June 2002
Volume 43, Issue 6
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Glaucoma  |   June 2002
Intraocular Distribution of 70-kDa Dextran after Subconjunctival Injection in Mice
Author Affiliations
  • Tae Woo Kim
    From the Glaucoma Center, University of California San Diego, La Jolla, California.
  • James D. Lindsey
    From the Glaucoma Center, University of California San Diego, La Jolla, California.
  • Makoto Aihara
    From the Glaucoma Center, University of California San Diego, La Jolla, California.
  • Todd L. Anthony
    From the Glaucoma Center, University of California San Diego, La Jolla, California.
  • Robert N. Weinreb
    From the Glaucoma Center, University of California San Diego, La Jolla, California.
Investigative Ophthalmology & Visual Science June 2002, Vol.43, 1809-1816. doi:
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      Tae Woo Kim, James D. Lindsey, Makoto Aihara, Todd L. Anthony, Robert N. Weinreb; Intraocular Distribution of 70-kDa Dextran after Subconjunctival Injection in Mice. Invest. Ophthalmol. Vis. Sci. 2002;43(6):1809-1816.

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

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Abstract

purpose. To investigate the intraocular distribution kinetics of 70-kDa dextran after subconjunctival injection.

methods. The right eye of 15 mice received a single subconjunctival injection of a 1.5-μL solution of 0.25% 70-kDa tetramethylrhodamine-dextran (TMR-D). The distribution of fluorescent labeling in eye sections was examined by fluorescence microscopy at 0.25, 1, 4, 24, or 72 hours after the injection. The brightness and homogeneity of fluorescence in the sclera, choroid, and retina were scored near the injection site, on the side of the globe opposite the injection site, and adjacent to the optic nerve head. Fluorescence intensity within the sclera and choroid adjacent to the optic nerve was assessed quantitatively by imaging densitometry.

results. TMR-D readily diffused transsclerally and dispersed throughout a large portion of the sclera, uvea, and cornea. Shortly after the injection, homogenous fluorescence was observed in the sclera and choroid on the same meridian as that of the injection site. This fluorescence gradually decreased in intensity with distance from the injection site. At the opposite meridian, fluorescence in the choroid was more intense than in the adjacent sclera and could be traced up to the ciliary muscle. TMR-D was also observed in the retinal and optic nerve vessels. The intensity of scleral and choroidal fluorescence adjacent to the optic nerve reached a maxima at 1 hour, and then decreased slowly, with half-lives of approximately 16 and 100 hours, respectively. Visible fluorescence was maintained at least until 72 hours in the sclera, choroid, iris, and cornea. Specific fluorescent labeling was never found in the contralateral eyes.

conclusions. Macromolecular 70-kDa dextran can be readily delivered to the mouse retina and uveal tissues by subconjunctival injection through transscleral diffusion, local hematogenous spread, and possibly movement through the uveoscleral outflow pathway. Subconjunctival injection may be a useful approach for delivering macromolecules to the retina and uvea.

Recent studies in a variety of experimental models suggest that large-molecular-weight compounds (>10 kDa) may be useful for treating a variety of chorioretinal disorders. Several anti-angiogenic agents, such as vascular endothelial growth factor (VEGF) receptor chimeric proteins, 1 tissue inhibitor of metalloproteinase (TIMP)-1 (28 kDa), 2 TIMP-2 (24 kDa), 3 pigment epithelium-derived factor (50 kDa), 4 and anti-VEGF antibodies (150 kDa) 5 can inhibit ocular neovascularization. Growth factors, such as brain-derived neurotrophic factor (27 kDa) and basic fibroblast growth factor (17 kDa) can rescue retinal ganglion cells 6 and photoreceptors, 7 respectively. However, targeted delivery of drugs to the choroid, retina, and optic nerve remains challenging. 
Topical delivery is not viable, because of long diffusional path length, rapid precorneal elimination by solution drainage to the lacrimal drainage system, normal or induced lacrimation, and corneal epithelial impermeability to molecules larger than 5 kDa. 8 Although systemic administration can deliver drugs to the posterior eye, the large systemic doses necessary are often associated with side effects. Repeated long-term intravitreal injections, as would be required for the chorioretinal disorders, run the risk of significant local complications, such as retinal detachment, endophthalmitis, and vitreous hemorrhage. 9 10 11  
Recently, Ambati et al. 12 demonstrated that macromolecules can be delivered to the posterior segment of the rabbit eye by subconjunctival infusion. Significant levels of bioactive protein were maintained in the rabbit choroid and retina after subconjunctival delivery by osmotic pump. Although their study clearly demonstrated the in vivo transscleral permeability of macromolecules, it did not determine the tissue routes through which the infused macromolecules dispersed. 
The present study was designed to investigate the kinetics of transscleral penetration and intraocular distribution of macromolecules after subconjunctival administration. For this purpose, a single dose of 70-kDa tetramethylrhodamine-dextran (TMR-D) was injected subconjunctivally into mice, and the tissue distribution over the course of 3 days was determined by histologic analysis. 
Methods
Subconjunctival Injection
Sixteen mice were used in the study. After anesthesia was induced with intraperitoneal injection of ketamine hydrochloride (100 mg/kg) and xylazine hydrochloride (9 mg/kg), the right eye of 15 mice received a subconjunctival injection at the superotemporal quadrant (Hamilton syringe; Hamilton Co., Reno, NV), containing a 1.5-μL solution of 70-kDa lysine-fixable TMR-D (D-1818; Molecular Probes, Eugene, OR). This compound is covalently linked to tissue during aldehyde fixation. TMR-D was dissolved with 0.1 M phosphate-buffered saline (PBS) at a concentration of 2.5 mg/mL. One mouse was used as a baseline control without subconjunctival injection. All animal protocols in this experiment conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Histologic Procedures and Fluorescence Microscopy
At 0.25, 1, 4, 24, or 72 hours after subconjunctival injection, animals were killed by CO2 inhalation and perfused transcardially through the left ventricle with 0.1 M PBS (pH 7.4) followed by 2% formaldehyde and 1.25% glutaraldehyde in 0.1 M PBS (pH 7.4). Twenty minutes after perfusion, the superotemporal cornea was marked with a permanent marking pen to maintain geographic orientation, and the eyes were enucleated and immersed in the same fixative at 4°C for 4 hours. The eyes were then placed in plastic molds that were half filled with tissue-freezing medium (TFM, Triangle Biomedical Sciences, Durham, NC) and were covered with more TFM. The molds were snap frozen by immersion in a 2-methylbutane and dry ice mixture, and the frozen tissue was sectioned axially at 12 μm on a cryostat. The sections were sequentially mounted onto positively charged slides (Positive-charged Microscope Slides; BioGenex, San Ramon, CA) and dried overnight. The slides were immersed in a solution of sodium borohydride (NaBH4, 1% in PBS; Sigma Chemical Co., St. Louis, MO) for 15 minutes to reduce the background fluorescence. 13 Immersion in NaBH4 was performed in reduced light on a shaker table under a fume hood and followed by several rinses in PBS. Coverslips were applied with a nonfluorescent mounting medium (Fluoromount-G; Southern Biotechnology Associates, Birmingham, AL). The slides were examined with fluorescence microscopy and photographed using a cooled digital camera (SPOT Digital Camera System; Diagnostic Instruments, Sterling Heights, MI). Fluorescence labeling at various intraocular tissues was evaluated for the brightness of the fluorescence by a subjective grading scale as follows: absent, 0; very dim, 1; moderately dim, 2; moderately bright, 3; bright, 4; and highly bright, 5. Fluorescence was also assessed for the homogeneity of the labeling as follows: sparsely punctate, a; densely punctate, b; moderately homogenous, c; evenly homogenous, d. For the grading of scleral and choroidal labeling, the equators of both proximal (on the same side as the injection site) and distal (on the side opposite the injection site) meridian and posterior polar area adjacent to the optic nerve in the proximal meridian were examined. For the grading of fluorescence in the cornea, only the central cornea was examined. The grading of ciliary body and iris was performed in both the proximal and distal meridians (Fig 1) . At each time point, three mice were studied. Eye sections of one mouse that had not received subconjunctival injection were examined as a baseline control. 
Fluorescence Intensity Measurement in the Sclera
To assess the fluorescent labeling quantitatively, fluorescence intensity was measured at the sclera and choroid on the digitally obtained image with ×400 magnification, with an image analyzer (Image-Pro, ver. 3.0.1; Media Cybernetics, Silver Spring, MD). The intensity was measured at the SC2-CH2 region (Fig. 1) . One measurement with a 20 × 60-pixel window was performed at the sclera. The intensity of choroidal fluorescence was measured at three stromal columns around the bright spot with a 16 × 16-pixel window (the smallest window available), and the mean value was calculated. 
Results
Subconjunctival Injection
A noticeable subconjunctival bleb was produced by the injection of 1.5 μL of fluid. Minor leakage from the injection site was observed immediately after the injection. The eyes of animals that had received subconjunctival injection of TMR-D did not show any sign of inflammation (hyperemia or loss of media clarity) for up to 72 hours. Therefore, it is unlikely that significant inflammation was induced by the injection. 
Low-Magnification Analysis
The distribution of fluorescence in ocular tissues was investigated at 0.25, 4, 24, and 72 hours after subconjunctival injection of TMR-D. In low-magnification views of whole globe sections at each time point, it was observed that TMR-D diffused rapidly and distributed over large areas of both intra- and extraocular tissues (Fig. 2) . By 15 minutes, the TMR-D had spread widely into many structures on the same side of the eye as the injection site (proximal meridian). By 4 hours, fluorescence was readily observed within large areas of tissues in both the proximal and distal meridians. Over the next 68 hours, the intensity of the fluorescence gradually declined, although labeled structures were visible in both the proximal and distal meridians at 72 hours after the TMR-D injection. 
High-Magnification Analysis
The identification of fluorescence label within specific ocular structures was more clearly discerned in examination at high magnification. Both the brightness and homogeneity of the fluorescence within most intraocular structures were scored in three animals at each time point and appear in Table 1 . Only the fluorescence brightness within the retinal and optic nerve blood vessels was scored, because it was difficult to judge homogeneity of fluorescence in these small structures. 
Fifteen Minutes.
Homogenous fluorescence was observed along the sclera, the choroid, and cornea in the meridian proximal to the injection site (Fig. 3A) . In the choroidal stroma, the fluorescence in the middle of the stromal column was more intense than at the edges adjacent to vascular components. Fluorescence was also observed in the distal meridian (Fig. 3C) . Unlike the proximal meridian, the fluorescence in the distal choroid and ciliary body was more intense than that in the adjacent sclera, where the fluorescence was barely visible. Moderately bright choroidal fluorescence was traced up to the ciliary muscles along the choroid and suprachoroidal space. No regional difference in fluorescence intensity at the stromal column was observed in the distal meridian. The ciliary body contained strong fluorescence around the blood vessels remote from the sclera, both in the proximal and distal meridians. 
One Hour.
Compared with 15 minutes, the fluorescence extended more to the distal meridian in the sclera and choroid (Table 1) . The iris was labeled, as was the ciliary body (Fig. 4A) . Fluorescence was also visible in the retinal vessels (Fig. 4C) . In the optic nerve, weak fluorescence was noted in the blood vessels (Fig. 4E) . At the parenchyma near the choroid, fluorescence was dimly visible, but it was difficult to differentiate from the bright choroidal labeling. 
Four Hours.
Moderately bright punctate fluorescence (arrows) was present in the sclera (arrows) and choroid (arrowheads) in the proximal meridian (Fig 5A) , as well as in the distal meridian (Fig 5C) . In contrast, diffuse fluorescence was present in the proximal meridian only. Dim fluorescence was consistently visible in the retinal blood vessels. In the optic nerve blood vessels, the fluorescence was visible in only one eye (Table 1)
Twenty-Four Hours.
Punctate fluorescence was present in the iris, ciliary body, choroid, and sclera at similar intensity in both the proximal and distal meridians (Table 1) . The fluorescence in the iris and along the blood vessels in the ciliary body is shown in Fig 6A . The punctate fluorescence observed in the sclera, choroid and retinal vessels is shown in Fig 6C . No fluorescence was observed in the optic nerve vessels (Table 1)
Seventy-Two Hours.
Punctate fluorescence was visible in the sclera, choroid, and iris (Table 1 , Fig 7A ). In contrast, no fluorescence was visible in the ciliary body (Fig. 7C) . Bright punctate fluorescence was observed over homogenous fluorescence in the corneal stroma, and no fluorescence was observed in the corneal epithelium (Fig. 7E) . The retinal vessels showed no fluorescence. 
Control Eyes
Very dim fluorescence from the erythrocytes was noted in the contralateral eye (Fig. 8A) and was also observed in the eye of a nontreated control mouse (Fig. 8C) . No other fluorescence was observed in the control sections. 
Fluorescence Intensity at the Sclera and Choroid: Time Profiles
The intensity of scleral and choroidal fluorescence was measured using digital imaging software and the result was expressed as a percentage of the brightest intensity measurement in the sclera near the injection site at 15 minutes. Brightness in the sclera and choroid reached maxima at 1 hour, then decreased slowly, with half-lives of approximately 16 hours and 100 hours, respectively (Fig. 9)
Discussion
Transscleral delivery has been considered to be a viable alternative for the intraocular delivery of macromolecules, because sclera has a large and accessible surface area, a high degree of hydration rendering it conducive to water-soluble substances, and a hypocellularity with an attendant paucity of proteolytic enzymes and protein-binding sites. 14 15 16 17 Recently, several in vitro experiments have demonstrated that the sclera is permeable to large molecules—-an up to 150-kDa antibody. 16 18 19 The present study showed that TMR-D readily penetrates the sclera and disperses through many intraocular tissues after a single subconjunctival injection. Therefore, it appears that transscleral influx and intrachoroidal dispersion of macromolecules in the mouse eye is not precluded by either transscleral aqueous flow or by choroidal blood flow. 
Histochemical counterstaining would help to identify various tissue types within the sections. However, it also would be likely to obscure the results. Because standard histochemical stains are either weakly or strongly fluorescent, counterstaining might introduce confusing fluorescent signals into the section that would be difficult to distinguish from TMR-D fluorescence. Because the fluorescent signals in the present study often were weak, it was important to avoid the generation of potentially confusing fluorescent signals. Thus, the present study was confined to examination of fluorescence and corresponding bright-field images of noncounterstained sections. Another important consideration is whether there was any separation of the fluorescent tag from the dextran in tissues or biological fluids. Several studies of fluorescent dextrans indicate that such separation is virtually nonexistent, 20 21 although specific experiments to investigate this were not conducted in the present study. 
Within 1 hour, the choroid showed homogenous labeling, with the most intense label in the middle of the stromal column. This finding suggests that the direct diffusion from the sclera is the major route of delivery to the choroid. In contrast, choroidal fluorescence was more intense in the distal meridian than in the adjacent sclera, suggesting that TMR-D had not diffused from the sclera. Further, the fluorescence was tracable up to the ciliary muscle along the choroid or suprachoroidal space, which suggests that TMR-D was dispersed through the uveoscleral outflow. 22 Because the corneal endothelium does not constitute a significant barrier for water-soluble molecules, 23 it is possible that the macromolecules enter the anterior chamber after diffusion to the corneal stroma from the sclera and drain to the choroid after uveoscleral outflow. Alternatively, the TMR-D in the proximal choroid may diffuse to the distal meridian choroid through the choroidal stroma at a faster rate than through the sclera. It is likely that the punctate staining corresponds most often to small blood vessels. Clearly identified larger blood vessels that contained fluorescence were often observed in the retina, optic nerve, and ciliary body. At times, it also may correspond to macrophages that have phagocytosed some of the labeled dextran, although this has not been shown conclusively. 
The fluorescence in the retinal and optic nerve vessels indicated that the hematogenous pathway also contributed to the intraocular dispersion of TMR-D. Perhaps, TMR-D penetrated the arterial wall in the retrobulbar region and mixed with the blood stream as the blood was about to enter the retina or optic nerve. The high concentration of TMR-D adjacent to the injection site could have been enough to deposit label in these blood vessels. Once the TMR-D entered the systemic circulation, massive dilution would occur. This could explain the absence of fluorescence observed in the contralateral eyes. The high-density fluorescence around the blood vessels at the ciliary body at 15 minutes after injection suggests that TMR-D in the ciliary body may also have derived in part from local ciliary body circulation. Another possibility is that the presence of fluorescence within vascular elements may represent a route of clearance for the injected dextran. 
One concern is whether subconjunctivally delivered macromolecules can reach biologically relevant concentrations in the choroid or other intraocular tissues. A recent study found that subconjunctival delivery of immunoglobulin (150 kDa) through an osmotic minipump achieved sufficient concentration in the choroid to exert a biological effect. 12 The present study extends these findings by demonstrating that the ciliary body and iris had fluorescence comparable to that in the choroid after subconjunctival administration of another macromolecule, fluorescent 70-kDa dextran (Table 1) . Moreover, small amounts of fluorescence were observed in portions of the optic nerve head. 
It is well known that dextrans even smaller than 70 kDa cannot penetrate the blood–retinal 24 or blood–brain barriers. 25 In contrast, neurotrophic factors are known to penetrate the blood–brain barrier. 26 27 Because both the inner blood–retinal barrier and blood–brain barriers are constituted by tight junctional complexes of the capillary endothelium, 28 it may be possible to assume that the neurotrophic factors can also penetrate the retinal capillaries. The current observation of fluorescence in the retinal vessels after subconjunctival injection suggests that the neurotrophic factors may be successfully delivered to retinal ganglion cells by a subconjunctival approach. 
In the present study, TMR-D dispersed to the iris and ciliary body after subconjunctival injection. This suggests that macromolecules can be used to treat iris abnormalities. For example, antiangiogenic molecules, such as anti-VEGF antibodies, TIMPs, and PEDF can be delivered to the ciliary body and iris through a subconjunctival approach in patients with retinal ischemia. Along with the interventions to stabilize the retina, adjunctive treatment with antiangiogenic agents in this manner may be useful for inhibiting the development of neovascular glaucoma in patients with ischemic retinal disorders. 
Several studies have demonstrated that horseradish peroxidase can diffuse from the peripapillary choroid into different parts of the optic nerve head region and also into the receptor layer of the sensory retina adjacent to Kuhnt’s intermediary tissue after intravenous administration. 29 30 31 In the present study, it seems that some TMR-D leaked to optic nerve parenchyma from the adjacent choroid, but it was difficult to differentiate this from the blurred fluorescence of the bright choroidal labeling. Further study is needed to elucidate whether 70-kDa dextran can penetrate the choroid to the adjacent optic nerve. 
Because most diseases in which the macromolecules may be beneficial are chronic, repeated long-term delivery is necessary. Some type of sustained-release delivery system would be useful to reduce the frequency of administration. A wide variety of sustained-release drug delivery systems are available, including subconjunctivally injectable modalities, such as microspheres, 32 liposomes, 33 34 35 36 37 and in situ–forming polymeric gels. 38 Biodegradable scleral implants 39 can also be useful, although placement necessitates a surgical procedure. The current observation of rapid intraocular distribution and slow elimination kinetics suggest that sustained-release delivery systems may result in an effective intraocular drug accumulation to maintain sufficient effect. Further, recent studies in our laboratory have demonstrated that scleral permeability to macromolecules can be increased by adjunctive prostaglandin exposure. 40 41 42 Concurrent administration of prostaglandin may enable more effective transscleral delivery of macromolecules. 
In conclusion, our findings show that macromolecular 70-kDa dextran can readily disperse to many intraocular tissues throughout the globe after subconjunctival injection. This may occur through a combination of transscleral diffusion, local hematogenous spread, and possibly movement through the uveoscleral outflow pathway. With the use of a suitable sustained-release delivery system, transscleral delivery with a subconjunctival approach may provide a successful modality for administering various macromolecules for the treatment of a variety of ocular diseases. 
 
Figure 1.
 
Schematic diagram showing locations where the fluorescent labeling was examined for grading. CB, ciliary body; CH, choroid; IR, iris; SC, sclera; ON, optic nerve.
Figure 1.
 
Schematic diagram showing locations where the fluorescent labeling was examined for grading. CB, ciliary body; CH, choroid; IR, iris; SC, sclera; ON, optic nerve.
Figure 2.
 
Distribution of the fluorescence in whole-globe sections at 15 minutes (A) and 4 hours (B), 24 hours (C), and 72 hours (D) after subconjunctival injection of TMR-D. Arrows: ciliary body; arrowheads: optic nerve head. Magnification, ×14.
Figure 2.
 
Distribution of the fluorescence in whole-globe sections at 15 minutes (A) and 4 hours (B), 24 hours (C), and 72 hours (D) after subconjunctival injection of TMR-D. Arrows: ciliary body; arrowheads: optic nerve head. Magnification, ×14.
Table 1.
 
Intraocular Distribution of Fluorescence after Subconjunctival Injection of TMR-D
Table 1.
 
Intraocular Distribution of Fluorescence after Subconjunctival Injection of TMR-D
Time (h) Distribution of Fluorescence
SC1 SC2 SC3 CH1 CH2 CH3 CB1 CB2 IR1 IR2 RV ONV CO
0.25 5d 3d4a 1 2d 2d3a 3c 3b 2b3a 0 2a 0 0 2c
0.25 5d 4c 1 2d 2d 3c 3b 3b 3b 3b 2 1 2c
0.25 5d 3d 1 2d3a 2c 3c 2d3b 2d3c 0 2d 2 1 2c
1 4c5d 3c4b 2d 4d 3b4b 3c 2d3a 2d3b 2d4a 3a 3 1 2c
1 3d4a 4c 2c 2c3b 3c4b 2c 2d3b 2d3c 2d3b 2d3a 3 1 2c
1 4d 4c5a 3a 3d4b 3d 3c 2d 2d 3b 3a 3 1 2c
4 3d4b 3d5b 2d3b 2d3b 2d3b 2b 3a 3a 3b 3b 3 1 3d4a
4 3d4b 3d5b 2d3b 2d3b 2d3b 2b 3b 2c 3b 3b 3 0 2d3a
4 3d4b 2d4b 3b 2d3b 2d3b 2b 2d3b 2a 2d3b 3b 3 0 3d4a
24 2d3b 3b4b 3a 3b 3b 2a 2b 2b 3b 3b 3 0 2d3b
24 3d4b 2d4a 2d3b 3b 2d3b 2a 2a 2c3b 3b 3b 3 0 3d4a
24 3b 3b 3b 3b 3b 2b 2c 2c3b 3b 3b 3 0 3d4a
72 3b 3b 3a 3b 3b 2a 2c 0 3b 3b 0 0 2d3b
72 2d3b 3b 3a 3b 3b 3a 2a 3a 3b 3b 2 0 3d4b
72 3b 3b 3a 3b 3b 2a 2a 0 3b 3b 0 0 2d3b
Figure 3.
 
Fluorescence (A, C) and light (B, D) microscopic images at 15 minutes after subconjunctival injection of TMR-D. (A) Homogenous fluorescent staining was observed along the sclera, the choroid, and cornea in the meridian proximal to the injection site. Note that the fluorescence in the middle of the choroidal stromal column was more intense than in the vascular side (top inset, arrows). Moderately bright fluorescence was observed along the blood vessels in the ciliary body. Note that the fluorescence was more intense around the ciliary vessels remote from the sclera (bottom inset, arrowheads). (C) In the distal meridian, moderately bright fluorescence was observed along the choroid and around the blood vessels in the ciliary body (inset, arrows). The fluorescence in the ciliary body was more intense around the vessels remote from the sclera. The choroidal fluorescence could be easily traced up to the ciliary muscle (inset, arrowheads). CB, ciliary body; CJ, conjunctiva; CO, cornea; IR, iris; LE, lens; RE, retina; SC, sclera; ( Image not available ), Schlemm canal. Magnification: (AD) ×38; (A, upper inset) ×65; (A, lower inset) ×170; (C, inset) ×82; (D, inset) ×82.
Figure 3.
 
Fluorescence (A, C) and light (B, D) microscopic images at 15 minutes after subconjunctival injection of TMR-D. (A) Homogenous fluorescent staining was observed along the sclera, the choroid, and cornea in the meridian proximal to the injection site. Note that the fluorescence in the middle of the choroidal stromal column was more intense than in the vascular side (top inset, arrows). Moderately bright fluorescence was observed along the blood vessels in the ciliary body. Note that the fluorescence was more intense around the ciliary vessels remote from the sclera (bottom inset, arrowheads). (C) In the distal meridian, moderately bright fluorescence was observed along the choroid and around the blood vessels in the ciliary body (inset, arrows). The fluorescence in the ciliary body was more intense around the vessels remote from the sclera. The choroidal fluorescence could be easily traced up to the ciliary muscle (inset, arrowheads). CB, ciliary body; CJ, conjunctiva; CO, cornea; IR, iris; LE, lens; RE, retina; SC, sclera; ( Image not available ), Schlemm canal. Magnification: (AD) ×38; (A, upper inset) ×65; (A, lower inset) ×170; (C, inset) ×82; (D, inset) ×82.
Figure 4.
 
Fluorescence (A, C, E) and light (B, D, F) microscopic images at 1 hour after subconjunctival injection of TMR-D. (A, B) Diffuse and bright punctate labeling was observed in the iris and the ciliary body of the proximal meridian. (C, D) Fluorescence also was found in the retinal vessels (C, arrows). (E, F) In the optic nerve, weak fluorescence was found in the blood vessels (E, small arrows). At the parenchyma near the choroid, dimly visible fluorescence was noted (E, large arrows), but it was difficult to differentiate from the blurred fluorescence of the highly bright choroidal labeling. Brighter fluorescence was observed along the central retinal vessel (E, arrowheads). CB, ciliary body; CH, choroid; CO, cornea; IR, iris; ON, optic nerve; RE, retina; SC, sclera. Magnification, ×150.
Figure 4.
 
Fluorescence (A, C, E) and light (B, D, F) microscopic images at 1 hour after subconjunctival injection of TMR-D. (A, B) Diffuse and bright punctate labeling was observed in the iris and the ciliary body of the proximal meridian. (C, D) Fluorescence also was found in the retinal vessels (C, arrows). (E, F) In the optic nerve, weak fluorescence was found in the blood vessels (E, small arrows). At the parenchyma near the choroid, dimly visible fluorescence was noted (E, large arrows), but it was difficult to differentiate from the blurred fluorescence of the highly bright choroidal labeling. Brighter fluorescence was observed along the central retinal vessel (E, arrowheads). CB, ciliary body; CH, choroid; CO, cornea; IR, iris; ON, optic nerve; RE, retina; SC, sclera. Magnification, ×150.
Figure 5.
 
Fluorescence (A, C) and light (B, D) microscopic images 4 hours after subconjunctival injection of TMR-D. Moderately bright punctate fluorescence was present in the sclera (arrows) and choroid (arrowheads) in the proximal meridian (A, B) as well as, in the distal meridian (C, D). In contrast, diffuse fluorescence was present in the proximal meridian only. CB, ciliary body; IR, iris; RE, retina; SC, sclera. Magnification: (AD) ×38; (A, inset) ×280; (C, D, inset) ×76.
Figure 5.
 
Fluorescence (A, C) and light (B, D) microscopic images 4 hours after subconjunctival injection of TMR-D. Moderately bright punctate fluorescence was present in the sclera (arrows) and choroid (arrowheads) in the proximal meridian (A, B) as well as, in the distal meridian (C, D). In contrast, diffuse fluorescence was present in the proximal meridian only. CB, ciliary body; IR, iris; RE, retina; SC, sclera. Magnification: (AD) ×38; (A, inset) ×280; (C, D, inset) ×76.
Figure 6.
 
Fluorescence (A, C) and light (B, D) microscopic images at 24 hours after subconjunctival injection of TMR-D. (A, B) The fluorescence was visible at the iris (A, arrowheads) and along the blood vessel wall at the ciliary body (A, arrows). (C, D) At the posterior pole, fluorescence was observed in the sclera (C, small arrows), choroid (C, arrowheads), and retinal vessels (C, large arrows). Magnification, ×150. CB, ciliary body; CO, cornea; IR, iris; ON, optic nerve; RE, retina.
Figure 6.
 
Fluorescence (A, C) and light (B, D) microscopic images at 24 hours after subconjunctival injection of TMR-D. (A, B) The fluorescence was visible at the iris (A, arrowheads) and along the blood vessel wall at the ciliary body (A, arrows). (C, D) At the posterior pole, fluorescence was observed in the sclera (C, small arrows), choroid (C, arrowheads), and retinal vessels (C, large arrows). Magnification, ×150. CB, ciliary body; CO, cornea; IR, iris; ON, optic nerve; RE, retina.
Figure 7.
 
Fluorescence (A, C, E) and light (B, D, F) microscopic images at 72 hours after subconjunctival injection of TMR-D. (A, B) Punctate fluorescence was still visible at the sclera (A, arrows) and choroid (A, arrowheads). (C, D) Punctate fluorescence also was observed in the iris (C, arrowheads). Fluorescence was not visible in the ciliary body. (E, F) Punctate fluorescence was observed, with homogenous fluorescence in the corneal stroma. No fluorescence was observed in the epithelium. Magnification, ×150. CB, ciliary body; CH, choroid; CO, cornea; CS, corneal stroma; EP, corneal epithelium; IR, iris; RE, retina; SC, sclera.
Figure 7.
 
Fluorescence (A, C, E) and light (B, D, F) microscopic images at 72 hours after subconjunctival injection of TMR-D. (A, B) Punctate fluorescence was still visible at the sclera (A, arrows) and choroid (A, arrowheads). (C, D) Punctate fluorescence also was observed in the iris (C, arrowheads). Fluorescence was not visible in the ciliary body. (E, F) Punctate fluorescence was observed, with homogenous fluorescence in the corneal stroma. No fluorescence was observed in the epithelium. Magnification, ×150. CB, ciliary body; CH, choroid; CO, cornea; CS, corneal stroma; EP, corneal epithelium; IR, iris; RE, retina; SC, sclera.
Figure 8.
 
Fluorescence (A, C) and light (B, D) microscopic images of the contralateral eye at 1 hour (A, B) after subconjunctival injection of TMR-D and the eye of a nontreated control mouse (C, D). Specific fluorescence was never observed, other than dimly visible fluorescence in the erythrocytes (arrows). Magnification, ×150.
Figure 8.
 
Fluorescence (A, C) and light (B, D) microscopic images of the contralateral eye at 1 hour (A, B) after subconjunctival injection of TMR-D and the eye of a nontreated control mouse (C, D). Specific fluorescence was never observed, other than dimly visible fluorescence in the erythrocytes (arrows). Magnification, ×150.
Figure 9.
 
Fluorescence intensity at the posterior polar sclera and choroid. Intensity is expressed as a percentage of the brightest intensity measurement in the sclera near the injection site at 0.25 hours after the TMR-D injection.
Figure 9.
 
Fluorescence intensity at the posterior polar sclera and choroid. Intensity is expressed as a percentage of the brightest intensity measurement in the sclera near the injection site at 0.25 hours after the TMR-D injection.
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Figure 1.
 
Schematic diagram showing locations where the fluorescent labeling was examined for grading. CB, ciliary body; CH, choroid; IR, iris; SC, sclera; ON, optic nerve.
Figure 1.
 
Schematic diagram showing locations where the fluorescent labeling was examined for grading. CB, ciliary body; CH, choroid; IR, iris; SC, sclera; ON, optic nerve.
Figure 2.
 
Distribution of the fluorescence in whole-globe sections at 15 minutes (A) and 4 hours (B), 24 hours (C), and 72 hours (D) after subconjunctival injection of TMR-D. Arrows: ciliary body; arrowheads: optic nerve head. Magnification, ×14.
Figure 2.
 
Distribution of the fluorescence in whole-globe sections at 15 minutes (A) and 4 hours (B), 24 hours (C), and 72 hours (D) after subconjunctival injection of TMR-D. Arrows: ciliary body; arrowheads: optic nerve head. Magnification, ×14.
Figure 3.
 
Fluorescence (A, C) and light (B, D) microscopic images at 15 minutes after subconjunctival injection of TMR-D. (A) Homogenous fluorescent staining was observed along the sclera, the choroid, and cornea in the meridian proximal to the injection site. Note that the fluorescence in the middle of the choroidal stromal column was more intense than in the vascular side (top inset, arrows). Moderately bright fluorescence was observed along the blood vessels in the ciliary body. Note that the fluorescence was more intense around the ciliary vessels remote from the sclera (bottom inset, arrowheads). (C) In the distal meridian, moderately bright fluorescence was observed along the choroid and around the blood vessels in the ciliary body (inset, arrows). The fluorescence in the ciliary body was more intense around the vessels remote from the sclera. The choroidal fluorescence could be easily traced up to the ciliary muscle (inset, arrowheads). CB, ciliary body; CJ, conjunctiva; CO, cornea; IR, iris; LE, lens; RE, retina; SC, sclera; ( Image not available ), Schlemm canal. Magnification: (AD) ×38; (A, upper inset) ×65; (A, lower inset) ×170; (C, inset) ×82; (D, inset) ×82.
Figure 3.
 
Fluorescence (A, C) and light (B, D) microscopic images at 15 minutes after subconjunctival injection of TMR-D. (A) Homogenous fluorescent staining was observed along the sclera, the choroid, and cornea in the meridian proximal to the injection site. Note that the fluorescence in the middle of the choroidal stromal column was more intense than in the vascular side (top inset, arrows). Moderately bright fluorescence was observed along the blood vessels in the ciliary body. Note that the fluorescence was more intense around the ciliary vessels remote from the sclera (bottom inset, arrowheads). (C) In the distal meridian, moderately bright fluorescence was observed along the choroid and around the blood vessels in the ciliary body (inset, arrows). The fluorescence in the ciliary body was more intense around the vessels remote from the sclera. The choroidal fluorescence could be easily traced up to the ciliary muscle (inset, arrowheads). CB, ciliary body; CJ, conjunctiva; CO, cornea; IR, iris; LE, lens; RE, retina; SC, sclera; ( Image not available ), Schlemm canal. Magnification: (AD) ×38; (A, upper inset) ×65; (A, lower inset) ×170; (C, inset) ×82; (D, inset) ×82.
Figure 4.
 
Fluorescence (A, C, E) and light (B, D, F) microscopic images at 1 hour after subconjunctival injection of TMR-D. (A, B) Diffuse and bright punctate labeling was observed in the iris and the ciliary body of the proximal meridian. (C, D) Fluorescence also was found in the retinal vessels (C, arrows). (E, F) In the optic nerve, weak fluorescence was found in the blood vessels (E, small arrows). At the parenchyma near the choroid, dimly visible fluorescence was noted (E, large arrows), but it was difficult to differentiate from the blurred fluorescence of the highly bright choroidal labeling. Brighter fluorescence was observed along the central retinal vessel (E, arrowheads). CB, ciliary body; CH, choroid; CO, cornea; IR, iris; ON, optic nerve; RE, retina; SC, sclera. Magnification, ×150.
Figure 4.
 
Fluorescence (A, C, E) and light (B, D, F) microscopic images at 1 hour after subconjunctival injection of TMR-D. (A, B) Diffuse and bright punctate labeling was observed in the iris and the ciliary body of the proximal meridian. (C, D) Fluorescence also was found in the retinal vessels (C, arrows). (E, F) In the optic nerve, weak fluorescence was found in the blood vessels (E, small arrows). At the parenchyma near the choroid, dimly visible fluorescence was noted (E, large arrows), but it was difficult to differentiate from the blurred fluorescence of the highly bright choroidal labeling. Brighter fluorescence was observed along the central retinal vessel (E, arrowheads). CB, ciliary body; CH, choroid; CO, cornea; IR, iris; ON, optic nerve; RE, retina; SC, sclera. Magnification, ×150.
Figure 5.
 
Fluorescence (A, C) and light (B, D) microscopic images 4 hours after subconjunctival injection of TMR-D. Moderately bright punctate fluorescence was present in the sclera (arrows) and choroid (arrowheads) in the proximal meridian (A, B) as well as, in the distal meridian (C, D). In contrast, diffuse fluorescence was present in the proximal meridian only. CB, ciliary body; IR, iris; RE, retina; SC, sclera. Magnification: (AD) ×38; (A, inset) ×280; (C, D, inset) ×76.
Figure 5.
 
Fluorescence (A, C) and light (B, D) microscopic images 4 hours after subconjunctival injection of TMR-D. Moderately bright punctate fluorescence was present in the sclera (arrows) and choroid (arrowheads) in the proximal meridian (A, B) as well as, in the distal meridian (C, D). In contrast, diffuse fluorescence was present in the proximal meridian only. CB, ciliary body; IR, iris; RE, retina; SC, sclera. Magnification: (AD) ×38; (A, inset) ×280; (C, D, inset) ×76.
Figure 6.
 
Fluorescence (A, C) and light (B, D) microscopic images at 24 hours after subconjunctival injection of TMR-D. (A, B) The fluorescence was visible at the iris (A, arrowheads) and along the blood vessel wall at the ciliary body (A, arrows). (C, D) At the posterior pole, fluorescence was observed in the sclera (C, small arrows), choroid (C, arrowheads), and retinal vessels (C, large arrows). Magnification, ×150. CB, ciliary body; CO, cornea; IR, iris; ON, optic nerve; RE, retina.
Figure 6.
 
Fluorescence (A, C) and light (B, D) microscopic images at 24 hours after subconjunctival injection of TMR-D. (A, B) The fluorescence was visible at the iris (A, arrowheads) and along the blood vessel wall at the ciliary body (A, arrows). (C, D) At the posterior pole, fluorescence was observed in the sclera (C, small arrows), choroid (C, arrowheads), and retinal vessels (C, large arrows). Magnification, ×150. CB, ciliary body; CO, cornea; IR, iris; ON, optic nerve; RE, retina.
Figure 7.
 
Fluorescence (A, C, E) and light (B, D, F) microscopic images at 72 hours after subconjunctival injection of TMR-D. (A, B) Punctate fluorescence was still visible at the sclera (A, arrows) and choroid (A, arrowheads). (C, D) Punctate fluorescence also was observed in the iris (C, arrowheads). Fluorescence was not visible in the ciliary body. (E, F) Punctate fluorescence was observed, with homogenous fluorescence in the corneal stroma. No fluorescence was observed in the epithelium. Magnification, ×150. CB, ciliary body; CH, choroid; CO, cornea; CS, corneal stroma; EP, corneal epithelium; IR, iris; RE, retina; SC, sclera.
Figure 7.
 
Fluorescence (A, C, E) and light (B, D, F) microscopic images at 72 hours after subconjunctival injection of TMR-D. (A, B) Punctate fluorescence was still visible at the sclera (A, arrows) and choroid (A, arrowheads). (C, D) Punctate fluorescence also was observed in the iris (C, arrowheads). Fluorescence was not visible in the ciliary body. (E, F) Punctate fluorescence was observed, with homogenous fluorescence in the corneal stroma. No fluorescence was observed in the epithelium. Magnification, ×150. CB, ciliary body; CH, choroid; CO, cornea; CS, corneal stroma; EP, corneal epithelium; IR, iris; RE, retina; SC, sclera.
Figure 8.
 
Fluorescence (A, C) and light (B, D) microscopic images of the contralateral eye at 1 hour (A, B) after subconjunctival injection of TMR-D and the eye of a nontreated control mouse (C, D). Specific fluorescence was never observed, other than dimly visible fluorescence in the erythrocytes (arrows). Magnification, ×150.
Figure 8.
 
Fluorescence (A, C) and light (B, D) microscopic images of the contralateral eye at 1 hour (A, B) after subconjunctival injection of TMR-D and the eye of a nontreated control mouse (C, D). Specific fluorescence was never observed, other than dimly visible fluorescence in the erythrocytes (arrows). Magnification, ×150.
Figure 9.
 
Fluorescence intensity at the posterior polar sclera and choroid. Intensity is expressed as a percentage of the brightest intensity measurement in the sclera near the injection site at 0.25 hours after the TMR-D injection.
Figure 9.
 
Fluorescence intensity at the posterior polar sclera and choroid. Intensity is expressed as a percentage of the brightest intensity measurement in the sclera near the injection site at 0.25 hours after the TMR-D injection.
Table 1.
 
Intraocular Distribution of Fluorescence after Subconjunctival Injection of TMR-D
Table 1.
 
Intraocular Distribution of Fluorescence after Subconjunctival Injection of TMR-D
Time (h) Distribution of Fluorescence
SC1 SC2 SC3 CH1 CH2 CH3 CB1 CB2 IR1 IR2 RV ONV CO
0.25 5d 3d4a 1 2d 2d3a 3c 3b 2b3a 0 2a 0 0 2c
0.25 5d 4c 1 2d 2d 3c 3b 3b 3b 3b 2 1 2c
0.25 5d 3d 1 2d3a 2c 3c 2d3b 2d3c 0 2d 2 1 2c
1 4c5d 3c4b 2d 4d 3b4b 3c 2d3a 2d3b 2d4a 3a 3 1 2c
1 3d4a 4c 2c 2c3b 3c4b 2c 2d3b 2d3c 2d3b 2d3a 3 1 2c
1 4d 4c5a 3a 3d4b 3d 3c 2d 2d 3b 3a 3 1 2c
4 3d4b 3d5b 2d3b 2d3b 2d3b 2b 3a 3a 3b 3b 3 1 3d4a
4 3d4b 3d5b 2d3b 2d3b 2d3b 2b 3b 2c 3b 3b 3 0 2d3a
4 3d4b 2d4b 3b 2d3b 2d3b 2b 2d3b 2a 2d3b 3b 3 0 3d4a
24 2d3b 3b4b 3a 3b 3b 2a 2b 2b 3b 3b 3 0 2d3b
24 3d4b 2d4a 2d3b 3b 2d3b 2a 2a 2c3b 3b 3b 3 0 3d4a
24 3b 3b 3b 3b 3b 2b 2c 2c3b 3b 3b 3 0 3d4a
72 3b 3b 3a 3b 3b 2a 2c 0 3b 3b 0 0 2d3b
72 2d3b 3b 3a 3b 3b 3a 2a 3a 3b 3b 2 0 3d4b
72 3b 3b 3a 3b 3b 2a 2a 0 3b 3b 0 0 2d3b
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