highest rate of reduction occurred during the first 45 minutes in culture; subsequent small reductions were statistically insignificant when compared with the 45-minute time point. For full-thickness retinas, a small reduction in ONL thickness occurred over time but was not significantly different from controls. (C) ONL cell layering. A small but significant decrease in the number of ONL cell layers occurred in both preparations only after 24 and 48 hours in culture (P < 0.05). Data reported as mean ± SEM; n = 8 eyes of four animals.
Figure 9.
 
Effect of gelatin on photoreceptor sheets. Gelatin-embedded photoreceptor sheets from the superior eyecup were incubated at 37°C for 30 minutes to allow the gelatin to melt. The culture medium was then replaced, and sheets were maintained in culture at 4°C for about 15 minutes to 24 hours. Controls were photoreceptor sheets maintained at 4°C without gelatin removal, sheets maintained at 37°C, and intact full-thickness retinas fixed immediately after removal from the eye. (A) SV2 labeling. Loss of gelatin (37°C to 4°C group) did not significantly increase SV2 labeling in the ONL of photoreceptor sheets maintained in culture at 4°C even after 24 hours, compared with sheets in which gelatin was not removed (4°C group). Both groups showed significantly less SV2 labeling (*P < 0.001) compared with sheets maintained at 37°C for 24 hours (37°C group). SV2 labeling was significantly higher in all specimens after 24 hours in culture, compared with intact retina fixed immediately after removal from the eye (controls, P < 0.001). (B) ONL thickness. A significant reduction in ONL thickness was observed in the groups from which gelatin support had been removed (37°C to 4°C, 37°C groups, *P < 0.001) after 15 to 30 minutes and 24 hours. Thickness was preserved in photoreceptor sheets embedded in gelatin and maintained at 4°C for 15 to 30 minutes. After 24 hours, there was a significant reduction in ONL thickness compared with the control (*P < 0.001). The least reduction overall was seen in specimens maintained in culture at 4°C with gelatin support. (C) ONL cell layering. The only significant reduction in ONL cell layering compared with controls was seen in photoreceptor sheets maintained at 37°C (*P < 0.05). Data are reported as mean ± SEM; n = 5 eyes of five animals.
Figure 9.
 
Effect of gelatin on photoreceptor sheets. Gelatin-embedded photoreceptor sheets from the superior eyecup were incubated at 37°C for 30 minutes to allow the gelatin to melt. The culture medium was then replaced, and sheets were maintained in culture at 4°C for about 15 minutes to 24 hours. Controls were photoreceptor sheets maintained at 4°C without gelatin removal, sheets maintained at 37°C, and intact full-thickness retinas fixed immediately after removal from the eye. (A) SV2 labeling. Loss of gelatin (37°C to 4°C group) did not significantly increase SV2 labeling in the ONL of photoreceptor sheets maintained in culture at 4°C even after 24 hours, compared with sheets in which gelatin was not removed (4°C group). Both groups showed significantly less SV2 labeling (*P < 0.001) compared with sheets maintained at 37°C for 24 hours (37°C group). SV2 labeling was significantly higher in all specimens after 24 hours in culture, compared with intact retina fixed immediately after removal from the eye (controls, P < 0.001). (B) ONL thickness. A significant reduction in ONL thickness was observed in the groups from which gelatin support had been removed (37°C to 4°C, 37°C groups, *P < 0.001) after 15 to 30 minutes and 24 hours. Thickness was preserved in photoreceptor sheets embedded in gelatin and maintained at 4°C for 15 to 30 minutes. After 24 hours, there was a significant reduction in ONL thickness compared with the control (*P < 0.001). The least reduction overall was seen in specimens maintained in culture at 4°C with gelatin support. (C) ONL cell layering. The only significant reduction in ONL cell layering compared with controls was seen in photoreceptor sheets maintained at 37°C (*P < 0.05). Data are reported as mean ± SEM; n = 5 eyes of five animals.
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