In Vivo Imaging and Counting of Rat Retinal Ganglion Cells Using a Scanning Laser Ophthalmoscope

Abstract

purpose. To determine whether a scanning laser ophthalmoscope (SLO) is useful for in vivo imaging and counting of rat retinal ganglion cells (RGCs).

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.

Ocular hypertension, ischemia-reperfusion, and optic nerve crush rodent models have helped elucidate the pathophysiology of glaucoma and other optic neuropathies. ¹ In these models, the number of retinal ganglion cells (RGCs) is usually counted in retinal flatmounts that are prepared after labeling of RGCs, then the changes of RGCs are evaluated by comparing the equivalent retinal positions in experimental and control eyes. ^{2 3 4} However, inconsistency of staining efficacy or inherent variability of RGC density ⁵ prevents detection of small differences in RGC survival between experimental and control eyes. 

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

Animals

Male Brown Norway rats, 12 weeks of age and weighing 200 to 250 g, were used. Six rats (group 1) were used for in vivo RGC imaging by SLO over time (before and 1, 2, and 4 weeks after unilateral optic nerve crush) and to compare the SLO images and retinal flatmount images at 4 weeks after optic nerve crush. The comparison of both images in the nonsurgical eyes served as baseline data. In addition, 12 rats (group 2) were used to compare the images of SLO and retinal flatmounts at 1 and 2 weeks after optic nerve crush (6 rats for each time point). Furthermore, three rats (group 3) for each time point (baseline and 1, 2, and 4 weeks after optic nerve crush) were used for the histochemical analyses of microglia. 

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 10× 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 1 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 3 4 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. 

Image Overlay Analysis

To distinguish RGCs from other types of cells, which presumably were microglial cells that became fluorescent by phagocytosis of cell debris of RGCs after optic nerve crush, an image overlay analysis was performed. The cell positions in the SLO images taken after optic nerve crush were compared with those at baseline in the analysis. Because RGCs are not mobile and are stably labeled by retrograde transport of DiA 2 months after dye injection, fluorescent cells that have emerged in a location where no fluorescence was observed before should be other types of cells. With the image analysis software (Photoshop; Adobe Systems, Inc.), the SLO images recorded before optic nerve crush had their brightness inverted and were overlaid as a 50% opaque layer onto the SLO images of the same retinal area taken after axonal injury. Theoretically, if the precrush image were identical with the postcrush image, the composite image, which is produced by the overlay with perfect alignment of the two images, would become homogeneously gray. Slight misalignment of identical spots in the two images produces a shadowing effect around the spots. In contrast, fluorescent spots that disappeared or emerged after the crush should appear as black or white spots in the composite images. The image overlay system was used in the rats in group 1 (for control and 4 weeks after the crush) and group 2 (for 1 and 2 weeks after the crush). The composite SLO images were compared with the identical retinal area in the flatmounts. RGC counts in SLO images were determined by subtracting the number of newly emerged fluorescent cells from total cell counts. 

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 microglia ¹⁵ 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 × 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

Fluorescent RGCs stained with DiA were visible in vivo with an SLO with argon blue laser illumination and the optical filter sets for fluorescein angiography (Fig. 1) . Although the retinal area that can be viewed in a single SLO frame was limited, it was not difficult to obtain a retinal image of a desired area by aligning the direction of the rat eye manually. The entire image of an SLO frame could not be focused well simultaneously. However, at least 0.1 mm² of retinal area could be focused well simultaneously in an SLO frame. The retinal area used for cell counting in retinas of group 1 in the flatmount preparation was 0.30 ± 0.15 mm² (n = 12). The baseline labeled cell density (cell number/retinal area) of the selected retinal areas in both eyes of group 1 rats was 1967 ± 377 cells/mm² (n = 12). 

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 criteria ^{13 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) . 

To determine the relationship of microglia to the newly emerged DiA fluorescence after the optic nerve crush, we stained microglia in the retinal flatmounts with isolectin B4 after taking images of DiA fluorescence in SLO and retinal flatmounts. The counts of lectin-labeled microglial cell bodies were 28.3 ± 1.5, 71.3 ± 2.5, 115.3 ± 13.3, and 66.0 ± 10.4/mm² at baseline and 1, 2, and 4 weeks after optic nerve crush, respectively (Fig. 7) . In agreement with a previous report, ¹⁶ optic nerve crush significantly increased the number of microglia, which peaked 2 weeks after the crush. Furthermore, a point-by-point comparison of the overlaid composite images of SLO and lectin-stained retinal flatmounts showed that newly emerged fluorescent spots in SLO images colocalized mostly with microglial processes and occasionally in the vicinity of microglial cell bodies (Fig. 8) . The colocalization was observed 1, 2, and 4 weeks after optic nerve crush. 

Discussion

Imaging of living RGCs in vivo has definite advantages for counting RGCs over conventional microscopic observation in flatmount retina preparations. In the histologic assessment, the number of RGCs in the selected sampling areas (usually <10% of the entire retina) are averaged and compared between eyes of the same or different rats. Accordingly, the number of RGCs and their distribution in the retina are assumed to be the same in the right and left eyes and in different animals. Also, consistent labeling of RGCs is another essential premise that can never be proven exactly until the total number of RGCs is counted. However, Danias et al. ⁵ pointed out the inherent problem of RGC counting by a sampling method in retinal flatmounts. They reported considerable variability of RGC density in different eyes of Wistar rats by counting RGCs in the entire retina and estimated that nearly 20 animals are necessary for the detection of a 20% difference between control and experimental eyes when 25% of the retinal area is used for cell counting. However, in vivo imaging allows repeated counting of RGCs in the same retinal area without being affected by the variability of staining and density of RGCs in different retinas. Therefore, if the same sampling procedure were used to detect the difference between pre- and posttreatment status, in vivo imaging would need fewer animals than histologic methods. 

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. 

 Supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture, Japan.

 Submitted for publication June 7, 2005; revised October 27, 2005, and February 23, 2006; accepted May 15, 2006.

 Disclosure: T. Higashid e, None; I. Kawaguchi, None; S. Ohkubo, None; H. Takeda, None; K. Sugiyam a, 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.

 

Figure 2.

 

Representative photographs showing the changes of DiA-labeled cells in the same retinal area of control (A–E) and experimental (F–J) eyes. (A–D, F–I) SLO images; (E, J) flatmount images. The optic nerve of the right eye was crushed intraorbitally with a clip. The left eye served as the untreated control. SLO imaging of the same retinal area of control and experimental eyes was performed before (A, F) and at 1 (B, G), 2 (C, H), and 4 (D, I) weeks after axonal injury. Retinal flatmounts of control (E) and experimental (J) eyes were prepared after the SLO imaging at postcrush week 4, and the same retinal areas of SLO images were examined by fluorescence microscopy. Arrows: small spots of punctate fluorescence representing cell debris of dead RGCs. Scale bar, 100 μm.

Figure 5.

 

Comparison of SLO image overlay system and retinal flatmount in control (A–D) and postcrush (1, E–H; 2, I–L; and 4, M–P weeks) retinas. (A, E, I, M) baseline SLO images; (B, F, J, N) overlaid composite SLO images produced from images on both sides; (C, G, K, O) SLO images taken immediately before preparation of retinal flatmounts; (D, H, L, P) identical retinal flatmount area to SLO images of C, G, K, and O, respectively. Newly emerged fluorescent spots were not present in control retinas, but were observed in postcrush retinas (F, J, N, arrows). Identical cells to those indicated are marked by arrows in G, H, K, L, O, and P. Arrows in retinal flatmounts indicate cells with sizes and shapes different from those of typical RGCs. Scale bar, 100 μm.

Figure 1.

 

Representative SLO image of normal rat retina showing retinal ganglion cells (RGCs) retrogradely labeled with DiA. SLO imaging was performed with an argon blue laser (488 nm) and optical filter sets for fluorescein angiography. Labeled RGCs are visible as white spots.

Figure 3.

 

The DiA-labeled cell density after optic nerve crush determined by SLO. The relative density of labeled cells in selected retinal areas of an SLO image at each time point was determined in each eye, with labeled cell density of the SLO image taken at baseline being 100%, and the results were averaged. Data are presented as the mean ± SD (n = 6 each). *P < 0.01 (Wilcoxon signed rank test), **P < 0.01 (Wilcoxon rank sum test).

Figure 4.

 

Image overlay analysis of SLO images in control (A–F) and experimental (G–L) eyes. The SLO image recorded before optic nerve crush (A, G) had its brightness inverted (B, H) and was overlaid as a 50% opaque layer, onto the SLO image of the same retinal area taken before (C, I) and 1 (D, J), 2 (E, K), and 4 (F, L) weeks after axonal injury. Although the composite image of the two identical images became homogeneously gray when they were perfectly aligned (C, I, left half), a shadowing effect around the spots appeared when the spots were slightly misaligned (C, I, right half). Fluorescent spots that disappeared or emerged in the second image should appear as black or white spots in the composite image, respectively. In control retina, few black and white spots were observed, aside from the shadowing effect (D–F). Postcrush retina showed many spots, indicating disappearing RGCs (black) and emerging white non-RGC cells (J–L). The position of white spots changed over time, reflecting the movement of the cells. Arrowheads and arrows: examples of black and white spots, respectively.

Figure 6.

 

RGC counts by SLO and in retinal flatmounts. RGC counts in SLO images were determined by subtracting the number of newly emerged fluorescent cells from total cell counts. Morphologic criteria for discriminating non-RGC cells from RGCs were applied to RGC counting in retinal flatmounts. RGC counts by SLO are not significantly different from those in retinal flatmounts. Data are presented as the mean ± SD (n = 6 each).

Figure 7.

 

Lectin-stained microglia in retinal flatmounts. Representative photographs of retinal flatmounts at baseline (A), and 1 (B), 2 (C), and 4 (D) weeks after optic nerve crush. Lectin-stained cell bodies (arrows) and processes (arrowheads) of microglia increased after optic nerve injury, which peaked 2 weeks after the crush. Retinal vessels are also stained by lectin. Scale bar, 100 μm.

Figure 8.

 

Example of point-by-point comparison of newly emerged fluorescent spots in SLO images and lectin-stained microglia. (A) Newly emerged fluorescent spots were marked by arrows in the overlaid composite images of baseline (C) and 2 weeks after optic nerve crush (B). (B, C, arrows) Identical positions to those in (A). The same retinal positions highlighted by arrows in (B) were carefully marked on the retinal flatmount showing DiA fluorescence 2 weeks after the crush (E). Identical arrow positions in (E) can be located on the lectin-stained flatmount (D) by making a superimposed image (F) of (D) and (E). Blood vessels showing lectin binding facilitate the correct positioning of the two images. By comparing arrows in (A) and (D), newly emerged fluorescent spots in SLO images appear to colocalize mostly with microglial processes and occasionally within the vicinity of microglial cell bodies (*). Scale bar, 100 μm.