| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
From the Department of Ophthalmology, Kanazawa University Graduate School of Medical Science, Kanazawa, Japan.
 |
Abstract |
|---|
 Top Abstract MethodsResultsDiscussionReferences |
|---|
METHODS. RGCs of Brown Norway rats were retrogradely labeled bilaterally with the fluorescent dye 4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodine (DiA). The unilateral optic nerve was crushed intraorbitally with a clip. RGCs were imaged in vivo with an SLO with an argon blue laser (488 nm) and optical filter sets for fluorescein angiography, before and 1, 2, and 4 weeks after the crush. Fluorescent cells were also counted in retinal flatmounts at baseline and 1, 2, and 4 weeks after the crush. An image overlay analysis was performed to check cell positions in the SLO images over time. Lectin histochemical analysis was performed to determine the relationship of microglia to the newly emerged DiA fluorescence detected by image overlay analysis after the optic nerve crush.
RESULTS. Fluorescent RGCs were visible in vivo with an SLO. RGC survival decreased gradually after the crush. In the retina after the optic nerve crush, newly emerged DiA fluorescence detected by image overlay analysis corresponded to fluorescent cells morphologically different from RGCs in the retinal flatmount and was colocalized mostly with lectin-stained microglial processes. RGC counts by SLO were comparable to those in retinal flatmounts.
CONCLUSIONS. The SLO is useful for in vivo imaging of rat RGCs and therefore may be a valuable tool for monitoring RGC changes over time in various rat models of RGC damage.
Examination of RGC changes in vivo over time may overcome this drawback. Only a few papers have reported successful imaging of rat RGCs in vivo with a confocal laser scanning microscope,6 a fluorescence microscope,7 or conventional infrared fundus photography.8 Recently, in vivo imaging of RGC apoptosis using a confocal laser scanning microscope has been reported.9 However, imaging techniques using a confocal laser scanning microscope or a fluorescence microscope have not been widely adopted because of the limited availability of the equipment and the necessary special modifications of the microscope. Counts of RGCs made with infrared fundus photography have not been published. Therefore, a new methodology for in vivo imaging of RGCs is needed.
The scanning laser ophthalmoscope (SLO) is widely used in fluorescein and indocyanine green angiography to examine human retinal diseases. Furthermore, the SLO has been reported to be useful for evaluation of the retinal nerve fiber layer and the optic disc of human glaucomatous eyes.10 11 The SLO can also be used for imaging rat retinas to study retinal microcirculation.12 In this study, we wanted to determine whether the SLO is useful for in vivo imaging and counting of rat RGCs by using the optic nerve crush model.
|
Methods |
|---|
|
TopAbstractMethodsResultsDiscussionReferences |
|---|
All animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Experiments were conducted on rats anesthetized by intraperitoneal injection of pentobarbital sodium (65 mg/kg).
Retrograde Fluorescent Staining of RGCs
Retrograde staining of RGCs of both eyes was achieved by injecting a fluorescent dye into the superior colliculus bilaterally. Rats were placed in a stereotactic apparatus (Narishige Co. Ltd., Tokyo, Japan), and the skin of the skull was incised. The brain surface was exposed by perforating the parietal bone with a dental drill to facilitate dye injection. A fluorescent dye, 4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodine (DiA; Invitrogen, Eugene, OR), was dissolved in dimethylformamide (Sigma-Aldrich, St. Louis, MO) at a concentration of 10 µg/mL. The dye solution (2.0 µL) was injected at a point 5.5 mm caudal to the bregma and 1.5 mm lateral to the midline on both sides to a depth of 4.5 mm from the surface of the skull, in accordance with the results of preliminary experiments to identify the position of the superior colliculus.
In Vivo Imaging of RGCs with the SLO
The eyes were dilated with 0.5% tropicamide and 0.5% phenylephrine hydrochloride eye drops (Santen Pharmaceuticals, Osaka, Japan). To preserve corneal clarity throughout the experiment, we placed a custom-made contact lens with a radius of curvature of 2.75 mm and a diameter of 5.0 mm (Kyoto Contact Lens, Kyoto, Japan) on the cornea after topical anesthesia with 0.4% oxybuprocaine hydrochloride eye drops (Santen Pharmaceuticals). A rat was placed on a heating pad (Deltaphase Isothermal Pad; Braintree Scientific, Inc., Braintree, MA), and its head was gently held manually to keep the eye in position for viewing the fundus with an SLO (SLO 101; Rodenstock Instruments, Munich, Germany). Under illumination with an argon blue laser (488 nm), we focused on the retinal surface by changing the refractive values in the SLO setting, visualizing fluorescent RGCs through the optical filter sets for fluorescein angiography. Dynamic fundus images with a field angle of 40° were recorded in the center of the fundus and also in the midperipheral area. The SLO images were digitized by an analog-to-digital video converter (Canopus ADVC-300; Canopus Co., Ltd., Kobe, Japan) and saved as DV-AVI files on a computer (VAIO VGC-RA50S; Sony Corp., Tokyo, Japan), running commercial computer software (DV GatePlus; Sony Corp.). In vivo images of RGCs were obtained before and 1, 2, and 4 weeks after optic nerve crush.
Intraorbital Optic Nerve Crush
Intraorbital optic nerve crush was performed 2 months after the retrograde staining of RGCs because we observed a gradual increase in the intensity and the area of RGC staining for up to 2 months after dye injection (data not shown). The conjunctiva of the right eye was incised in the superotemporal quadrant to expose the optic nerve by careful blunt dissection under an operating microscope. The optic nerve was crushed 2 mm behind the globe for 30 seconds with a 60-g clip (Micro Vascular Clip; Roboz Surgical Instrument Co., Gaithersburg, MD). Special care was taken not to damage the blood supply to the eye. The fundus was monitored by indirect ophthalmoscopy, and immediate recovery of retinal blood supply was observed in each eye after removal of the clip. The left eye had no operation.
Retinal Flatmounts
In group 1 rats, both eyes were subjected to retinal flatmount preparation 4 weeks after the optic nerve crush. In group 2 and 3 rats, the retinal flatmounts were prepared from the right eye.
Immediately after in vivo imaging of RGCs, the eyes were enucleated after administration of an overdose of anesthesia by an intraperitoneal injection of pentobarbital sodium. The anterior segments were removed, and the resultant posterior eye cups were fixed in 4% paraformaldehyde and 0.1 M phosphate-buffered saline (PBS) for 1 hour at room temperature. After six radial cuts were made in the periphery of the eye cup, the retina was carefully separated from the retinal pigment epithelium. A small marking cut was placed in the peripheral corner of the superior retinal portion for the correct identification of the retinal orientation. The retina was then flatmounted on a glass slide, covered with an antifade mounting solution (Vectashield; Vector Laboratories, Burlingame, CA) and glass coverslip, and kept in the dark at 4°C until microscopic observation.
RGCs in the retinal flatmounts were examined with a fluorescence microscope (Nikon Eclipse TE300; Nikon Corp., Kanagawa, Japan) equipped with a filter set (excitation filter 450 to 490 nm; barrier filter 520 nm; B-2A; Nikon) and 10x objective. RGC images were recorded as JPEG files with a digital cooled charge-coupled device camera (DS-5Mc-L1; Nikon) and were stored on a computer (Dimension 8300; Dell Inc., Round Rock, TX).
Image Analysis for Cell Counting
A retinal area 0.5 to 1.5 mm from the center of the optic disc, where cells were intensely stained and were well focused in SLO images throughout the experimental period, was selected for each eye. Still images of the selected retinal area were created from the DV-AVI files of dynamic SLO images by using video-editing software (Premiere 6.5; Adobe Systems Inc., Mountain View, CA). The identical retinal area of the same eye was determined in SLO images obtained at different times and also in the flatmount images by using image-editing software (Photoshop, ver. 6.0; Adobe Systems Inc.). The number of labeled cells in the selected area of each image was counted manually in a masked fashion by the same investigator. Small spots of punctate fluorescence observed in the flatmounted images after the optic nerve crush were not included in the cell count because they represented cell debris of dead RGCs (see Fig. 2J , arrows).13 When counting RGCs only, we applied the morphologic criteria for discriminating non-RGC cells from RGCs.13 14 Cells with irregular shape, intense DiA staining, and smaller or larger size than typical RGCs (see Fig. 5D ) were considered to be non-RGC cells such as microglia (see Fig. 5 , arrows in 5H , 5L , 5P ). The size of the retinal area for RGC counting was measured in the flatmount images using image-analysis software (Image-Pro Express 4.0; Media Cybernetics, Inc., Silver Spring, MD), and the density of labeled cells in each image was calculated.
|
|
|
|
|
Histochemical Analysis of Microglia
For the group 3 rats, SLO imaging of RGCs was performed at baseline and at the time when the retinal flatmounts were to be prepared. After obtaining images of DiA-labeled RGCs with an SLO in vivo and with a fluorescence microscope (Axioplan 2; Carl Zeiss GmbH, Jena, Germany) with a filter set (equivalent to B-2A; Nikon) in the flatmounted retina, we performed histochemical staining of microglia15 in the flatmounted retina. The glass coverslip covering the retinal flatmounts was carefully removed. The retina was floated in PBS, flatmounted on a nylon membrane (Hybond-N+; GE Healthcare, Piscataway, NJ), and blocked with 0.5% bovine serum albumin in PBS and 1.0% Triton X-100 at room temperature for 1 hour. The retina was then incubated with 20 µg/mL of biotin-conjugated isolectin B4 from Griffonia simplicifolia (Sigma-Aldrich, Inc.) in PBS with 0.5% bovine serum albumin and 0.5% Triton X-100 at 4°C overnight. After it was washed in PBS, the retina was treated with 10 µg/mL of Alexa Fluor 350-conjugated avidin (NeutrAvidin; Invitrogen) in PBS with 0.5% bovine serum albumin and 0.1% Triton X-100 at room temperature for 2 hours. The retina was then washed in PBS, carefully peeled from the nylon membrane, flatmounted on a glass slide, and covered with antifade medium (Vectashield; Vector Laboratories) and a glass coverslip. Fluorescence of Alexa Fluor 350 labeling was examined using a fluorescence microscope (Axioplan; Carl Zeiss GmbH) with an appropriate filter set for the dye. The cell density of the microglia was measured at four retinal locations (500 x 500 µm2 each, 1–1.5 mm from the center of the optic disc) per eye and averaged.
Statistical Analysis
The relative density of labeled cells in the selected retinal area of an SLO image at each time point or of the flatmount image was determined for each eye with the labeled cell density of the SLO image taken before the optic nerve crush being 100%. Differences in cell densities were statistically analyzed by the Wilcoxon rank sum test for comparison between control and crushed eyes and by the Wilcoxon signed rank test for comparison of SLO images taken at different time points and of cell densities between SLO and flatmount images. For the comparison of image overlay analysis and retinal flatmounts, differences in RGC counts by SLO and those in retinal flatmounts were also examined by the Wilcoxon signed rank test. One-way ANOVA tests and post hoc tests were used to assess changes of microglial cell densities caused by optic nerve crush. P < 0.05 was considered statistically significant. Data are expressed as the mean ± SD.
|
Results |
|---|
|
TopAbstractMethodsResultsDiscussionReferences |
|---|
After the optic nerve crush, a decrease in fluorescent cells in selected retinal areas of the SLO image was significant at 1 week and progressed at 2 and 4 weeks (Figs. 2 3) . The number of labeled cells in the untreated control eyes remained stable during the experimental period (Figs. 2 3) . To evaluate the validity of cell counts in the SLO images, the number of labeled cells was also determined in retinal flatmounts. The relative density of DiA-labeled cells in SLO images (with that in retinal flatmounts being 100%) was 101.5% ± 9.9%, 98.2% ± 4.8%, 91.3% ± 4.4%, and 99.6% ± 12.0% at baseline and 1, 2, and 4 weeks after optic nerve crush, respectively. Cell counts by SLO were not significantly different from those in retinal flatmounts. However, details of cell structure were less clear in SLO images than in flatmount images (Fig. 2) . Furthermore, most of the punctate fluorescence, which we considered to be cell debris of dead RGCs, observed in the flatmount images of postcrush retina was not clearly visible with the SLO (Fig. 2 , arrows). Thus, the resolution and sensitivity of fluorescence in SLO images were lower than those in flatmount images.
To identify non-RGC cells which became fluorescent after the optic nerve crush, we performed an image overlay analysis. The overlay composite images of control retinas showed few black or white spots, indicating that few deaths followed by phagocytosis of RGCs occurred in the control retinas during the experimental period (Fig. 4) . In contrast, a significant number of black and white spots appeared in the composite images of postcrush retina. The position of white spots changed over time, reflecting the movement of the cells (Fig. 4) . Thus, fluorescent spots in SLO images of postcrush retina (Fig. 2) consisted of both surviving RGCs and emerging non-RGC cells.
By comparing the composite SLO images and retinal flatmounts, we determined that the newly emerged fluorescent spots in the SLO images after the optic nerve crush corresponded with fluorescent cells with shapes and sizes different from RGCs in retinal flatmounts (Fig. 5) . Therefore, we determined RGC counts in SLO images by subtracting the number of newly emerged fluorescent cells from total cells. The results were compared with RGC counts by retinal flatmounts with application of morphologic criteria13 14 to discriminate non-RGC cells from RGCs. There were no significant differences between RGC counts by SLO and those by retinal flatmounts in control and postcrush retinas (Fig. 6) .
|
|
|
|
Discussion |
|---|
|
TopAbstractMethodsResultsDiscussionReferences |
|---|
In this study, we used an SLO and repeatedly imaged RGCs successfully in vivo. Visualization of fluorescent cells in vivo with an SLO was reported by Nishiwaki et al.12 In their study, fluorescent leukocytes stained with acridine orange were counted in rat retinas in SLO images. Similarly, fluorescent RGCs were countable in SLO images in this study.
For long-term in vivo monitoring of RGCs, carbocyanines are suitable as tracers because they are nontoxic, their labeling is intense, and labeling is stable, with survival times of up to 1 year.17 18 Among carbocyanines, DiI (1-1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), and 4Di-10Asp (4-(4-(didecylamino)styryl)-N-methylpyridinium iodine) have been used for retrograde labeling of RGCs to count RGCs in retinal flatmounts.3 4 18 Naskar et al.4 reported the visualization of RGCs in vivo by retrograde staining with 4Di-10Asp and fluorescence microscopy. DiA is a carbocyanine dye that is widely used as an anterograde and retrograde neuronal tracer in vivo. We chose DiA as a tracer for the SLO imaging because it has an appropriate absorption and fluorescence emission property for the argon laser and optical filter sets equipped for fluorescein angiography with an SLO. However, the labeled cell density with carbocyanines is lower than that with a hydrophilic tracer (Fluorogold; Fluorochrome, Denver, CO), probably due to lower solubility of the lipophilic dyes.19 20 The density of DiA-labeled cells in SLO images of normal rat retinas was comparable to reported densities of RGCs stained with other carbocyanines.4 18 19 20 Furthermore, the extent of decrease in of labeled cells after axonal injury agreed with that in previous reports,21 22 and the number of labeled cells in control eyes was constant during the experimental period. Thus, DiA is a suitable tracer for in vivo imaging of RGCs with an SLO.
We encountered several problems in SLO imaging of RGCs. First, the whole area of a single SLO frame (40°) could not be focused well simultaneously because of the image distortion by the contact lens and the spherical property of the eye. Although the minimum well-focused area was larger than the area for RGC counting in retinal flatmounts in other studies,2 4 19 23 modification of a contact lens to enlarge the well-focused area would improve the quality of SLO images and save recording and analysis time. Second, peripheral fundus areas were difficult to image with an SLO. We selected a sampling area for RGC counting 0.5 to 1.5 mm from the center of the optic disc because of the ease of focusing. However, the adult rat retina extends more than 5 mm from the center of the optic disc.5 Therefore, an SLO is not suitable for evaluation of RGCs in the peripheral retina.
Finally, SLO image quality was not as good as that of retinal flatmounts. Although the cell density measured in SLO images was not significantly different from that determined in flatmount images, the resolution and sensitivity of fluorescence in SLO images were lower than those in flatmount images. As reported previously, cells stained by retrograde labeling are not necessarily RGCs, because other types of cells may become labeled by phagocytosis of fluorescent RGC debris.4 15 24 Morphologic criteria were defined to distinguish RGCs from other types of cells for counting RGCs in the retinal flatmount.13 14 25 However, the SLO images of labeled cells were not good enough for discrimination of cell types. Thus, it may be difficult to distinguish RGCs from microglial cells morphologically and to count RGCs accurately in postcrush retina in SLO images.
To discriminate RGCs from other types of cells in SLO images, we developed a new image overlay analysis. The overlaid composite images of pre- and postcrush SLO images showed that new fluorescent spots emerged and moved after the optic nerve crush. A point-by-point comparison of the SLO image overlay and the retinal flatmount in the identical retinal area revealed that the newly emerged fluorescent spots in SLO images after the crush corresponded to non-RGC cells. Therefore, it is reasonable to determine RGC counts in SLO images by subtracting the number of newly emerged fluorescent cells from total cell counts. Furthermore, similar RGC counts by SLO and retinal flatmounts indicate that the image overlay system is useful to count RGCs in SLO images.
As newly emerged fluorescent spots are thought to be microglia that became labeled by phagocytosis of dead RGCs, we compared the SLO image overlay and lectin staining of microglia in retinal flatmounts. Colocalization of most of the new fluorescent spots with microglial processes indicated that image overlay analysis can be useful to monitor the activity of microglia after optic nerve injury.
In conclusion, the SLO is useful for in vivo imaging and counting of rat RGCs and may be a valuable tool for monitoring RGC changes over time in various rat models of RGC damage. This approach will be useful for studying the pathology of optic neuropathy including glaucoma and for developing new therapies.
|
Footnotes |
|---|
Submitted for publication June 7, 2005; revised October 27, 2005, and February 23, 2006; accepted May 15, 2006.
Disclosure: T. Higashide, None; I. Kawaguchi, None; S. Ohkubo, None; H. Takeda, None; K. Sugiyama, None
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: Tomomi Higashide, Department of Ophthalmology, Kanazawa University Graduate School of Medical Science, 13-1 Takara-machi, Kanazawa, Ishikawa 9208641, Japan; eyetomo{at}kenroku.kanazawa-u.ac.jp.
|
References |
|---|
|
TopAbstractMethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  C K S Leung, J D Lindsey, L Chen, Q Liu, and R N Weinreb Longitudinal profile of retinal ganglion cell damage assessed with blue-light confocal scanning laser ophthalmoscopy after ischaemic reperfusion injury Br. J. Ophthalmol., July 1, 2009; 93(7): 964 - 968. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Nagata, T. Higashide, S. Ohkubo, H. Takeda, and K. Sugiyama In Vivo Quantitative Evaluation of the Rat Retinal Nerve Fiber Layer with Optical Coherence Tomography Invest. Ophthalmol. Vis. Sci., June 1, 2009; 50(6): 2809 - 2815. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Murata, M. Aihara, Y.-N. Chen, T. Ota, J. Numaga, and M. Araie Imaging Mouse Retinal Ganglion Cells and Their Loss In Vivo by a Fundus Camera in the Normal and Ischemia-Reperfusion Model Invest. Ophthalmol. Vis. Sci., December 1, 2008; 49(12): 5546 - 5552. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. K.-s. Leung, J. D. Lindsey, J. G. Crowston, C. Lijia, S. Chiang, and R. N. Weinreb Longitudinal Profile of Retinal Ganglion Cell Damage after Optic Nerve Crush with Blue-Light Confocal Scanning Laser Ophthalmoscopy Invest. Ophthalmol. Vis. Sci., November 1, 2008; 49(11): 4898 - 4902. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. C. Gray, R. Wolfe, B. P. Gee, D. Scoles, Y. Geng, B. D. Masella, A. Dubra, S. Luque, D. R. Williams, and W. H. Merigan In Vivo Imaging of the Fine Structure of Rhodamine-Labeled Macaque Retinal Ganglion Cells Invest. Ophthalmol. Vis. Sci., January 1, 2008; 49(1): 467 - 473. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
