April 2012
Volume 53, Issue 4
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Retina  |   April 2012
Creatine Transporter Immunolocalization in Aged Human and Detached Retinas
Author Affiliations & Notes
  • Clairton F. de Souza
    From theDepartment of Ophthalmology, University of Auckland, Auckland, New Zealand;
    Department of Optometry and Vision Science, University of Auckland, Auckland, New Zealand;
    New Zealand National Eye Centre, University of Auckland, Auckland, New Zealand
  • Michael Kalloniatis
    Department of Optometry and Vision Science, University of Auckland, Auckland, New Zealand;
    New Zealand National Eye Centre, University of Auckland, Auckland, New Zealand
    Centre for Eye Health, University of New South Wales, Sydney, Australia; and
    School of Optometry and Vision Science, University of New South Wales, Sydney, Australia.
  • David L. Christie
    Molecular, Cellular, and Developmental Biology, School of Biological Sciences, University of Auckland, Auckland, New Zealand;
  • Philip J. Polkinghorne
    From theDepartment of Ophthalmology, University of Auckland, Auckland, New Zealand;
    New Zealand National Eye Centre, University of Auckland, Auckland, New Zealand
  • Charles N. J. McGhee
    From theDepartment of Ophthalmology, University of Auckland, Auckland, New Zealand;
    New Zealand National Eye Centre, University of Auckland, Auckland, New Zealand
  • Monica L. Acosta
    Department of Optometry and Vision Science, University of Auckland, Auckland, New Zealand;
    New Zealand National Eye Centre, University of Auckland, Auckland, New Zealand
  • Corresponding author: Monica Acosta, Department of Optometry and Vision Science, University of Auckland, Private Bag 92019, Auckland, New Zealand; m.acosta@auckland.ac.nz  
Investigative Ophthalmology & Visual Science April 2012, Vol.53, 1936-1945. doi:10.1167/iovs.11-8462
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      Clairton F. de Souza, Michael Kalloniatis, David L. Christie, Philip J. Polkinghorne, Charles N. J. McGhee, Monica L. Acosta; Creatine Transporter Immunolocalization in Aged Human and Detached Retinas. Invest. Ophthalmol. Vis. Sci. 2012;53(4):1936-1945. doi: 10.1167/iovs.11-8462.

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      © 2016 Association for Research in Vision and Ophthalmology.

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Abstract

Purpose.: To identify the distribution of creatine transporter (CRT) in the aged human retina and how this expression pattern is modified after retinal detachment.

Methods.: An affinity-purified antibody raised against the CRT was used in the immunohistochemical investigation. The anti-CRT antibody was colocalized with neuronal markers (calbindin, parvalbumin, Islet-1, calretinin, GAD67, Go-alpha), glia markers (glutamine synthetase, glial fibrillary acid protein), and a blood vessel basal membrane marker (laminin). Confocal microscopy was used to visualize the labeling patterns in retinal sections. The level of CRT expression was determined in the retina using a semiquantification method.

Results.: Immunohistochemical assessment of CRT expression in the normal aged retina revealed strong labeling in photoreceptor synaptic terminals and in inner and outer segments. Labeling was also observed on subpopulations of amacrine cells and ganglion cells as well as in the outer and inner plexiform layers. CRT labeling was observed in blood vessels, although was absent in glial cells. In retinal detachment, the CRT labeling pattern was maintained, although there was an apparent decrease in labeling in inner retina and an increase in CRT expression in photoreceptors.

Conclusions.: CRT is expressed in areas of intense metabolic activity, such as photoreceptors, selected cells in the inner retina, and sites of creatine transport into the retina (inner retinal blood vessels and retinal pigment epithelium). The absence of CRT to Müller cells harmonizes with the concept that glial cells are a biosynthetic source of creatine but not a source of creatine to other retinal neurons. The increased immunolabeling of CRT localized to the outer retina in retinal detachment suggests a regional metabolic remodeling occurring in photoreceptor cells.

Introduction
Impaired energy metabolism plays an important role in the pathogenesis and progression of any neurological insult. Creatine and its high-energy phosphorylated analogue, phosphocreatine, are essential molecules that maintain ATP levels in high-energy demanding tissues such as the retina and the brain. 1,2 Creatine level is maintained through uptake from diet and endogenous biosynthesis. 3,4 A specific plasma membrane creatine transporter (CRT) is required for the cellular uptake of creatine from the blood. 5,6 CRT is critical in normal brain function, as mutations in the CRT gene (SLC6A8) lead to X-linked mental retardation, epilepsy, developmental delays in speech and language, seizures, and autistic behavior. 710 As an energy buffer, creatine is an efficient neuroprotective agent in animal models of neurodegeneration and brain injury. 1,3,1115 Creatine supplementation is currently under trial in human subjects with neurological pathologies such as Parkinson's and Alzheimer's diseases that have known bioenergetic deficits. 1625 Maintenance of ATP supply is of fundamental importance for the function of the retina. Hence, it is expected that the CRT transporter should be localized to areas of high energy demand in this tissue. 
Retinal detachment is associated with altered energy metabolism and oxygen tension 26,27 because of the hypoxic environment for photoreceptor cells as they are separated from the pigmented epithelium. The hypoxic insult is followed by photoreceptor cell death, triggering cellular remodeling of the glia and inner retinal neurons. 2831 Although metabolic regulation is expected in remaining cells to compensate for the anaerobic environment, the role of high-energy phosphate transporters, such as CRT, in the hypoxic environment remains to be determined. 
Immunocytochemical studies have shown CRT localization to both luminal and abluminal faces of the rat retinal blood vessel endothelial cells and retinal pigmented epithelium. 32 It has also been detected in retinal neurons of different vertebrates 33 ; however, CRT expression in human tissues has not been characterized. This study refers to the detection of CRT expression in the normal and detached human retina as a way of evaluating creatine regulation in retinal degeneration conditions. 
Methods
Collection of Human Normal and Detached Retina Samples
Retinal tissue samples were obtained after informed consent from patients. The procedures were in accordance with the tenets of the Declaration of Helsinki and approved by the Institutional Review Committee of the University of Auckland and Auckland District Health Board. Specimens used as normal aged retinas were obtained from three Caucasian patients (2 women aged 70 years old and a male individual aged 87 years old) subjected to enucleation for uveal melanoma with no other previous treatment. Human retina obtained from enucleated eyes owing to uveal melanoma has been used in other studies. 3437 The retinal samples were collected after enucleation from the opposite side of the tumor. A detailed description of these samples can be found in de Souza et al. 38 Three rhegmatogenous retinal detachment (RRD) specimens were from patients aged 54 to 62 years. The samples were characterized in a separate publication and used here to demonstrate CRT distribution in a diseased retina. 65 The patient demographic information is shown in Table 1. We used samples from mid-peripheral retina (area within 7 mm from the ora serrata) to illustrate the patterns of CRT expression in aged normal retina and comparison with retinal detachment sections. 
Table 1
 
Donor Subject Demographics
Table 1
 
Donor Subject Demographics
Donor Age Sex Ethnicity
1. Control 70 Female Caucasian
2. Control 70 Female Caucasian
3. Control 87 Male Caucasian
4. Retinal detachment (2 wk) 62 Male Caucasian
5. Retinal detachment (1 mo) 62 Male Caucasian
6. Retinal detachment (2 mo) 54 Male Caucasian
Retinal specimens were fixed in a mixture of 4% paraformaldehyde and 0.01% glutaraldehyde in phosphate buffer for 30 minutes. Tissues were washed in PBS, pH 7.4, before cryoprotection by immersion in graded series of sucrose up to 30% wt/vol in PBS. Then, the tissues were embedded in optimum cutting temperature medium (OCT; PELCO International, Redding, CA) and sectioned at 16 μm with a Leica CM3050 S cryostat (Heidelberg, Germany). 
Immunohistochemistry
Immunohistochemical procedures for detection of creatine transporter were conducted as previously reported. 33,39,40 Briefly, retinal vertical sections were incubated for 1 hour in a blocking solution containing 6% goat serum (Sigma, St. Louis, MO), 1% BSA (Sigma), 0.5% Triton-X-100 (Fisher Scientific Inc., Fairlawn, NJ). Retinal sections were double labeled by overnight incubation at room temperature with primary antibodies. The rabbit polyclonal antibody was raised against a purified COOH-terminal fragment of the bovine CRT protein, expressed as a fusion protein with glutathione-S-transferase (GST). 41 Antibody specificity for retinal tissue has been previously reported by our group. 33,42 Details of primary antibodies used in this study are described in Table 2. Primary and secondary antibodies were diluted in a buffer containing 3% goat serum, 1% bovine serum, 0.5% Triton X-100, and 0.05% thimerasol in PBS (phosphate buffer solution, pH 7.4). Secondary antibodies were applied to the tissues for 3 hours and consisted of goat anti-mouse conjugated with Alexa TM 594 (Molecular Probes, Eugene, OR), goat anti-rat conjugated with Alexa TM 594 (Molecular Probes), goat anti-rabbit antibody conjugated to Alexa 488 (Molecular Probes), and goat anti-rat antibody conjugated to Alexa 488. An exception was the use of one primary antibody (GFAP) conjugated to Cy3 (Sigma). All secondary antibodies were used at a 1:500 concentration. Slides were washed and cover slipped using antifading reagent (Prolong Gold; Invitrogen, Eugene, OR) containing 4′, 6-diamidino-2-phenylindole (DAPI) and sealed with nail polish. Negative controls were performed by omission of the primary or secondary antibodies otherwise maintaining the protocol steps. 
Table 2.
 
Primary Antibodies Used in This Study
Table 2.
 
Primary Antibodies Used in This Study
Antibody Host Source Working Concentration
Creatine transporter Rabbit Assoc. Prof. David Christie (University of Auckland, NZ) 1:100
Calbindin D28K Mouse Sigma-Aldrich, St. Louis, MO (C9848) 1:1000
Calretinin Mouse BD Biosciences, San Jose, CA (610,908) 1:1000
Parvalbumin Mouse Sigma (P3088) 1:1000
Islet-1 Mouse Developmental Studies Hybridoma Bank at the University of Iowa, Iowa City, IA 1:500
Go-protein (α subunit) Go-alpha Mouse Chemicon International Inc., Billerica, MA (MAB327) 1:500
Glutamate decarboxylase (GAD67) Mouse Chemicon (AB5992) 1:500
Glutamine synthetase (GS) Mouse BD Biosciences (610,518) 1:1000
GFAP-Cy3 Mouse Sigma (C9205) 1:3000
Laminin Rat Abcam, Cambridge, UK (ab79057) 1:200
MAP-2 Mouse Novus Biologicals (NB300-143) 1:200
Tyrosine hydroxylase (TH) Rabbit Chemicon (AB152) 1:1000
Confocal Microscopy
Single- and double-labeled retinal sections were visualized and images captured using an Olympus confocal scanning laser microscope (FV1000; Olympus, Tokyo, Japan). High-resolution scanning was performed using a ×40 or ×60 oil objective lens (plan apochromat). Scans were collected with z-axis sequence with a step size between 0.5 and 1.0 μm per frame and visualized in a monitor with 1024 × 1024 pixel resolution. The final images corresponded to a maximal intensity projection of 12 μm stack of scans from a selected area of the retinal specimen. The brightness and contrast of final figures were equally adjusted by using Adobe Photoshop (Adobe Systems, Mountain View, CA). 
Semiquantitative Analysis of CRT Immunolabeling
We used a semiquantification approach to determine the level of CRT immunolabeling (expressed in pixel value), within detached retina compared with mid-peripheral nondetached retina. Suitable retinal images randomly selected from two control retina and two RRD retinas, processed under identical conditions and with confocal images of identical characteristics, were converted to gray scale using the Image J program (National Institutes of Health, Bethesda, MD). Color images were converted into 8-bit grayscale by using the split-channel function. The level of immunoreactivity is therefore expressed over a 256-pixel value range. Three different retinal areas were assessed: CRT immunoreactivity over the whole retina, outer nuclear layer, and within cone photoreceptors. The overall aim was to determine the difference in pixel value for CRT immunoreactivity in normal and detached areas. To overcome the error introduced by background immunolabeling, the following method was used when comparing the images from control and RRD retina. By thresholding the immunoreactivity, we determined the area that was immunoreactive and used this value to normalize the pixel value for CRT immunoreactivity. In this way, the average pixel value reflected the average CRT immunoreactivity in labeled retinal areas alone from images acquired under identical settings. The difference in pixel value was calculated by subtracting the RRD pixel value from the control pixel value (control – RRD). Seven images from the three control and three retinal detachment samples were used in the assessment. The average area analyzed was approximately 600,000 pixels per image. The pixel value for cone photoreceptors can be directly assessed within photoreceptor somata. We made use of the unique morphological appearance of cone photoreceptors to determine the level of CRT immunolabeling in a 100-pixel area within 5 to 10 cone photoreceptors in four different regions from aged normal and the 2-weeks retinal detachment samples. 
Results
Immunolocalization of CRT in Human Retina
To determine the identity of the CRT-labeled cells, we colocalized CRT immunoreactivity with a range of cell markers. The calcium-binding protein calbindin is a known marker for cone photoreceptors, horizontal cells, and subpopulations of bipolar cells (DB3 OFF-bipolar cells), amacrine cells, and ganglion cells in the human retina (Fig. 1C). 4345 Strong CRT labeling was found colocalizing with calbindin-positive photoreceptor outer and inner segments and synaptic terminals (Figs. 1A–D, arrowhead). We observed CRT on the calbindin-labeled photoreceptor cell axon (Figs. 1A–D, thick arrow). A type of amacrine cell with mitral shape and lobular appendage, suggestive to be AII amacrine cells, colocalized with CRT labeling (Figs. 1B–D). CRT labeling was also observed in the inner plexiform layer (IPL) and nerve fiber layer (NFL), as well as on calbindin-negative ganglion cells (Figs. 1B–D, small arrow). Moreover, there was a uniform expression of CRT in the retinal pigmented epithelium (RPE) cells (Fig. 1F, arrows), that could not be clearly discriminated from the background fluorescence in the RPE when secondary antibody alone was added to the tissue (Fig.1E). 
Figure 1.
 
Immunofluorescence labeling in human aged normal retina. Images were captured using confocal microscopy. DAPI was used as counter-staining (A). Creatine transporter (B) colocalizes with calbindin (C). Strong immunoreactivity coexpressed in cones (arrows), photoreceptor synaptic terminals (arrowheads), AII amacrine cells, and outer and inner plexiform layers. (E) DAPI labeling of a negative control for the immunocytochemical procedure where secondary antibody was applied in the absence of a primary antibody. (F) The presence of CRT immunoreactivity at the RPE. (Scale bar: 20 μm.) HC, horizontal cell; AC, amacrine cell; OPL, outer plexiform layer; IPL, inner plexiform layer; GCL, ganglion cell layer.
Figure 1.
 
Immunofluorescence labeling in human aged normal retina. Images were captured using confocal microscopy. DAPI was used as counter-staining (A). Creatine transporter (B) colocalizes with calbindin (C). Strong immunoreactivity coexpressed in cones (arrows), photoreceptor synaptic terminals (arrowheads), AII amacrine cells, and outer and inner plexiform layers. (E) DAPI labeling of a negative control for the immunocytochemical procedure where secondary antibody was applied in the absence of a primary antibody. (F) The presence of CRT immunoreactivity at the RPE. (Scale bar: 20 μm.) HC, horizontal cell; AC, amacrine cell; OPL, outer plexiform layer; IPL, inner plexiform layer; GCL, ganglion cell layer.
To determine if CRT was expressed in retinal glial cells, we colocalized CRT with glutamine synthetase (GS), a marker of Müller cells (Figs. 2A–F). GS expression was noted throughout Müller cell from their end-feet to the outer limiting membrane. The absence of CRT expression in Müller cells was evident in the GS/CRT double-labeled retina (Figs. 2D–F). CRT negative processes of Müller cells were found traversing the neurosensory retina (Figs. 2C, 2D, yellow arrow), whereas GS labeling was seen around inner retinal cells or in projections that run in parallel to CRT labeling in the outer plexiform and outer nuclear layers (Figs. 2D–F, white arrow). In Figure 2E and Figure 2F we show that there is no overlap between GS and CRT in both plexiform layers. 
Figure 2.
 
Confocal microscopy images of human aged normal retina immunolabeled with CRT and GS. DAPI used as counter-staining (A). CRT labeling (B) and the Müller cell marker GS (C) revealed lack of colocalization of both signals. (E) and (F) inserts correspond to a magnification of outlined areas in (D). (Scale bars: A–D, 20 μm; E and F, 15 μm.) OPL, outer plexiform layer; IPL, inner plexiform layer; GCL, ganglion cell layer.
Figure 2.
 
Confocal microscopy images of human aged normal retina immunolabeled with CRT and GS. DAPI used as counter-staining (A). CRT labeling (B) and the Müller cell marker GS (C) revealed lack of colocalization of both signals. (E) and (F) inserts correspond to a magnification of outlined areas in (D). (Scale bars: A–D, 20 μm; E and F, 15 μm.) OPL, outer plexiform layer; IPL, inner plexiform layer; GCL, ganglion cell layer.
CRT Immunoreactive Cells in Inner Retina
We used a range of cellular markers for identification of the inner retina cell types colocalizing with CRT. CRT-positive cells represent several cell populations. In Figure 1, we show the expression of CRT in AII amacrine cells, although there was no colocalization with horizontal cells, and DB3 OFF bipolar cells labeled with calbindin (Figs. 1B–D). Another calcium-binding protein, parvalbumin, was used as a marker for horizontal cells; yet, there was no expression of CRT in these cells (Figs. 3A–C, arrow). Nonetheless, parvalbumin also labels ganglion cells, which colocalized with CRT (Figs. 3A–C, arrowheads). Moreover, we used the antibodies Islet-1 and Go-alpha to label ON rod and cone bipolar cells. 44 Among Go-alpha–positive bipolar and amacrine cells, there was absence of colocalization with CRT (Figs. 3D–F, arrows); however, we found some sparse colocalization with Ilet-1–positive cells located in the outer sector of the inner nuclear layer (INL), corresponding to the ON bipolar cells (Figs. 3G–I, arrowhead). 
Figure 3.
 
Immunolabeling of CRT and cellular markers of inner retinal neurons. (A–C) Colocalization of parvalbumin (PV) and CRT shows CRT in ganglion cells (arrows), but not in horizontal cells (yellow arrows). (D–F) ON bipolar cells were identified by Go-alpha where there was no colocalization. (G–I) Few islet-1 cells expressed CRT (arrowhead). (J–L) In GAD67 amacrine cells there was some CRT colocalization. (M–O) Calretinin (CR) showed complete colocalization with CRT in the mid-peripheral human retina. (P–R) Some MAP-2 positive cells expressed CRT. (Scale bar: 20 μm.) OPL, outer plexiform layer; IPL, inner plexiform layer; GCL, ganglion cell layer.
Figure 3.
 
Immunolabeling of CRT and cellular markers of inner retinal neurons. (A–C) Colocalization of parvalbumin (PV) and CRT shows CRT in ganglion cells (arrows), but not in horizontal cells (yellow arrows). (D–F) ON bipolar cells were identified by Go-alpha where there was no colocalization. (G–I) Few islet-1 cells expressed CRT (arrowhead). (J–L) In GAD67 amacrine cells there was some CRT colocalization. (M–O) Calretinin (CR) showed complete colocalization with CRT in the mid-peripheral human retina. (P–R) Some MAP-2 positive cells expressed CRT. (Scale bar: 20 μm.) OPL, outer plexiform layer; IPL, inner plexiform layer; GCL, ganglion cell layer.
There were other CRT-positive amacrine cells not labeled with calbindin. To determine the neurochemical signature of these CRT immunoreactive amacrine cells, we used GAD67, calretinin and MAP-2 antibodies, which are markers of GABAergic, AII, and tyrosine hydroxylase/dopaminergic amacrine cells, respectively. 4648 We found partial colocalization between GAD67 and CRT-positive amacrine cells (Figs. 3J–L), mostly at large amacrine cells whose morphology suggest them to be the tyrosine hydroxylase (TH)-immunopositive amacrine cells. In fact, this notion is corroborated by the similar partial colocalization between CRT and MAP-2 (Figs. 3P–R). Accordingly, we confirmed the immunolabeling of TH amacrine cells with MAP-2 by double labeling with these two markers (Figs. 4A–C). In contrast, extensive colocalization was evident in calretinin and CRT-labeled amacrine cells (Figs. 3M–O, arrowheads). In primate peripheral retinas, essentially all calretinin-positive cells in the INL are AII amacrine cells, 47 therefore human AII amacrine cells express CRT. Calbindin and calretinin also labeled the stratification of these amacrine cells in the inner plexiform layer (Figs. 1B–D and 3M–O) where colocalization with CRT was observed. 
Figure 4.
 
Double immunofluorescence of aged normal retinas with markers of amacrine cell types MAP-2 (A), TH (B), and a blood vessel basal membrane marker, laminin (D) and CRT (E). MAP-2 labeled big neurons corresponding to amacrine cells (arrow) and a specific labeling pattern in the IPL (arrowhead), whereas CRT specifically labeled the soma of these cells (B–C). Cell nuclei (blue) were labeled with DAPI. Multiple and punctiform expression pattern of CRT detected in a blood vessel (bv) at the INL (F). (Scale bars: 10 μm.) IPL, inner plexiform layer; GCL, ganglion cell layer.
Figure 4.
 
Double immunofluorescence of aged normal retinas with markers of amacrine cell types MAP-2 (A), TH (B), and a blood vessel basal membrane marker, laminin (D) and CRT (E). MAP-2 labeled big neurons corresponding to amacrine cells (arrow) and a specific labeling pattern in the IPL (arrowhead), whereas CRT specifically labeled the soma of these cells (B–C). Cell nuclei (blue) were labeled with DAPI. Multiple and punctiform expression pattern of CRT detected in a blood vessel (bv) at the INL (F). (Scale bars: 10 μm.) IPL, inner plexiform layer; GCL, ganglion cell layer.
Previous work demonstrated CRT expression at the inner blood retinal barrier, where CRT is regulating creatine entry. 4,6,32 CRT immunolabeling was also evident in the retina blood vessels. Laminin, a blood vessel basal membrane marker, was used to outline the vascular structures of the inner retina. CRT was observed with variable expression in blood vessels located in the INL and ganglion cell layer (Figs. 4 D–F). 
CRT Expression in Retinal Detachment
To determine whether in retinal detachment there are changes in creatine transport involving the glial cells, we assessed the CRT expression pattern and used glial fibrillary acid protein (GFAP) as a marker of astrocytes or activated Müller cells. In normal aged retina (Figs. 5A–C), astrocytes are mainly located in the NFL (Fig. 5A, double arrows). Despite the strong CRT labeling observed in the NFL, the GFAP-positive astrocyte processes did not show colocalization with CRT in normal retina (Figs. 5A–F, double arrows), suggesting that CRT expression in the NFL layers corresponded to a bundle of ganglion cell axons. To confirm this observation, we individualized the series of confocal microscopy images (0.45-μm thick each) from the final superimposed images stack. There was no colocalization between CRT and GFAP (Figs. 5D–F). 
Figure 5.
 
Confocal images of normal aged (A–F) and detached retinas (G–O). Immunolabeling of GFAP in the normal aged retinas is shown in the astrocyte processes in the NFL (double arrows, A–C) not colocalizing with CRT (merged images in C). (D–F) Single confocal image sections (0.45μm) showing absence of colocalization between CRT and GFAP. (G–I) GFAP is upregulated and found in the hypertrophic trunks of Müller cells (double arrows) in retinal detachment without colocalization with CRT. (J–L) In retinal detachment specimens, CRT is expressed in calretinin cells in inner retina. (M–O) The labeling pattern of CRT and GAD67 is maintained in detached retina. (Scale bars: 20 μm.) OPL, outer plexiform layer; IPL, inner plexiform layer; GCL, ganglion cell layer.
Figure 5.
 
Confocal images of normal aged (A–F) and detached retinas (G–O). Immunolabeling of GFAP in the normal aged retinas is shown in the astrocyte processes in the NFL (double arrows, A–C) not colocalizing with CRT (merged images in C). (D–F) Single confocal image sections (0.45μm) showing absence of colocalization between CRT and GFAP. (G–I) GFAP is upregulated and found in the hypertrophic trunks of Müller cells (double arrows) in retinal detachment without colocalization with CRT. (J–L) In retinal detachment specimens, CRT is expressed in calretinin cells in inner retina. (M–O) The labeling pattern of CRT and GAD67 is maintained in detached retina. (Scale bars: 20 μm.) OPL, outer plexiform layer; IPL, inner plexiform layer; GCL, ganglion cell layer.
Three retinal detachment specimens were analyzed for the pattern of CRT expression. All three samples presented identical pattern of CRT distribution despite differences in the age of patients and duration of detachment. Figure 5 is representative of the CRT expression pattern found in all three RRD samples. In the detachment samples, there was expression of CRT in the outer retina, mainly in the outer nuclear layer and photoreceptor inner segments (Figs. 5H, 5K, and 5N). 
Photoreceptor outer segment loss is a widespread phenomenon after retinal detachment; however, in areas where they were present, the difference in pixel value between RRD and normal outer nuclear layer (ONL) (ie, rods) reflected increased CRT immunoreactivity (Table 3). We found that although the overall retinal CRT immunoreactivity was reduced, there was an overall increase within the ONL. Also, when specific comparisons were made, we corroborated that a specific CRT pixel value increase was observed within morphologically recognized cone photoreceptors. Table 3 shows five examples where CRT immunoreactivity was compared between samples from control and RRD retinas. A positive pixel value indicates that the level of immunoreactivity was overall higher in the control retina, whereas a negative pixel value means the RRD retina displayed a higher level of CRT immunoreactivity in the ONL (Table 3). In fact, when specific comparisons were made within cone photoreceptors, the RRD retina displayed a relatively higher level of CRT immunoreactivity (Table 3). 
Table 3.
 
Difference in Immunoreactivity Found between Control and RRD Samples
Table 3.
 
Difference in Immunoreactivity Found between Control and RRD Samples
Difference in IMR (pixel value) [Control-RRD]
Whole Retina Outer Nuclear Layer Cone Photoreceptors
Example 1 24 −69 −39
Example 2 26 −50 −99
Example 3 88 −64 −149
Example 4 93 −46 −71
Example 5 78 −77 −134
An important manifestation of anatomical remodeling of the retina after detachment is the hypertrophy and proliferation of Müller cell processes, which is evident by upregulation of GFAP. Figures 5G–I show GFAP-positive hypertrophic Müller cells spanning the entire retina but no colocalization with CRT is observed (double arrows). Conversely, CRT colocalization pattern in the inner retina with cell markers of inner retinal neurons was preserved. Figures 5J–L illustrate the colocalization pattern found in retinal detachment between CRT and calretinin amacrine cells. The immunolabeling pattern of CRT and GAD67 was also maintained in the inner retina (Figs. 5M–O). Therefore, the hypoxic conditions in the outer retina did not alter the immunocytochemical expression of CRT and the different macromolecular markers in inner retinal cells. 
Discussion
We have shown the presence of CRT with a specific labeling pattern in photoreceptors and selected neurons, but absent in glial cells in the human retina. In detached retina, increased immunolabeling of CRT in the ONL suggests a regional metabolic remodeling occurring in photoreceptor cells. 
CRT Distribution Pattern in the Retina
The distribution of CRT expression in the human retina shares similarities to the pattern of labeling described in the retina of other vertebrate species. 33 CRT is expressed by photoreceptors, inner retinal neurons, blood vessels, and retinal pigment epithelium (RPE). The interspecies presence of CRT in all three retinal neuronal orders and in the retinal capillaries suggests that the CRT transports creatine into photoreceptors and neurons at the blood retinal barriers (BRBs). Accordingly, Nakashima et al. 32 documented CRT expression in a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB) using RT-PCR and Western blot. They also demonstrated that creatine transporter at the inner BRB (iBRB) works by continuously providing creatine at a near-maximum transport rate. The RPE cells represent the outer BRB in the holangiotic human retina. Our observation of partial CRT increase in human RPE, corroborated by its finding in other vertebrate retinas, suggests facilitated creatine transport into photoreceptors. This transport model is comparable to the transport systems of lactate and glucose into the retina with monocarboxylate transporter 1 and glucose transporter 1 via both iBRB and RPE. 49,50  
Preferential CRT immunolabeling to specific neurons in the inner retina (dopaminergic, AII amacrine cells and some ganglion cells) represents a conundrum. The vicinity of some neurons to blood vessels could explain the random yet specific CRT labeling to amacrine cells; however, the fact that all AII amacrine cells express CRT and that immunolabeling is observed also in the plexiform layers suggests that the hypothesis is incorrect. We propose an alternative explanation in that the CRT immunoreactivity identifies neurons that are resistant to disturbances of the energy metabolism facilitated by the provision of creatine. Evidence shows that amacrine cells are not equally susceptible to retinal ischemia or excitotoxicity. 5154 Variation in number and types of glutamate receptors as well as the expression of receptors that may provide neuroprotection (e.g., purinergic receptors) 55,56 helps to explain the variable tolerance of neurons to insult. Furthermore, structural changes in amacrine cells subsequent to deafferentation and input losses may also corroborate for variable vulnerability of amacrine cells to excitotoxic or ischemic insults. In addition, AII amacrine cells have been shown not to possess glycogen stores, thus suggesting that these cells may rely on creatine for energy. 57  
CRT Distribution in Müller Cells
We have shown that in the human retina, similar to other vertebrates, Müller cells do not express the CRT. Glial cells have an established role in delivering nutrients to retinal neurons and providing lactate to photoreceptors. 57,58 Therefore, our observation that CRT is absent from human glia suggests that either (1) within Müller cells there is no creatine/phosphocreatine system to preserve their ATP levels or (2) that they have a local source of creatine biosynthesis. The latter hypothesis was strongly reinforced by the demonstration with RT-PCR, Western blot, and HPLC of the creatine precursor enzyme S-adenosyl- l -methionine:N-guanidinoacetate methyltransferase (GAMT) in retinal Müller cells. 5 Immunocytochemical colocalization of GAMT with glutamine synthetase further confirmed Müller cells as a local source of creatine biosynthesis. 5 Nevertheless, the combined evidence for creatine biosynthesis by Müller cells and the absence of CRT in these retinal glial cells raises the question about if and how the trafficking of creatine between Müller cells and neurons occurs. One possibility is that the production of creatine by Müller cells solely serves their own metabolic needs without provision to other retinal cells. 
We also verified the absence of CRT expression in Müller cells after retinal detachment. The detachment-induced hypoxic insult causes hypertrophy and proliferation of Müller cell processes. In this hypoxic environment, there was increased CRT expression specific to photoreceptors. The overall reduction in CRT immunoreactivity complies with other studies; however, we are showing within the ONL an increase in the RRD tissue, likely because of the protein moving into the somata of photoreceptors. These suggest an increased demand for ATP synthesis from anaerobic metabolism in photoreceptors; however, it is also likely that the increased expression of CRT in the ONL and decrease in the INL represent a redistribution of CRT protein owing to remodeling of the outer segments and disruption of its compartmentalization. This redistribution could be similar to what occurs to the rod photoreceptor protein rhodopsin, which is aberrantly expressed in the ONL after retinal detachment 30 ; however, because phosphocreatine/creatine regenerate ATP significantly faster than glycolysis and oxidative phosphorylation, 59 the increased CRT expression in the sector correspondent to hypoxic insult in detached samples reinforces the concept that the creatine/phosphocreatine shuttle system plays an important role in the retinal energy homeostasis in damaging conditions. 
Metabolic Diseases
The relevance of creatine metabolism and transport is demonstrated by the cumulative evidence of its neuroprotective properties and use in major clinical trials for neurodegenerative pathologies, such as Parkinson's and Huntington's disease and amyotrophic lateral sclerosis. 11,12,15,16,25,60,61 The increased expression and function of CRT mRNA in the blood brain barrier under hyperammonemic conditions 62 suggest that CRT plays an important role in neuroprotective mechanisms in neurological disorders. For example, the deprivation of creatine because of the loss of photoreceptor contact with the RPE combined with disruption in creatine biosynthesis by a reactive and proliferative Müller cell, further support the need for creatine uptake in the detached retina. 
Moxon-Lester et al. 63 showed a significant reduction in the CRT immunolabeling of rat retinas in an acute retinal ischemia experiment. Acute ischemia is a much more severe degeneration than retinal detachment. Yet, disrupted biosynthesis of creatine by hyperornithinemia affects the choroid and retina in patients with gyrate atrophy who have a reduced creatine supply from the blood and a compromised supply from the Müller cells 64 ; however, retinal detachment represents a localized and less severe hypoxic and hypoglycemic insult where the inner retinal circulation is intact and the outer retina may still obtain nutrients in the subretinal fluid by diffusion from the choroid depending on the height and duration of the detachment. 
References
Béard E Braissant O . Synthesis and transport of creatine in the CNS: importance for cerebral functions. J Neurochem. 2010;115:297–313. [CrossRef] [PubMed]
Wyss M Kaddurah-Daouk R . Creatine and creatinine metabolism. Physiol Rev. 2000;80:1107–1203. [PubMed]
Klein AM Ferrante RJ . The neuroprotective role of creatine. Subcell Biochem. 2007;46:205–243. [PubMed]
Tachikawa M Hosoya KI Ohtsuki S Terasaki T . A novel relationship between creatine transport at the blood-brain and blood-retinal barriers, creatine biosynthesis, and its use for brain and retinal energy homeostasis. Subcell Biochem. 2007;46:83–98. [PubMed]
Nakashima T Tomi M Tachikawa M Watanabe M Terasaki T Hosoya K . Evidence for creatine biosynthesis in Müller glia. Glia. 2005;52:47–52. [CrossRef] [PubMed]
Ohtsuki S . New aspects of the blood–brain barrier transporters; its physiological roles in the central nervous system. Biol Pharm Bull. 2004;27:1489–1496. [CrossRef] [PubMed]
Bizzi A Bugiani M Salomons GS X linked creatine deficiency syndrome: A novel mutation in creatine transporter gene SLC6A8. Ann Neurol. 2002;52:227–231. [CrossRef] [PubMed]
Salomons GS Van Dooren SJM Verhoeven NM X-linked creatine-transporter gene (SLC6A8) defect: a new creatine-deficiency syndrome. Am J Hum Genet. 2001;68:1497–1500. [CrossRef] [PubMed]
Schulze A . Creatine deficiency syndromes. Mol Cell Biochem. 2003;244:143–150. [CrossRef] [PubMed]
Stockler S Schutz PW Salomons GS . Cerebral creatine deficiency syndromes: clinical aspects, treatment and pathophysiology. Subcell Biochem. 2008;46:149–166.
Brustovetsky N Brustovetsky T Dubinsky JM . On the mechanisms of neuroprotection by creatine and phosphocreatine. J Neurochem. 2001;76:425–434. [CrossRef] [PubMed]
Klivenyi P Ferrante RJ Matthews RT Neuroprotective effects of creatine in a transgenic animal model of amyotrophic lateral sclerosis. Nat Med. 1999;5:347–350. [CrossRef] [PubMed]
Malcon C Kaddurah-Daouk R Beal MF . Neuroprotective effects of creatine administration against NMDA and malonate toxicity. Brain Res. 2000;860:195–198. [CrossRef] [PubMed]
Perasso L Adriano E Ruggeri P Burov S Gandolfo C Balestrino M . In vivo neuroprotection by a creatine-derived compound: phosphocreatine-Mg-complex acetate. Brain Res. 2009;1285:158–163. [CrossRef] [PubMed]
Prass K Royl G Lindauer U Improved reperfusion and neuroprotection by creatine in a mouse model of stroke. J Cereb Blood Flow Metab. 2006;27:452–459. [CrossRef] [PubMed]
Bender A Koch W Elstner M Creatine supplementation in Parkinson disease: a placebo-controlled randomized pilot trial. Neurology. 2006;67:1262–1264. [CrossRef] [PubMed]
Bender A Auer DP Merl T Creatine supplementation lowers brain glutamate levels in Huntington's disease. J Neurol. 2005;252:36–41. [CrossRef] [PubMed]
Dedeoglu A Kubilus JK Yang L Ferrante KL Hersch SM Beal MF . Creatine therapy provides neuroprotection after onset of clinical symptoms in Huntington's disease transgenic mice. J Neurochem. 2003;85:1359–1367. [CrossRef] [PubMed]
Ferrante RJ Andreassen OA Jenkins BG Neuroprotective effects of creatine in a transgenic mouse model of Huntington's disease. J Neurosci. 2000;20:4389–4397. [PubMed]
Hersch S Gevorkian S Marder K Creatine in Huntington disease is safe, tolerable, bioavailable in brain and reduces serum 8OH2′dG. Neurology. 2006;66:250–252. [CrossRef] [PubMed]
Matthews RT Yang L Jenkins BG Neuroprotective effects of creatine and cyclocreatine in animal models of Huntington's disease. J Neurosci. 1998;18:156–163. [PubMed]
Ryu H Rosas HD Hersch SM Ferrante RJ . The therapeutic role of creatine in Huntington's disease. Pharmacol Ther. 2005;108:193–207. [CrossRef] [PubMed]
Tabrizi S Blamire A Manners D Creatine therapy for Huntington's disease: clinical and MRS findings in a 1-year pilot study. Neurology. 2003;61:141–142. [CrossRef] [PubMed]
Verbessem P Lemiere J Eijnde B Creatine supplementation in Huntington's disease: a placebo-controlled pilot trial. Neurology. 2003;61:925–930. [CrossRef] [PubMed]
Yang L Calingasan NY Wille EJ Combination therapy with coenzyme Q10 and creatine produces additive neuroprotective effects in models of Parkinson's and Huntington's diseases. J Neurochem. 2009;109:1427–1439. [CrossRef] [PubMed]
Lewis G Mervin K Valter K Limiting the proliferation and reactivity of retinal Müller cells during experimental retinal detachment: the value of oxygen supplementation. Am J Ophthalmol. 1999;128:165–172. [CrossRef] [PubMed]
Mervin K Valter K Maslim J Lewis G Fisher S Stone J . Limiting photoreceptor death and deconstruction during experimental retinal detachment: the value of oxygen supplementation. Am J Ophthalmol. 1999;128:155–164. [CrossRef] [PubMed]
Cook B Lewis GP Fisher SK Adler R . Apoptotic photoreceptor degeneration in experimental retinal detachment. Invest Ophthalmol Vis Sci. 1995;36:990–996. [PubMed]
Fisher SK Lewis GP . Müller cell and neuronal remodeling in retinal detachment and reattachment and their potential consequences for visual recovery: a review and reconsideration of recent data. Vis Res. 2003;43:887–897. [CrossRef]
Fisher SK Lewis GP Linberg KA Verardo MR . Cellular remodeling in mammalian retina: results from studies of experimental retinal detachment. Prog Retin Eye Res. 2005;24:395–431. [CrossRef] [PubMed]
Lewis GP Linberg KA Fisher SK . Neurite outgrowth from bipolar and horizontal cells after experimental retinal detachment. Invest Ophthalmol Vis Sci. 1998;39:424–434. [PubMed]
Nakashima T Tomi M Katayama K Blood to retina transport of creatine via creatine transporter (CRT) at the rat inner blood–retinal barrier. J Neurochem. 2004;89:1454–1461. [CrossRef] [PubMed]
Acosta ML Kalloniatis M Christie DL . Creatine transporter localization in developing and adult retina: importance of creatine to retinal function. Am J Physio Cell Physiol. 2005;289:C1015–1023. [CrossRef]
Hannibal J Hindersson P Ostergaard J Melanopsin is expressed in PACAP-containing retinal ganglion cells of the human retinohypothalamic tract. Invest Ophthalmol Visual Sci. 2004;45:4202–4209. [CrossRef]
Spitznas M Bornfeld N . The architecture of the most peripheral retinal vessels. Albrecht Von Graefes Arch Klin Exp Ophthalmol. 1977;203:217–229. [CrossRef] [PubMed]
Tornqvist K Ehinger B . Peptide immunoreactive neurons in the human retina. Invest Ophthalmol Vis Sci. 1988;29:680–686. [PubMed]
Van Haesendonck E Missotten L . A subgroup of bipolar cells in human retina is GABA-immunoreactive. Neurosci Lett. 1993;161:187–190. [CrossRef] [PubMed]
de Souza CF Kalloniatis M Polkinghorne PJ McGhee CN Acosta ML . Functional activation of glutamate ionotropic receptors in the human peripheral retina. Exp Eye Res. 2012;94:71–84. [CrossRef] [PubMed]
Dodd J Birch N Waldvogel HJ Christie DL . Functional and immunocytochemical characterization of the creatine transporter in rat hippocampal neurons. J Neurochem. 2010;115:684–693. [CrossRef] [PubMed]
Mak C Waldvogel H Dodd J Immunohistochemical localisation of the creatine transporter in the rat brain. Neuroscience. 2009;163:571–585. [CrossRef] [PubMed]
Dodd JR Christie DL . Cysteine 144 in the third transmembrane domain of the creatine transporter is located close to a substrate-binding site. J Biol Chem. 2001;276:46983–46988. [CrossRef] [PubMed]
Acosta ML Bumsted OB Keely M Tan SS Kalloniatis M . Emergence of cellular markers and functional ionotropic glutamate receptors on tangentially dispersed cells in the developing mouse retina. J Comp Neurol. 2008;506:506–523. [CrossRef] [PubMed]
Chiquet C Dkhissi-Benyahya O Cooper HM . Calcium-binding protein distribution in the retina of strepsirhine and haplorhine primates. Brain Res Bull. 2005;68:185–194. [CrossRef] [PubMed]
Haverkamp S Haeseleer F Hendrickson A . A comparison of immunocytochemical markers to identify bipolar cell types in human and monkey retina. Vis Neurosci. 2003;20:589–600. [CrossRef] [PubMed]
Nag TC Wadhwa S . Calbindin and parvalbumin immunoreactivity in the developing and adult human retina. Brain Res Dev Brain Res. 1996;93:23–32. [CrossRef] [PubMed]
Gábriel R Wilhelm M Straznicky C . Microtubule-associated protein 2 (MAP2)-immunoreactive neurons in the retina of Bufo marinus : colocalisation with tyrosine hydroxylase and serotonin in amacrine cells. Cell Tissue Res. 1992;269:175–182. [CrossRef] [PubMed]
Mills SL Massey SC . AII amacrine cells limit scotopic acuity in central macaque retina: a confocal analysis of calretinin labeling. J Comp Neurol. 1999;411:19–34. [CrossRef] [PubMed]
Nguyen-Legros J Versaux-Botteri C Savy C . Dopaminergic and GABAergic retinal cell populations in mammals. Microsc Res Tech. 1997;36:26–42. [CrossRef] [PubMed]
Chidlow G Wood JPM Graham M Osborne NN . Expression of monocarboxylate transporters in rat ocular tissues. Am J Physiol Cell Physiol. 2005;288:C416–428. [CrossRef] [PubMed]
Gerhart DZ Leino RL Drewes LR . Distribution of monocarboxylate transporters MCT1 and MCT2 in rat retina. Neuroscience. 1999;92:367–375. [CrossRef] [PubMed]
Dijk F Kamphuis W . An immunocytochemical study on specific amacrine cell subpopulations in the rat retina after ischemia. Brain Res. 2004;1026:205–217. [CrossRef] [PubMed]
Osborne NN Casson RJ Wood JPM Chidlow G Graham M Melena J . Retinal ischemia: mechanisms of damage and potential therapeutic strategies. Prog Retin Eye Res. 2004;23:91–147. [CrossRef] [PubMed]
Sun D Bui BV Vingrys AJ Kalloniatis M . Alterations in photoreceptor bipolar cell signaling following ischemia/reperfusion in the rat retina. J Comp Neurol. 2007;505:131–146. [CrossRef] [PubMed]
Sun D Vingrys AJ Kalloniatis M . Metabolic and functional profiling of the ischemic/reperfused rat retina. J Comp Neurol. 2007;505:114–130. [CrossRef] [PubMed]
Larsen AK Osborne NN . Involvement of adenosine in retinal ischemia. Studies on the rat. Invest Ophthalmol Vis Sci. 1996;37:2603–2611. [PubMed]
Zhang X Zhang M Laties AM Mitchell CH . Balance of purines may determine life or death of retinal ganglion cells as A3 adenosine receptors prevent loss following P2X7 receptor stimulation. J Neurochem. 2006;98:566–575. [CrossRef] [PubMed]
Rungger-Brändle E Kolb H Niemeyer G . Histochemical demonstration of glycogen in neurons of the cat retina. Invest Ophthalmol Vis Sci. 1996;37:702–715. [PubMed]
Poitry-Yamate CL Poitry S Tsacopoulos M . Lactate released by Müller glial cells is metabolized by photoreceptors from mammalian retina. J Neurosci. 1995;15:5179–5191. [PubMed]
Wallimann T Wyss M Brdiczka D Nicolay K Eppenberger HM . Intracellular compartmentation, structure and function of creatine kinase isoenzymes in tissues with high and fluctuating energy demands: the ‘phosphocreatine circuit' for cellular energy homeostasis. Biochem J. 1992;281:21. [PubMed]
Béard E Braissant O . Synthesis and transport of creatine in the CNS: importance for cerebral functions. J Neurochem. 2010;115:297–313. [CrossRef] [PubMed]
Brosnan JT Brosnan ME . Creatine: endogenous metabolite, dietary, and therapeutic supplement. Nutrition. 2007;27:241–261. [CrossRef]
Bélanger M Asashima T Ohtsuki S Yamaguchi H Ito S Terasaki T . Hyperammonemia induces transport of taurine and creatine and suppresses claudin-12 gene expression in brain capillary endothelial cells in vitro. Neurochem Int. 2007;50:95–101. [CrossRef] [PubMed]
Moxon-Lester L Takamoto K Colditz PB Barnett NL . S-Adenosyl-l-methionine restores photoreceptor function following acute retinal ischemia. Vis Neurosci. 2009;26:429–441. [CrossRef] [PubMed]
Sipilä I Simell O Arjomaa P . Gyrate atrophy of the choroid and retina with hyperornithinemia. Deficient formation of guanidinoacetic acid from arginine. J Clin Invest. 1980;66:684. [CrossRef] [PubMed]
de Souza CF Kalloniatis M Polkinghome PJ McGhee CN Acosta ML . Functional and anatomical remodeling in human retinal detachment. Exp Eye Res. 2012;97:(1) 73–89. [CrossRef] [PubMed]
Footnotes
 Disclosure: C.F. de Souza, None; M. Kalloniatis, None; D.L. Christie, None; P.J. Polkinghorne, None; C.N.J. McGhee, None; M.L. Acosta, None
Footnotes
 Supported in part by the New Zealand Optometric Vision Research Foundation (NZOVRF) reference number 3625663.
Figure 1.
 
Immunofluorescence labeling in human aged normal retina. Images were captured using confocal microscopy. DAPI was used as counter-staining (A). Creatine transporter (B) colocalizes with calbindin (C). Strong immunoreactivity coexpressed in cones (arrows), photoreceptor synaptic terminals (arrowheads), AII amacrine cells, and outer and inner plexiform layers. (E) DAPI labeling of a negative control for the immunocytochemical procedure where secondary antibody was applied in the absence of a primary antibody. (F) The presence of CRT immunoreactivity at the RPE. (Scale bar: 20 μm.) HC, horizontal cell; AC, amacrine cell; OPL, outer plexiform layer; IPL, inner plexiform layer; GCL, ganglion cell layer.
Figure 1.
 
Immunofluorescence labeling in human aged normal retina. Images were captured using confocal microscopy. DAPI was used as counter-staining (A). Creatine transporter (B) colocalizes with calbindin (C). Strong immunoreactivity coexpressed in cones (arrows), photoreceptor synaptic terminals (arrowheads), AII amacrine cells, and outer and inner plexiform layers. (E) DAPI labeling of a negative control for the immunocytochemical procedure where secondary antibody was applied in the absence of a primary antibody. (F) The presence of CRT immunoreactivity at the RPE. (Scale bar: 20 μm.) HC, horizontal cell; AC, amacrine cell; OPL, outer plexiform layer; IPL, inner plexiform layer; GCL, ganglion cell layer.
Figure 2.
 
Confocal microscopy images of human aged normal retina immunolabeled with CRT and GS. DAPI used as counter-staining (A). CRT labeling (B) and the Müller cell marker GS (C) revealed lack of colocalization of both signals. (E) and (F) inserts correspond to a magnification of outlined areas in (D). (Scale bars: A–D, 20 μm; E and F, 15 μm.) OPL, outer plexiform layer; IPL, inner plexiform layer; GCL, ganglion cell layer.
Figure 2.
 
Confocal microscopy images of human aged normal retina immunolabeled with CRT and GS. DAPI used as counter-staining (A). CRT labeling (B) and the Müller cell marker GS (C) revealed lack of colocalization of both signals. (E) and (F) inserts correspond to a magnification of outlined areas in (D). (Scale bars: A–D, 20 μm; E and F, 15 μm.) OPL, outer plexiform layer; IPL, inner plexiform layer; GCL, ganglion cell layer.
Figure 3.
 
Immunolabeling of CRT and cellular markers of inner retinal neurons. (A–C) Colocalization of parvalbumin (PV) and CRT shows CRT in ganglion cells (arrows), but not in horizontal cells (yellow arrows). (D–F) ON bipolar cells were identified by Go-alpha where there was no colocalization. (G–I) Few islet-1 cells expressed CRT (arrowhead). (J–L) In GAD67 amacrine cells there was some CRT colocalization. (M–O) Calretinin (CR) showed complete colocalization with CRT in the mid-peripheral human retina. (P–R) Some MAP-2 positive cells expressed CRT. (Scale bar: 20 μm.) OPL, outer plexiform layer; IPL, inner plexiform layer; GCL, ganglion cell layer.
Figure 3.
 
Immunolabeling of CRT and cellular markers of inner retinal neurons. (A–C) Colocalization of parvalbumin (PV) and CRT shows CRT in ganglion cells (arrows), but not in horizontal cells (yellow arrows). (D–F) ON bipolar cells were identified by Go-alpha where there was no colocalization. (G–I) Few islet-1 cells expressed CRT (arrowhead). (J–L) In GAD67 amacrine cells there was some CRT colocalization. (M–O) Calretinin (CR) showed complete colocalization with CRT in the mid-peripheral human retina. (P–R) Some MAP-2 positive cells expressed CRT. (Scale bar: 20 μm.) OPL, outer plexiform layer; IPL, inner plexiform layer; GCL, ganglion cell layer.
Figure 4.
 
Double immunofluorescence of aged normal retinas with markers of amacrine cell types MAP-2 (A), TH (B), and a blood vessel basal membrane marker, laminin (D) and CRT (E). MAP-2 labeled big neurons corresponding to amacrine cells (arrow) and a specific labeling pattern in the IPL (arrowhead), whereas CRT specifically labeled the soma of these cells (B–C). Cell nuclei (blue) were labeled with DAPI. Multiple and punctiform expression pattern of CRT detected in a blood vessel (bv) at the INL (F). (Scale bars: 10 μm.) IPL, inner plexiform layer; GCL, ganglion cell layer.
Figure 4.
 
Double immunofluorescence of aged normal retinas with markers of amacrine cell types MAP-2 (A), TH (B), and a blood vessel basal membrane marker, laminin (D) and CRT (E). MAP-2 labeled big neurons corresponding to amacrine cells (arrow) and a specific labeling pattern in the IPL (arrowhead), whereas CRT specifically labeled the soma of these cells (B–C). Cell nuclei (blue) were labeled with DAPI. Multiple and punctiform expression pattern of CRT detected in a blood vessel (bv) at the INL (F). (Scale bars: 10 μm.) IPL, inner plexiform layer; GCL, ganglion cell layer.
Figure 5.
 
Confocal images of normal aged (A–F) and detached retinas (G–O). Immunolabeling of GFAP in the normal aged retinas is shown in the astrocyte processes in the NFL (double arrows, A–C) not colocalizing with CRT (merged images in C). (D–F) Single confocal image sections (0.45μm) showing absence of colocalization between CRT and GFAP. (G–I) GFAP is upregulated and found in the hypertrophic trunks of Müller cells (double arrows) in retinal detachment without colocalization with CRT. (J–L) In retinal detachment specimens, CRT is expressed in calretinin cells in inner retina. (M–O) The labeling pattern of CRT and GAD67 is maintained in detached retina. (Scale bars: 20 μm.) OPL, outer plexiform layer; IPL, inner plexiform layer; GCL, ganglion cell layer.
Figure 5.
 
Confocal images of normal aged (A–F) and detached retinas (G–O). Immunolabeling of GFAP in the normal aged retinas is shown in the astrocyte processes in the NFL (double arrows, A–C) not colocalizing with CRT (merged images in C). (D–F) Single confocal image sections (0.45μm) showing absence of colocalization between CRT and GFAP. (G–I) GFAP is upregulated and found in the hypertrophic trunks of Müller cells (double arrows) in retinal detachment without colocalization with CRT. (J–L) In retinal detachment specimens, CRT is expressed in calretinin cells in inner retina. (M–O) The labeling pattern of CRT and GAD67 is maintained in detached retina. (Scale bars: 20 μm.) OPL, outer plexiform layer; IPL, inner plexiform layer; GCL, ganglion cell layer.
Table 1
 
Donor Subject Demographics
Table 1
 
Donor Subject Demographics
Donor Age Sex Ethnicity
1. Control 70 Female Caucasian
2. Control 70 Female Caucasian
3. Control 87 Male Caucasian
4. Retinal detachment (2 wk) 62 Male Caucasian
5. Retinal detachment (1 mo) 62 Male Caucasian
6. Retinal detachment (2 mo) 54 Male Caucasian
Table 2.
 
Primary Antibodies Used in This Study
Table 2.
 
Primary Antibodies Used in This Study
Antibody Host Source Working Concentration
Creatine transporter Rabbit Assoc. Prof. David Christie (University of Auckland, NZ) 1:100
Calbindin D28K Mouse Sigma-Aldrich, St. Louis, MO (C9848) 1:1000
Calretinin Mouse BD Biosciences, San Jose, CA (610,908) 1:1000
Parvalbumin Mouse Sigma (P3088) 1:1000
Islet-1 Mouse Developmental Studies Hybridoma Bank at the University of Iowa, Iowa City, IA 1:500
Go-protein (α subunit) Go-alpha Mouse Chemicon International Inc., Billerica, MA (MAB327) 1:500
Glutamate decarboxylase (GAD67) Mouse Chemicon (AB5992) 1:500
Glutamine synthetase (GS) Mouse BD Biosciences (610,518) 1:1000
GFAP-Cy3 Mouse Sigma (C9205) 1:3000
Laminin Rat Abcam, Cambridge, UK (ab79057) 1:200
MAP-2 Mouse Novus Biologicals (NB300-143) 1:200
Tyrosine hydroxylase (TH) Rabbit Chemicon (AB152) 1:1000
Table 3.
 
Difference in Immunoreactivity Found between Control and RRD Samples
Table 3.
 
Difference in Immunoreactivity Found between Control and RRD Samples
Difference in IMR (pixel value) [Control-RRD]
Whole Retina Outer Nuclear Layer Cone Photoreceptors
Example 1 24 −69 −39
Example 2 26 −50 −99
Example 3 88 −64 −149
Example 4 93 −46 −71
Example 5 78 −77 −134
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