August 2008
Volume 49, Issue 8
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Anatomy and Pathology/Oncology  |   August 2008
Change in Embryonic Eye Size and Retinal Cell Proliferation following Intravitreal Injection of Glycosaminoglycans
Author Affiliations
  • Willi Halfter
    From the Department of Neurobiology, University of Pittsburgh, Pittsburgh, Pennsylvania.
Investigative Ophthalmology & Visual Science August 2008, Vol.49, 3289-3298. doi:10.1167/iovs.07-1421
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      Willi Halfter; Change in Embryonic Eye Size and Retinal Cell Proliferation following Intravitreal Injection of Glycosaminoglycans. Invest. Ophthalmol. Vis. Sci. 2008;49(8):3289-3298. doi: 10.1167/iovs.07-1421.

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

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Abstract

purpose. The vitreous body (VB) is a transparent, extracellular matrix structure that fills the vitreous cavity of the eye. Major constituents of the VB are hyaluronic acid (HA) and glycosaminoglycans (GAGs), both of which are highly charged, linear carbohydrate polymers. The present experiments investigate a possible role of HA and GAGs in regulating eye size during development and investigate whether changes in eye size are synchronized with cell proliferation in the retina.

methods. Chondroitin sulfate (CS), heparan sulfate (HS), heparin (Hep), dextran sulfate (DexS), and HA were injected into the vitreous cavity of embryonic day 5 chick embryos. Eye size was assessed 1 to 5 days after the injections. Cell counts and BrdU labeling established whether changes in eye size were paralleled by an increase or a decrease in retinal cell proliferation.

results. Injection of CS and HS led to an increase in eye size that was accompanied by a similar increase in retinal cell numbers. Hep and DexS injections led to a decrease in eye size that had no impact on cell proliferation in the retina. HA application had no effect on eye size.

conclusions. The changes in eye size after intravitreal application of GAGs demonstrate that the composition of the VB can play an important role in the regulation of eye size during embryogenesis. The fact that retinal cell proliferation is elevated after an increase in eye size indicates a regulatory role of eye size for retinal cell numbers.

The vitreous is a gelatinous extracellular matrix structure of the eye. Although tiny in mice, it accounts for up to 50% of total eye weight in chick and humans. More than 90% of the vitreous is water, which is retained by several negatively charged polymers in the vitreous body (VB). 1 2 3 One of these polymers is hyaluronic acid (HA), 1 2 3 a linear carbohydrate composed of repeating disaccharide units. HA is synthesized by a single-cell surface enzyme, hyaluronan synthase. The glycosaminoglycan (GAG) side chains of proteoglycans, also highly charged carbohydrate polymers, represent the other water-binding VB constituents in VB. 4 5 In contrast to HA, GAGs are covalently bound to a core protein, and they are assembled in the Golgi apparatus by glycosyltransferases that add monosaccharide and disaccharide units to the hydroxyl groups of serines within specific recognition sites of the core proteins. The most prominent proteoglycans of the VB extracellular matrix (ECM) are 2 chondroitin sulfate proteoglycans (CSPG), collagen IX and versican. 6 7 8 9 Collagen IX and versican are also found in cartilage or connective tissue, where they are responsible for tissue swelling by the retention of large quantities of water. 10 11 12 Three heparan sulfate proteoglycans have been detected in embryonic vitreous—agrin, perlecan, and collagen XVIII—all which are major constituents of ocular basement membranes. 13 14 15  
Abnormal eye development caused by mutations shows that proteoglycans and their GAG side chains are essential in ocular morphogenesis. For example, the deletion of the GAG-attachment segment of versican leads to autosomal dominant Wagner syndrome, a rare condition associated with congenital vitreoretinal degeneration caused by early-onset vitreal liquefaction. 16 Further, an autosomal recessive mutation of the proteoglycan collagen IX leads to moderate to high myopia, possibly because of VB liquefaction, 17 and the deletion of collagen XVIII in Knobloch syndrome leads to congenital high myopia with a high rate of retinal detachment. 18 Overexpression of agrin in mice leads to abnormal ocular morphogenesis, 19 and targeted deletion of the GAG attachment sites of perlecan cause lens capsule damage and microphthalmia. 20  
Regulation of eye size has been almost exclusively associated with changes in the choroid and the sclera, and the current hypothesis states that negative defocus and the expanding or shrinking scleral ECM are responsible for the regulation of eye size. 21 22 23 24 25 26 The fact that mutations responsible for congenital myopia syndromes were linked to constituents that are abundant in VB but are only minor components in sclera or choroid suggests that the ECM of the vitreous may also play a role in determining eye size, particularly during development. In Stickler syndrome, for example, mutations of collagen II and V/XI cause early liquefaction of the vitreous, often followed by retinal detachment, 27 and mutations of collagen XVIII in Knobloch syndrome results in early-onset vitreal and retinal detachments resulting from a faulty vitreoretinal junction. 18  
Consistent with the hypothesis that the ECM of the vitreous has a role in regulating embryonic eye growth, we show that intraocular injections of GAGs, such as chondroitin sulfate, heparan sulfate, or heparin, lead to changes in eye size at developmental stages, when the sclera has not yet formed and the retina does not process visual information. We also show that an increase in eye size leads to a proportional upregulation of cell proliferation in the retina. 
Materials and Methods
Eye Injections and Histology
Chondroitin sulfate (CS), heparan sulfate (HS), heparin (Hep), and dextran sulfate (DexS) in DMEM at concentrations between 0.1 and 50 mg/mL were injected into the eyes of embryonic day (E) 5 chick embryos. One microliter of CS, HS, Hep, or DexS was injected into the right eye, and the left eye served as the control. Two CS preparations were used: CS A from bovine trachea (C-8529; Sigma, St. Louis, MO) and CS 6-sulfate from shark cartilage (C-4384; Sigma). Heparan sulfate (H7640; Sigma) and heparin (H-3125; Sigma) were purified from bovine kidney and porcine intestine, respectively. Three different Hep batches were used with identical results. Two bacterial-derived DexS preparations with molecular weights of 9 to 20 kDa (D-6924; Sigma) or >500 kDa (D-8906; Sigma) were used. Controls included injections of PBS, 10 mg/mL rooster comb, or bacterial HA (H-5388 [Sigma]; 53747 [Fluka Biochemical, Ronkonkoma, NY]), 50 mg/mL each D-sorbitol (S-1876; Sigma), polyvinylpyrrolidone (PVP-10; Sigma), and dextran T500 (17–0320-01; Pfizer, New York, NY). The molecular weight for both HA preparations was 1400 kDa. 28 All solutions included FastGreen (Sigma) as a tracer, and all solutions were prepared fresh. CS and the other reagents injected into the E5 embryonic eyes were nontoxic, and the survival rate of injected embryos was greater than 90%, identical with the survival rate of embryos injected with PBS. Only surviving and morphologically normal animals were included in the evaluation of the experiments. 
Embryos were killed 1 to 5 days after the injections. Eye size was determined as described. For histology, the heads were fixed in 4% paraformaldehyde and cryoprotected in 25% sucrose. Twenty-micrometer cryostat sections through the experimental and the contralateral control eyes of each embryo were labeled with mAbs to islet 1, visinin, laminin1 (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), and N-CAM 29 to determine differentiation into the various synaptic and cellular layers of the retina. To detect retinal folding, a monoclonal antibody to nonmuscle myosin was used (mAb4A12). Its antigen specificity was established by expression cloning from a phage library of chick amnion cDNAs (accession code gb/M26510/CHKMYHN). Cellular myosin outlines the ventricular surface of the retinal neuroepithelium, and its detection visualized most obviously the folding of the retina. Secondary antibodies were from Jackson ImmunoResearch (West Gove, PA). Specimens were mounted in 90% glycerol and examined with an epifluorescence or a confocal microscope (Flowview; Olympus, Tokyo, Japan). For transmission electron microscopy, the vitreous bodies were dissected, fixed in 4% paraformaldehyde, blocked in 1% milk, and incubated with a monoclonal antibody to collagen IX (mAb 2B9), 30 followed by a peroxidase-labeled secondary antibody (Jackson ImmunoResearch). Bound antibodies were visualized with DAB, and the samples were postfixed in 2.5% glutaraldehyde followed by 1% OsO4
Measurements of Eye Size
Increases and decreases in eye size after HA and GAG injections were determined by comparing the weights of the experimental and control eyes of each embryo and the surface areas of the cross-sectional profiles of the eyes. Eyes were dissected from the periocular connective tissue and weighed on an analytic balance (Sartorius GmbH, Göttingen, Germany). Dissected eyes included the lens, the retina, the VB, and the retinal pigment epithelium. For cross-sectional area measurements and for determining axial length and horizontal diameters of the eyes, the heads of the embryos were fixed in 2.5% glutaraldehyde and 0.01% picric acid overnight. To avoid tissue shrinkage, the heads were cryostat sectioned without cryoprotection along the nasotemporal axis up to the centers of the lenses of both eyes. The remaining frozen tissue blocks were photographed under a dissecting microscope. 31 32 Cross-sectional areas and eye lengths or diameters were measured from the digital images by using a drawing tablet and an image analyzer (Sigma Scan; Systat Software, San Jose, CA). Results were expressed as mean ± SEM. Data were analyzed by Student’s t-test, and P < 0.05 was considered significant. Semi-thin sections of plastic-embedded eyes were stained with AzurII/methylenblue. 33  
Retinal Cell Counts and BrdU Labeling
To determine the total retinal cell counts, dissected retinas were incubated in trypsin/EDTA once (Invitrogen, Carlsbad, CA) for 25 minutes at 37°C. The trypsin solution was removed, and the retinas were dissociated into single-cell suspensions in 1 mL PBS/20% fetal calf serum/5 U DNaseI (Roche, Indianapolis, IN) by repeated pipetting. One hundred microliters of 4% paraformaldehyde was added, and single cells were counted after a 1:10 dilution in a counting chamber (Neubauers; Fisher Scientific, Pittsburgh, PA). To determine proliferation, 100 μL 5-bromo-2-deoxyuridine (BrdU) at a concentration of 5 mM was added on top of the chorioallantoic membrane of the embryos. Embryos were killed 1 hour after BrdU addition. BrdU-labeled retinal cells were detected and counted in single-cell suspensions. For BrdU, islet 1, and visinin staining, the single-cell suspensions were blocked with 2% bovine serum albumin and 0.05% Triton-X-100 for 5 minutes and then were incubated with monoclonal antibodies to BrdU (clone BU-1; Amersham Biosciences, Piscataway, NJ), islet 1, or visinin (Developmental Studies Hybridoma Bank; University of Maryland) overnight. Labeled cells were detected with a Cy-3–labeled goat anti–mouse antibody (Jackson ImmunoResearch) and counted in a counting chamber (Neubauer) at a dilution of 1:2.5 under epifluorescent illumination. 
SDS-PAGE and Western Blotting
VBs were dissected from control and heparin-injected eyes. For the analysis of total VB protein constituents, the samples were homogenized in the presence of 4 M urea and SDS sample buffer. To analyze only the insoluble gel portions of the vitreous, the vitreous samples were suspended in 1 mL PBS, and the gel was pelleted by centrifugation. The VB gels were washed at least 3 times, including a final wash with 1 mL of 4 M urea/PBS. Gel pellets from control and Hep-treated eyes were dissolved by boiling in equal volumes of 4 M urea/SDS sample buffer. After SDS-PAGE and Western blotting, the blots were incubated with a polyclonal antiserum to collagen II (Rockland, Gilbertsville, PA) or a monoclonal antibody to collagen IX (mAb 2B9). 30 The proteins were detected using alkaline phosphatase-conjugated secondary antibodies (Jackson ImmunoResearch, Shady Grove, PA) and NBT and BCIP as labeling reagents (Roche, Indianapolis, IN). 
Results
Increase in Eye Size after Intravitreal Injection of Chondroitin and Heparan Sulfate
Intravitreal injection of 10 mg/mL CS at E5 led to a clearly visible enlargement of the eye globes 5 days after CS application (Fig. 1A) . The increase in eye size was quantified by comparing cross-sectional areas (Fig. 1B)as well as axial length and equatorial diameters of experimental and control eyes (Fig. 1C) . As measured from micrographs of horizontally sectioned heads, the cross-sectional area of experimental eyes was on average 21% greater than that of the contralateral control eyes 5 days after CS injection (Fig. 1B ; n = 12; P < 0.01). Axial length was, on average, 17% longer (P < 0.01), whereas the horizontal diameter was 11% longer (Fig. 1C ; P < 0.01). Injection of PBS had no effect on eye size (Fig. 1B ; n = 5). 
An alternative, more convenient, way to quantify the differences in eye size was weighing the dissected eyes. Five days after injection of 10 mg/mL CS from trachea (CS1; n = 8) and shark fin (CS2; n = 6) at E5 resulted in increases in eye weight of experimental eyes over control eyes by 23% and 26%, respectively (Fig. 1D) . The 90% pure shark cartilage CS2 was slightly more potent than the 70% pure tracheal CS1 (Fig. 1D ; compare CS1 and CS2). HS injection also led to an increase in eye size (Fig. 1D ; n = 7). Yet, with an increase in eye weight by 13% at 10 mg/mL, HS was less potent than CS. 
For control purposes, eyes were injected with PBS (n = 9; Fig. 1D ) and the osmotically active polymers dextran (50 mg/mL; Fig. 1D ; n = 6), PVP (50 mg/mL; Fig. 1D ; n = 7), D-sorbitol (50 mg/mL; Fig. 1D ; n = 6), and HA (10 mg/mL; Fig. 1D ; n = 7). Two different preparations of HA were used (Fig. 1D ; compare columns for Hya1 and Hya2). The very high viscosity of HA solutions limited its concentration to maximum, 10 mg/mL, for the injection experiments. For all these control compounds, no statistically significant difference between the weights of the experimental and control eyes was found 5 days after injection at E5. 
A dose-response curve was obtained by comparing the weights of experimental and control eyes 5 days after injection of CS with concentrations from 1 to 50 mg/mL. Maximum increase in eye size was obtained with CS concentrations between 25 and 50 mg/mL (Fig. 2A) . Normalization of the experimental data, using the eye weight of the controls as 100%, resulted in a saturation curve with 7 mg/mL CS as approximately the half-maximum concentration for increase in eye size (Fig. 2B)
Accelerated eye growth after CS injection was detected already 1 day after CS injection. Cross-sections of the embryo heads showed that the injected eyes were visibly larger than the control eyes (Fig. 2C) . The time course of excessive eye growth after CS injection is shown in Figure 2D . One day after CS injection, the weights of the injected eyes were 32% greater than of the control eyes (Fig. 2D ; n = 8). Three and 5 days after CS injection, the average weights of the experimental eyes were 27% (n = 7) and 32% (n = 6) greater than of the control eyes. 
Decrease in Eye Size after Intraocular Injection of Heparin and Dextran Sulfate
Injection of Hep into the eyes of E5 chick embryos resulted in a decrease in eye size 5 days after Hep application. Comparing control and experimental eyes, the injected eyes were visibly smaller (Figs. 3A 3B) , and the reduction in eye size was up to 70%. A concentration curve showed that half-maximum concentration of Hep for eye size reduction was approximately 1.5 mg/mL (Fig. 3C) . A time-course study using weight measurements showed that size reductions were undetectable 1 day after injection but were obvious 3 days after Hep injection (Fig. 3D)
A reduction in eye size similar to that with Hep was achieved by injection of DexS. The DexS concentration necessary for half-maximum reduction in eye size was 0.8 mg/mL, close to 50% less than with heparin (Fig. 3E) . Experiments with differently sized DexS preparations (10 kDa vs. >500 kDa) at identical wt/vol concentrations showed that the reduction in eye size was greater with >500 kDa DexS than with 10 kDa DexS (Fig. 3F , columns 1 and 2). Experiments also showed the injection of dextran that lacked the sulfate groups and that carried no negative charge did not lead to a change in eye size, even at concentrations up to 50 mg/mL (Fig. 3F ; Dex, column 3). The experiments also showed that potential contaminants from the microbiologically produced DexS were not responsible for causing a reduction in eye size because DexS is synthesized from Dex, which has no effect on eye size whatsoever. 
To find the cause for eye size reduction by heparin, the treated eyes were examined in closer detail. Comparing experimental and control eyes 1 day after Hep injection showed wrinkles around the ciliary margin (Fig. 4A , right eye) that were never seen in the control eyes (Fig. 4A , left eye). Cross-sections revealed that the wrinkles were in fact detachments of the retina from the pigment epithelium. The detachments were found preferentially but not exclusively at the retinal margin (Figs. 4B 5A 5B) . Five days after Hep injections, the detached retinal segments showed prominent cell death, and the retina in the folded segments was unusually thin (Fig. 5B)
Because heparin binds to several growth factors and cell adhesion proteins that may lead to a generic reduction of tissue growth or abnormal histogenesis, cell and synaptic layers of the retina and major heparin-binding proteins of the eye, such as N-CAM and laminin-1, were localized by immunostaining. As shown in Figure 5C , the optic nerve fiber layer and the inner plexiform layers formed properly, and the expression of laminin-1 in the inner limiting membrane (ILM) and NCAM in the optic nerve fiber layer and the inner plexiform layer in the retina in Hep-treated eyes was normal (Figs. 4B 5C) . Laminin labeling also showed that the ILM was continuous and intact (Fig. 4B) . As shown by staining for islet 1 and visinin, the ganglion cells layer (Fig. 5D)and the photoreceptor layer (Fig. 5E)had formed properly as well. 
Hep was also injected into the ventricular cavity of E5 chick brains to determine whether Hep reduces tissue growth in other segments of the CNS. Results showed that injections of 5 μL of 2 mg/mL Hep had no effect on brain size 5 days after the injections (Fig. 6C; n = 7). For comparison, injection of 1 μL of the same concentration of Hep into E5 eyes (n = 7) resulted in a dramatic reduction in eye size (Figs. 6A 6B) . To systemically treat the embryos with Hep, 70 to 100 μL Hep at a concentration of 10 mg/mL was applied to the chorioallantoic membrane of E5 embryos. There were no alterations in body size, eye size, or survival of the Hep-treated embryos (n = 7) compared with embryos treated with PBS alone (n = 7; data not shown) 5 days after Hep application. 
Weight measurements of experimental and control eyes showed that the reduction in eye weight in the experimental eyes was paralleled by an even greater reduction in the weight of their VB gels; the weight of the retina was not affected (Fig. 7A) . The reduced vitreous gel indicated an interference of Hep with one of the VB ECM components. To investigate this further, the binding of VB proteins to heparin was examined. Incubation of VB with heparin Sepharose showed that collagen II and collagen IX were retained, and both proteins were released from the heparin Sepharose with only 1 M salt (Figs. 7B 7C) . The binding of Hep to collagen II and IX suggests that intravitreally injected Hep could interfere with collagen II fibrillogenesis or (and) binding of collagen IX to collagen II fibrils. Ultrastructural analysis showed that the collagen fibrils from the heparin-injected eyes (Fig. 7E)had less of the electron-dense ECM material associated with it than collagen II fibrils from the control eyes (Fig. 7D , arrows). Immunostaining confirmed that the collagen II fibrils from experimental eyes (Fig. 7G)had less collagen IX immunoreactivity associated with it than collagen II fibrils from the control eyes (Fig. 7F , arrows). Finally, Western blots showed that the concentration and banding pattern of collagen II and IX in total VB samples from Hep-treated eyes (Fig. 7H , lanes 2 for coll2 and coll9) and control eyes (Fig. 7H , lanes 1 for coll2 and coll9) were similar. The insoluble vitreous gel from heparin-treated eyes (Fig. 7H , lane 4 for col9), however, contained less collagen IX than the VB gel from the control eyes (Fig. 7H , lane 3 for coll9; n = 4). In contrast, there was more of the fully processed, low molecular-weight collagen II band 34 35 (Fig. 7H , star, lane 4 of coll2) in the heparin-treated eyes (Fig. 7H , lane 4 for col2; n = 4) compared with VB gels from the control eyes (Fig. 7H , lane 3 for col2). Thus, intraocular Hep affected VB gel formation in two ways, by decreasing the binding of collagen IX to collagen II fibrils and by promoting the processing of collagen II from the proform to the fully processed trimer. 
Retinal Growth in Response to Increased Eye Size
To determine whether an increase in eye size by CS injection or a decrease in eye size after Hep application results in a corresponding increase or decrease of retinal growth, retinal cell counts from experimental and control eyes were established. Consistent with data presented (Fig. 1) , injections of 30 mg/mL CS from trachea (CS1) or shark fin (CS2) resulted in increases in eye weight of 29% and 32% (Fig. 8A ; eye size). Cell counts showed a similar increase in retinal cell numbers in the experimental eyes (Fig. 8B , cell count). To find out whether the increase in retinal cell numbers was caused by increased proliferation, embryos were labeled with BrdU, and the labeled retinal cells were counted. As shown in Figure 8C(proliferation), an increase in eye size by injection of 30 mg/mL CS from shark cartilage (CS2) led to a 29% increase in BrdU-labeled cells in retinas from experimental eyes over retinas from control eyes. To find out whether the increase of retinal cells also led to a greater number of differentiated neurons, dissociated retinal cells were labeled with islet 1 and visinin antibodies to identify ganglion cells and photoreceptors. Cell counts of the labeled cells showed a 26% increase of ganglion cells and a 34% increase in photoreceptors in experimental over control retinas (Fig. 8D)
Consistent with results shown in Figure 3 , intraocular injection of 1 mg/mL Hep led to a 28% reduction in eye weight (Fig. 8A , eye size). Cell counts of these heparin-injected eyes showed that the retinal cell numbers of the experimental eyes were unchanged compared with the retinal cell number from the control eyes (Fig. 8B , cell count). Hep injection also had no effect on cell proliferation in the retina, as shown by BrdU-labeled retinal cell counts from experimental and control eyes (Fig. 8C ; proliferation). 
Discussion
Intraocular Injection of GAGs Leads to Changes in Eye Size
VB is a highly hydrated ECM with approximately 98% water wt/wt. Its protein constituents include several collagen family members, such as collagen types II, IX, V/XI, IV, and XVIII. 1 2 3 Collagen II forms the principal fibrillar network that confers the gel-like properties of the VB. 36 37 CSPG type IX collagen localizes to the surface of the collagen II fibrils and separates the individual collagen II fibrils from each other, 6 10 11 12 whereas collagen V/XI initiates fibrillogenesis of collagen II. 38 Collagens IV and XVIII are constituents of ILM and are preset in VB only during ILM assembly in embryogenesis. 9 The noncollagenous proteins in vitreous include versican and several noncollagenous ECM glycoproteins, such as fibronectin, tenascin, and fibrillin. 8 9 The space-forming proteins of the vitreous are collagen IX and versican because they retain large quantities of water through the long, negatively charged GAG chains. Collagen IX is the most abundant CSPG in VB. 6 7 8 9 It is, next to aggrecan, also the most prominent CSPG in cartilage. 10 11 12 The vitreous collagen IX is different from the cartilage version because it has a very long, 350-kDa versus 17-kDa, CS side chain 6 and, thus, a higher capacity for water retention. HA is yet another highly charged carbohydrate polymer that is abundant in vertebrate VB and that may contribute to embryonic intraocular turgor. 1 2 3 However, HA is absent in the chick VB, 37 and it appears that collagen IX and versican are sufficient to provide the water retention necessary to generate the intraocular pressure essential for eye growth. The finding that intraocular injection of HA had no effect on eye size in our experimental system (Fig. 1D)is consistent with the fact that normal embryonic eye growth can proceed in the absence of HA. 
Along with the hypothesis that the water-binding GAG side chains of CSPGs are the dominant space-forming constituents in VB, one would expect that the injection of additional CS may lead to a larger vitreous and thereby an increase in eye size. The present experiments are consistent with this hypothesis: the injection of CS and HS into E5 embryonic chick eyes led to a maximum 30% increase in eye size. As expected, injection of HS was not as potent as that of CS because a high capacity for water binding has been linked to CSPGs and not to HSPGs. Injection of noncharged polymers, such as PVP, sorbitol, and dextran, which are known to be osmotically active, did not change eye size, suggesting that only the injection of negatively charged polymers may result in changes of eye size. In that respect it was unexpected that the application of HA, also a charged GAG polymer, had no effect on eye size. 
The increase in eye size after CS injection occurred within 24 hours, and the eye enlargement was predominantly axial, thus resembling a myopic eye enlargement. Unlike the postnatal eye, in which vision and growth of the sclera play key roles in regulating eye size, 21 22 the CS-induced eye enlargements between E5 and E10 occurred before the completion of scleral development 32 and before the maturation of visual function in the retina. 39 The current finding that the VB contributes in regulating eye growth during embryogenesis is consistent with classical experiments by Coulombre et al. 40 showing that leaks in the vitreous cavity in embryonic chick eyes lead to the collapse of the eye globe. Further, previous experiments dissolving the ILM and the vitreous gel with collagenase showed that liquefaction of both intraocular ECM structures leads to an increase in eye size, 32 again linking early eye growth with the structure and composition of the intraocular ECM. 
By implicating the GAG constituents of the VB and the ILM as regulators for early embryonic eye growth, our results may explain why mutations of proteins associated with VB fibril assembly lead to congenital high myopia in humans. Mutations of collagen II, IX, and XVIII in Stickler syndrome 41 42 43 and Knobloch syndrome 18 44 45 are associated with changes in the structure of the vitreous or the vitreoretinal junction and lead to congenital high myopia. 
Decrease in Eye Size after Intraocular Injection of Heparin and Dextran Sulfate
Intraocular injection of Hep and DexS, at concentrations between 1 and 5 mg/mL, led to a dramatic decrease in eye growth. The reduction in eye size seems paradoxical because Hep and DexS are negatively charged polycarbohydrates that, like CS, should result in an increase rather than a decrease in eye size. However, in contrast to CS, Hep does not retain large quantities of water, and Hep does not contribute to tissue swelling. Rather, heparin binds to and anchors a variety of growth factors, cell adhesion molecules, and ECM proteins. The multitude of heparin interactions suggested that the reduction of eye size after Hep injection might be attributed to a generic inhibition of tissue growth, an abnormal expression of a heparin-binding cell adhesion protein, or a dysfunction in retinal histogenesis. However, the formation of synaptic, ganglion, and photoreceptor layers in the experimental eyes was normal, and N-CAM and laminin-1, two major cell adhesion proteins with heparin-binding sites, were normally expressed in the heparin-injected eyes. Further, intraventricular heparin injection did not lead to a reduction in brain size, and systemic application of heparin at high concentrations onto the chorioallantoic membrane did not affect embryonic growth and development. Finally, retinal proliferation was not affected by heparin, as shown by cell counts and BrdU labeling (Fig. 8) . All data combined indicate that the reduction in eye size after intraocular heparin injection was not caused by a generic block of cell proliferation or abnormal ocular morphogenesis. Further analysis showed that the expansion of the VB gel was most dramatically affected in heparin-injected eyes and suggested an interference with the ECM network assembly of the vitreous gel. Although collagen II synthesis was not inhibited, the binding of collagen IX to the collagen II fibrils was reduced by heparin. The interaction of collagen II and IX is essential in vitreous gel expansion, and its blockage would result in less water retention and a smaller vitreous gel. In addition, heparin injection led to an accelerated processing of collagen II from its proform to the fully cleaved, low-molecular trimer. 34 35 Based on the current data, we speculate that the decreased binding of collagen IX, combined with accelerated collagen II fibrillogenesis, leads to a condensed VB gel that has less water retention capability and therefore cannot sufficiently expand. The smaller VB then leads to smaller eye globes. 
Increases in Eye Size Result in Proportional Increases in Retinal Cell Numbers
The chance to experimentally increase or decrease eye size allowed us to address whether changes in eye size lead to corresponding changes in retinal cell proliferation. Cell counts showed that an increase in eye size by 30% led to a proportional increase in retinal cell numbers because of an increase in proliferation. In addition, immunostaining for islet 1 and visinin showed that larger than normal eyes had correspondingly greater numbers of ganglion cells and photoreceptors. The present data show that increasing eye size through intravitreal pressure leads to a corresponding increase of the retina by cell proliferation that includes all retinal cell types. Cell proliferation in response to mechanical strain has been best documented for the vascular system. 46 47 48 49 It is mediated by growth factors and activation of the ERK/MAPK pathway combined with increased nuclear import. 50  
On the other hand, decreasing eye size with heparin was not accompanied by a decrease in retinal cell numbers and retinal proliferation. Rather, the reduction in eye size by Hep application led to the folding and detachment of segments of the retina as the retina grew at a normal, default proliferation rate. Data also revealed that retina detachment led to cell death in the detached segments of the retina, showing that the connection of the retina with the pigment epithelium is already essential during early embryonic development. 
In summary, the present experiments show that eye size can be manipulated by injection of CS, Hep, and DexS into the embryonic vitreous. CS injections led to greater water retention in the vitreous cavity and thereby to a greater eyeball. The increase in eye size was synchronized by a higher proliferation of retinal cells. Heparin, on the other hand interfered with the assembly of the extracellular matrix of the vitreous and led to shrinkage of the vitreous. The experiments demonstrate that the volume of the vitreous may have an important role in determining eye size during development. 
 
Figure 1.
 
Increase in eye size after intravitreal injection of CS at E5. A cross-section of an E10 chick head (A) shows the size difference between the experimental (E) and the contralateral control eye 5 days after injection of 10 mg/mL CS. The increase in eye size was quantified by comparing the cross-sectional areas (B), axial and horizontal lengths (C), and the weights (D) of the experimental (E; green columns) and the contralateral (C; red columns) control eyes. (A) Axial length and horizontal diameter are indicated as A and H, respectively. Area measurements also showed that the injection of PBS has no effect on eye size (B; PBS). Weight measurements showed that injection of 10 mg/mL CS from trachea (CS1) or shark fin (CS2) or an injection of 10 mg/mL HS at E5 resulted in a 23%, 26%, and 13% increases in eyes size at E10 (D). Injection of PBS (D, PBS), Dex (50 mg/mL), HA from rooster comb (Hya1; 10 mg/mL) or bacteria (Hya2; 10 mg/mL), PVP (50 mg/mL), and Sor (50 mg/mL) had no effect on eye weights. Data are expressed as means, and error bars depict SEM. P < 0.01, indicated when the differences between experimental and control eyes were significant.
Figure 1.
 
Increase in eye size after intravitreal injection of CS at E5. A cross-section of an E10 chick head (A) shows the size difference between the experimental (E) and the contralateral control eye 5 days after injection of 10 mg/mL CS. The increase in eye size was quantified by comparing the cross-sectional areas (B), axial and horizontal lengths (C), and the weights (D) of the experimental (E; green columns) and the contralateral (C; red columns) control eyes. (A) Axial length and horizontal diameter are indicated as A and H, respectively. Area measurements also showed that the injection of PBS has no effect on eye size (B; PBS). Weight measurements showed that injection of 10 mg/mL CS from trachea (CS1) or shark fin (CS2) or an injection of 10 mg/mL HS at E5 resulted in a 23%, 26%, and 13% increases in eyes size at E10 (D). Injection of PBS (D, PBS), Dex (50 mg/mL), HA from rooster comb (Hya1; 10 mg/mL) or bacteria (Hya2; 10 mg/mL), PVP (50 mg/mL), and Sor (50 mg/mL) had no effect on eye weights. Data are expressed as means, and error bars depict SEM. P < 0.01, indicated when the differences between experimental and control eyes were significant.
Figure 2.
 
Concentration curves (A, B) and time-course study (D) of increased eye size after CS injection in E5 chick eyes. The size increase was quantified by comparing the weights of the experimental (Ex; red curves) and control eyes (Con; black curves) 5 days after CS injection (A). Normalizing the CS concentration curve by taking the control values as 100% (B) showed a maximum increase in eye size with CS concentrations between 30 and 50 mg/mL. As shown in a cross-section of an E6 head, an increase in eye size was already obvious 1 day after CS injection at E5 (C; Ex, experimental eye). A time-course study shows the increase in eye weights between experimental (green bars) and control (red bars) eyes 1 to 6 days after CS injection (D).
Figure 2.
 
Concentration curves (A, B) and time-course study (D) of increased eye size after CS injection in E5 chick eyes. The size increase was quantified by comparing the weights of the experimental (Ex; red curves) and control eyes (Con; black curves) 5 days after CS injection (A). Normalizing the CS concentration curve by taking the control values as 100% (B) showed a maximum increase in eye size with CS concentrations between 30 and 50 mg/mL. As shown in a cross-section of an E6 head, an increase in eye size was already obvious 1 day after CS injection at E5 (C; Ex, experimental eye). A time-course study shows the increase in eye weights between experimental (green bars) and control (red bars) eyes 1 to 6 days after CS injection (D).
Figure 3.
 
Intraocular injection of Hep or DexS at E5 led to a reduction in eye size at E10. A micrograph of a head (A) or a cross-section of a head (B) shows an obvious reduction in eye size after injection of 1 mg/mL (A) or 2 mg/mL Hep (B; E, experimental eye). A concentration curve comparing eye weights of experimental (E; red curve) and control (C; black curve) eyes shows a concentration-dependent decrease in eye size that plateaus around 2 mg/mL Hep. A time-course study (D) shows that size differences in experimental (E; green bars) and control (C; red bars) eyes are obvious only 3 days after Hep injection. Injection of DexS also results in a reduction of eye size, as shown in the concentration curve (E). Maximum decrease in eye size is reached at 1 mg/mL DexS. Reduction in eye size was greater with injections of a high molecular-weight DexS (MWt >500 kDa) than with a lower molecular-weight form (10 kDa; F, compare column pairs 1 and 2). Injection of 50 mg/mL nonsulfated dextran (Dex) had no effect on eye size (F, column pair 3).
Figure 3.
 
Intraocular injection of Hep or DexS at E5 led to a reduction in eye size at E10. A micrograph of a head (A) or a cross-section of a head (B) shows an obvious reduction in eye size after injection of 1 mg/mL (A) or 2 mg/mL Hep (B; E, experimental eye). A concentration curve comparing eye weights of experimental (E; red curve) and control (C; black curve) eyes shows a concentration-dependent decrease in eye size that plateaus around 2 mg/mL Hep. A time-course study (D) shows that size differences in experimental (E; green bars) and control (C; red bars) eyes are obvious only 3 days after Hep injection. Injection of DexS also results in a reduction of eye size, as shown in the concentration curve (E). Maximum decrease in eye size is reached at 1 mg/mL DexS. Reduction in eye size was greater with injections of a high molecular-weight DexS (MWt >500 kDa) than with a lower molecular-weight form (10 kDa; F, compare column pairs 1 and 2). Injection of 50 mg/mL nonsulfated dextran (Dex) had no effect on eye size (F, column pair 3).
Figure 4.
 
Folding and detachment of the retina after intraocular injection of Hep. One day after Hep injection at E5, the sizes of the experimental eyes were not visibly smaller, yet the heparin-injected eyes showed retinal infoldings at the ciliary margin (A, arrows at the right eye) that were never seen in the control eyes (A, left eye). A cross-section through an experimental eye 1 day after heparin injection (B) that was stained for laminin showed focal detachments of the retina, most prominently at the ciliary margin (arrow). R, retina; L, lens. Scale bar, 100 μm.
Figure 4.
 
Folding and detachment of the retina after intraocular injection of Hep. One day after Hep injection at E5, the sizes of the experimental eyes were not visibly smaller, yet the heparin-injected eyes showed retinal infoldings at the ciliary margin (A, arrows at the right eye) that were never seen in the control eyes (A, left eye). A cross-section through an experimental eye 1 day after heparin injection (B) that was stained for laminin showed focal detachments of the retina, most prominently at the ciliary margin (arrow). R, retina; L, lens. Scale bar, 100 μm.
Figure 5.
 
Detachment and folding of the retina 3 and 5 days after heparin injection. Three days after Hep injection at E5, the experimental eyes showed several retinal foldings (A, white arrows). The cross-section was stained with an antibody to nonmuscle myosin to label the ventricular surface of the retina (R) and to visualize the retinal folds. A plastic section (B) of a retinal fold (F) 5 days after Hep injection shows that many cells of the detached retina underwent cell death and that the folded retina was thinner than the attached retina (R) The section was stained with AzurII/methylenblue. 33 Staining of E10 retinal cross-sections with antibodies to N-CAM (C), islet 1 (D), and visinin (E) showed that intraocular heparin did not interfere with NCAM expression (C) or the formation of the optic nerve fiber layer (OFL; C), the inner plexiform layer (IPL; C), the ganglion cell (GC; D), or the photoreceptors layer (PR; E). Scale bars: (A) 100 μm; (BE) 50 μm.
Figure 5.
 
Detachment and folding of the retina 3 and 5 days after heparin injection. Three days after Hep injection at E5, the experimental eyes showed several retinal foldings (A, white arrows). The cross-section was stained with an antibody to nonmuscle myosin to label the ventricular surface of the retina (R) and to visualize the retinal folds. A plastic section (B) of a retinal fold (F) 5 days after Hep injection shows that many cells of the detached retina underwent cell death and that the folded retina was thinner than the attached retina (R) The section was stained with AzurII/methylenblue. 33 Staining of E10 retinal cross-sections with antibodies to N-CAM (C), islet 1 (D), and visinin (E) showed that intraocular heparin did not interfere with NCAM expression (C) or the formation of the optic nerve fiber layer (OFL; C), the inner plexiform layer (IPL; C), the ganglion cell (GC; D), or the photoreceptors layer (PR; E). Scale bars: (A) 100 μm; (BE) 50 μm.
Figure 6.
 
Intraocular injection of Hep leads to dramatic decrease in eye size (A, B), but injection of Hep into the ventricular space of the optic tectum did not lead to an alteration of brain size (C). One microliter Hep at a concentration of 2 mg/mL was injected into the right eye of E5 chick embryos, and the heads (A) and eyes (B) were photographed 5 days after injection. Note the dramatic decrease in eye size. Injection of 5 μL of 2 mg/mL Hep into the ventricular space of E5 chick embryos did not lead to an alteration in brain size at E10 (C). Experimental (E) and control (left) brains are shown side by side (C).
Figure 6.
 
Intraocular injection of Hep leads to dramatic decrease in eye size (A, B), but injection of Hep into the ventricular space of the optic tectum did not lead to an alteration of brain size (C). One microliter Hep at a concentration of 2 mg/mL was injected into the right eye of E5 chick embryos, and the heads (A) and eyes (B) were photographed 5 days after injection. Note the dramatic decrease in eye size. Injection of 5 μL of 2 mg/mL Hep into the ventricular space of E5 chick embryos did not lead to an alteration in brain size at E10 (C). Experimental (E) and control (left) brains are shown side by side (C).
Figure 7.
 
Intraocular injection of Hep at E5 inhibits vitreous gel expansion in E10 eyes. Intraocular injection of 1 mg/mL Hep leads to a reduction of eye size by 28% (A; eye), as demonstrated by comparing the weights of experimental (E; green columns) and control (C; red columns) eyes. The weight reduction in the VBs from these eyes (VB; A) was 49%, whereas the retina weights (retina) were not affected by heparin (A). Western blot analysis comparing VB extracts (C) and high salt eluates from heparin Sepharose demonstrates the binding of collagen II (B) and collagen IX (C) to heparin. Transmission electron micrographs show comparable collagen II fibril assembly in VB from eyes injected with Hep (E) and control eyes (D; Con). However, there was less ECM material associated with the fibrils in the experimental as with fibrils from the control VB (D, arrows). Immunolabeling for collagen IX showed less immunoreactivity associated with the collagen II fibrils from experimental (G; Hep) than from control (F; Con; arrows) eyes. Western blot analysis (H) of total VB showed similar concentration and banding patterns of collagen II (Coll2) and IX (Coll 9) in VB from heparin-injected eyes and control samples (lanes 1 and 2 for coll2 and coll9). However, the insoluble gel portion of the VBs from the experimental eyes (E) had less collagen IX and more of the fully processed collagen II than VB from control eyes (C; lanes 3 and 4 for coll2 and coll9). *Fully processed collagen II band (lane 4, coll2). Scale bar, 500 nm.
Figure 7.
 
Intraocular injection of Hep at E5 inhibits vitreous gel expansion in E10 eyes. Intraocular injection of 1 mg/mL Hep leads to a reduction of eye size by 28% (A; eye), as demonstrated by comparing the weights of experimental (E; green columns) and control (C; red columns) eyes. The weight reduction in the VBs from these eyes (VB; A) was 49%, whereas the retina weights (retina) were not affected by heparin (A). Western blot analysis comparing VB extracts (C) and high salt eluates from heparin Sepharose demonstrates the binding of collagen II (B) and collagen IX (C) to heparin. Transmission electron micrographs show comparable collagen II fibril assembly in VB from eyes injected with Hep (E) and control eyes (D; Con). However, there was less ECM material associated with the fibrils in the experimental as with fibrils from the control VB (D, arrows). Immunolabeling for collagen IX showed less immunoreactivity associated with the collagen II fibrils from experimental (G; Hep) than from control (F; Con; arrows) eyes. Western blot analysis (H) of total VB showed similar concentration and banding patterns of collagen II (Coll2) and IX (Coll 9) in VB from heparin-injected eyes and control samples (lanes 1 and 2 for coll2 and coll9). However, the insoluble gel portion of the VBs from the experimental eyes (E) had less collagen IX and more of the fully processed collagen II than VB from control eyes (C; lanes 3 and 4 for coll2 and coll9). *Fully processed collagen II band (lane 4, coll2). Scale bar, 500 nm.
Figure 8.
 
Eye weights (A), retinal cell counts (B), and retinal cell proliferation (C) of eyes injected with tracheal CS (CS1), shark cartilage CS (CS2), or Hep at E5. (A) The increase in eye size 5 days after CS injection was determined by comparing eye weights of control (red; C) and experimental (green; E) eyes. Total retinal cell counts (B) and counts of BrdU-labeled cells as a measure for cell proliferation (C) were established for the same eyes. In a separate set of experiments, the numbers of ganglion cells (GC) and photoreceptors (PR) after CS2 application were determined by anti-islet 1 and anti-visinin staining of single-cell suspensions (D). CS 1 and CS2 injections led to increases in eye size of 29% and 32% (A). The increase in eye size was paralleled by similar increases in retinal cell numbers (B), in the numbers of BrdU-labeled cells (C), and in ganglion cell and photoreceptor counts (D). Heparin injection led to a 28% reduction in eye size (A; Hep), but retinal cell numbers (B; Hep) and BrdU incorporation (C; Hep) were unchanged.
Figure 8.
 
Eye weights (A), retinal cell counts (B), and retinal cell proliferation (C) of eyes injected with tracheal CS (CS1), shark cartilage CS (CS2), or Hep at E5. (A) The increase in eye size 5 days after CS injection was determined by comparing eye weights of control (red; C) and experimental (green; E) eyes. Total retinal cell counts (B) and counts of BrdU-labeled cells as a measure for cell proliferation (C) were established for the same eyes. In a separate set of experiments, the numbers of ganglion cells (GC) and photoreceptors (PR) after CS2 application were determined by anti-islet 1 and anti-visinin staining of single-cell suspensions (D). CS 1 and CS2 injections led to increases in eye size of 29% and 32% (A). The increase in eye size was paralleled by similar increases in retinal cell numbers (B), in the numbers of BrdU-labeled cells (C), and in ganglion cell and photoreceptor counts (D). Heparin injection led to a 28% reduction in eye size (A; Hep), but retinal cell numbers (B; Hep) and BrdU incorporation (C; Hep) were unchanged.
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Figure 1.
 
Increase in eye size after intravitreal injection of CS at E5. A cross-section of an E10 chick head (A) shows the size difference between the experimental (E) and the contralateral control eye 5 days after injection of 10 mg/mL CS. The increase in eye size was quantified by comparing the cross-sectional areas (B), axial and horizontal lengths (C), and the weights (D) of the experimental (E; green columns) and the contralateral (C; red columns) control eyes. (A) Axial length and horizontal diameter are indicated as A and H, respectively. Area measurements also showed that the injection of PBS has no effect on eye size (B; PBS). Weight measurements showed that injection of 10 mg/mL CS from trachea (CS1) or shark fin (CS2) or an injection of 10 mg/mL HS at E5 resulted in a 23%, 26%, and 13% increases in eyes size at E10 (D). Injection of PBS (D, PBS), Dex (50 mg/mL), HA from rooster comb (Hya1; 10 mg/mL) or bacteria (Hya2; 10 mg/mL), PVP (50 mg/mL), and Sor (50 mg/mL) had no effect on eye weights. Data are expressed as means, and error bars depict SEM. P < 0.01, indicated when the differences between experimental and control eyes were significant.
Figure 1.
 
Increase in eye size after intravitreal injection of CS at E5. A cross-section of an E10 chick head (A) shows the size difference between the experimental (E) and the contralateral control eye 5 days after injection of 10 mg/mL CS. The increase in eye size was quantified by comparing the cross-sectional areas (B), axial and horizontal lengths (C), and the weights (D) of the experimental (E; green columns) and the contralateral (C; red columns) control eyes. (A) Axial length and horizontal diameter are indicated as A and H, respectively. Area measurements also showed that the injection of PBS has no effect on eye size (B; PBS). Weight measurements showed that injection of 10 mg/mL CS from trachea (CS1) or shark fin (CS2) or an injection of 10 mg/mL HS at E5 resulted in a 23%, 26%, and 13% increases in eyes size at E10 (D). Injection of PBS (D, PBS), Dex (50 mg/mL), HA from rooster comb (Hya1; 10 mg/mL) or bacteria (Hya2; 10 mg/mL), PVP (50 mg/mL), and Sor (50 mg/mL) had no effect on eye weights. Data are expressed as means, and error bars depict SEM. P < 0.01, indicated when the differences between experimental and control eyes were significant.
Figure 2.
 
Concentration curves (A, B) and time-course study (D) of increased eye size after CS injection in E5 chick eyes. The size increase was quantified by comparing the weights of the experimental (Ex; red curves) and control eyes (Con; black curves) 5 days after CS injection (A). Normalizing the CS concentration curve by taking the control values as 100% (B) showed a maximum increase in eye size with CS concentrations between 30 and 50 mg/mL. As shown in a cross-section of an E6 head, an increase in eye size was already obvious 1 day after CS injection at E5 (C; Ex, experimental eye). A time-course study shows the increase in eye weights between experimental (green bars) and control (red bars) eyes 1 to 6 days after CS injection (D).
Figure 2.
 
Concentration curves (A, B) and time-course study (D) of increased eye size after CS injection in E5 chick eyes. The size increase was quantified by comparing the weights of the experimental (Ex; red curves) and control eyes (Con; black curves) 5 days after CS injection (A). Normalizing the CS concentration curve by taking the control values as 100% (B) showed a maximum increase in eye size with CS concentrations between 30 and 50 mg/mL. As shown in a cross-section of an E6 head, an increase in eye size was already obvious 1 day after CS injection at E5 (C; Ex, experimental eye). A time-course study shows the increase in eye weights between experimental (green bars) and control (red bars) eyes 1 to 6 days after CS injection (D).
Figure 3.
 
Intraocular injection of Hep or DexS at E5 led to a reduction in eye size at E10. A micrograph of a head (A) or a cross-section of a head (B) shows an obvious reduction in eye size after injection of 1 mg/mL (A) or 2 mg/mL Hep (B; E, experimental eye). A concentration curve comparing eye weights of experimental (E; red curve) and control (C; black curve) eyes shows a concentration-dependent decrease in eye size that plateaus around 2 mg/mL Hep. A time-course study (D) shows that size differences in experimental (E; green bars) and control (C; red bars) eyes are obvious only 3 days after Hep injection. Injection of DexS also results in a reduction of eye size, as shown in the concentration curve (E). Maximum decrease in eye size is reached at 1 mg/mL DexS. Reduction in eye size was greater with injections of a high molecular-weight DexS (MWt >500 kDa) than with a lower molecular-weight form (10 kDa; F, compare column pairs 1 and 2). Injection of 50 mg/mL nonsulfated dextran (Dex) had no effect on eye size (F, column pair 3).
Figure 3.
 
Intraocular injection of Hep or DexS at E5 led to a reduction in eye size at E10. A micrograph of a head (A) or a cross-section of a head (B) shows an obvious reduction in eye size after injection of 1 mg/mL (A) or 2 mg/mL Hep (B; E, experimental eye). A concentration curve comparing eye weights of experimental (E; red curve) and control (C; black curve) eyes shows a concentration-dependent decrease in eye size that plateaus around 2 mg/mL Hep. A time-course study (D) shows that size differences in experimental (E; green bars) and control (C; red bars) eyes are obvious only 3 days after Hep injection. Injection of DexS also results in a reduction of eye size, as shown in the concentration curve (E). Maximum decrease in eye size is reached at 1 mg/mL DexS. Reduction in eye size was greater with injections of a high molecular-weight DexS (MWt >500 kDa) than with a lower molecular-weight form (10 kDa; F, compare column pairs 1 and 2). Injection of 50 mg/mL nonsulfated dextran (Dex) had no effect on eye size (F, column pair 3).
Figure 4.
 
Folding and detachment of the retina after intraocular injection of Hep. One day after Hep injection at E5, the sizes of the experimental eyes were not visibly smaller, yet the heparin-injected eyes showed retinal infoldings at the ciliary margin (A, arrows at the right eye) that were never seen in the control eyes (A, left eye). A cross-section through an experimental eye 1 day after heparin injection (B) that was stained for laminin showed focal detachments of the retina, most prominently at the ciliary margin (arrow). R, retina; L, lens. Scale bar, 100 μm.
Figure 4.
 
Folding and detachment of the retina after intraocular injection of Hep. One day after Hep injection at E5, the sizes of the experimental eyes were not visibly smaller, yet the heparin-injected eyes showed retinal infoldings at the ciliary margin (A, arrows at the right eye) that were never seen in the control eyes (A, left eye). A cross-section through an experimental eye 1 day after heparin injection (B) that was stained for laminin showed focal detachments of the retina, most prominently at the ciliary margin (arrow). R, retina; L, lens. Scale bar, 100 μm.
Figure 5.
 
Detachment and folding of the retina 3 and 5 days after heparin injection. Three days after Hep injection at E5, the experimental eyes showed several retinal foldings (A, white arrows). The cross-section was stained with an antibody to nonmuscle myosin to label the ventricular surface of the retina (R) and to visualize the retinal folds. A plastic section (B) of a retinal fold (F) 5 days after Hep injection shows that many cells of the detached retina underwent cell death and that the folded retina was thinner than the attached retina (R) The section was stained with AzurII/methylenblue. 33 Staining of E10 retinal cross-sections with antibodies to N-CAM (C), islet 1 (D), and visinin (E) showed that intraocular heparin did not interfere with NCAM expression (C) or the formation of the optic nerve fiber layer (OFL; C), the inner plexiform layer (IPL; C), the ganglion cell (GC; D), or the photoreceptors layer (PR; E). Scale bars: (A) 100 μm; (BE) 50 μm.
Figure 5.
 
Detachment and folding of the retina 3 and 5 days after heparin injection. Three days after Hep injection at E5, the experimental eyes showed several retinal foldings (A, white arrows). The cross-section was stained with an antibody to nonmuscle myosin to label the ventricular surface of the retina (R) and to visualize the retinal folds. A plastic section (B) of a retinal fold (F) 5 days after Hep injection shows that many cells of the detached retina underwent cell death and that the folded retina was thinner than the attached retina (R) The section was stained with AzurII/methylenblue. 33 Staining of E10 retinal cross-sections with antibodies to N-CAM (C), islet 1 (D), and visinin (E) showed that intraocular heparin did not interfere with NCAM expression (C) or the formation of the optic nerve fiber layer (OFL; C), the inner plexiform layer (IPL; C), the ganglion cell (GC; D), or the photoreceptors layer (PR; E). Scale bars: (A) 100 μm; (BE) 50 μm.
Figure 6.
 
Intraocular injection of Hep leads to dramatic decrease in eye size (A, B), but injection of Hep into the ventricular space of the optic tectum did not lead to an alteration of brain size (C). One microliter Hep at a concentration of 2 mg/mL was injected into the right eye of E5 chick embryos, and the heads (A) and eyes (B) were photographed 5 days after injection. Note the dramatic decrease in eye size. Injection of 5 μL of 2 mg/mL Hep into the ventricular space of E5 chick embryos did not lead to an alteration in brain size at E10 (C). Experimental (E) and control (left) brains are shown side by side (C).
Figure 6.
 
Intraocular injection of Hep leads to dramatic decrease in eye size (A, B), but injection of Hep into the ventricular space of the optic tectum did not lead to an alteration of brain size (C). One microliter Hep at a concentration of 2 mg/mL was injected into the right eye of E5 chick embryos, and the heads (A) and eyes (B) were photographed 5 days after injection. Note the dramatic decrease in eye size. Injection of 5 μL of 2 mg/mL Hep into the ventricular space of E5 chick embryos did not lead to an alteration in brain size at E10 (C). Experimental (E) and control (left) brains are shown side by side (C).
Figure 7.
 
Intraocular injection of Hep at E5 inhibits vitreous gel expansion in E10 eyes. Intraocular injection of 1 mg/mL Hep leads to a reduction of eye size by 28% (A; eye), as demonstrated by comparing the weights of experimental (E; green columns) and control (C; red columns) eyes. The weight reduction in the VBs from these eyes (VB; A) was 49%, whereas the retina weights (retina) were not affected by heparin (A). Western blot analysis comparing VB extracts (C) and high salt eluates from heparin Sepharose demonstrates the binding of collagen II (B) and collagen IX (C) to heparin. Transmission electron micrographs show comparable collagen II fibril assembly in VB from eyes injected with Hep (E) and control eyes (D; Con). However, there was less ECM material associated with the fibrils in the experimental as with fibrils from the control VB (D, arrows). Immunolabeling for collagen IX showed less immunoreactivity associated with the collagen II fibrils from experimental (G; Hep) than from control (F; Con; arrows) eyes. Western blot analysis (H) of total VB showed similar concentration and banding patterns of collagen II (Coll2) and IX (Coll 9) in VB from heparin-injected eyes and control samples (lanes 1 and 2 for coll2 and coll9). However, the insoluble gel portion of the VBs from the experimental eyes (E) had less collagen IX and more of the fully processed collagen II than VB from control eyes (C; lanes 3 and 4 for coll2 and coll9). *Fully processed collagen II band (lane 4, coll2). Scale bar, 500 nm.
Figure 7.
 
Intraocular injection of Hep at E5 inhibits vitreous gel expansion in E10 eyes. Intraocular injection of 1 mg/mL Hep leads to a reduction of eye size by 28% (A; eye), as demonstrated by comparing the weights of experimental (E; green columns) and control (C; red columns) eyes. The weight reduction in the VBs from these eyes (VB; A) was 49%, whereas the retina weights (retina) were not affected by heparin (A). Western blot analysis comparing VB extracts (C) and high salt eluates from heparin Sepharose demonstrates the binding of collagen II (B) and collagen IX (C) to heparin. Transmission electron micrographs show comparable collagen II fibril assembly in VB from eyes injected with Hep (E) and control eyes (D; Con). However, there was less ECM material associated with the fibrils in the experimental as with fibrils from the control VB (D, arrows). Immunolabeling for collagen IX showed less immunoreactivity associated with the collagen II fibrils from experimental (G; Hep) than from control (F; Con; arrows) eyes. Western blot analysis (H) of total VB showed similar concentration and banding patterns of collagen II (Coll2) and IX (Coll 9) in VB from heparin-injected eyes and control samples (lanes 1 and 2 for coll2 and coll9). However, the insoluble gel portion of the VBs from the experimental eyes (E) had less collagen IX and more of the fully processed collagen II than VB from control eyes (C; lanes 3 and 4 for coll2 and coll9). *Fully processed collagen II band (lane 4, coll2). Scale bar, 500 nm.
Figure 8.
 
Eye weights (A), retinal cell counts (B), and retinal cell proliferation (C) of eyes injected with tracheal CS (CS1), shark cartilage CS (CS2), or Hep at E5. (A) The increase in eye size 5 days after CS injection was determined by comparing eye weights of control (red; C) and experimental (green; E) eyes. Total retinal cell counts (B) and counts of BrdU-labeled cells as a measure for cell proliferation (C) were established for the same eyes. In a separate set of experiments, the numbers of ganglion cells (GC) and photoreceptors (PR) after CS2 application were determined by anti-islet 1 and anti-visinin staining of single-cell suspensions (D). CS 1 and CS2 injections led to increases in eye size of 29% and 32% (A). The increase in eye size was paralleled by similar increases in retinal cell numbers (B), in the numbers of BrdU-labeled cells (C), and in ganglion cell and photoreceptor counts (D). Heparin injection led to a 28% reduction in eye size (A; Hep), but retinal cell numbers (B; Hep) and BrdU incorporation (C; Hep) were unchanged.
Figure 8.
 
Eye weights (A), retinal cell counts (B), and retinal cell proliferation (C) of eyes injected with tracheal CS (CS1), shark cartilage CS (CS2), or Hep at E5. (A) The increase in eye size 5 days after CS injection was determined by comparing eye weights of control (red; C) and experimental (green; E) eyes. Total retinal cell counts (B) and counts of BrdU-labeled cells as a measure for cell proliferation (C) were established for the same eyes. In a separate set of experiments, the numbers of ganglion cells (GC) and photoreceptors (PR) after CS2 application were determined by anti-islet 1 and anti-visinin staining of single-cell suspensions (D). CS 1 and CS2 injections led to increases in eye size of 29% and 32% (A). The increase in eye size was paralleled by similar increases in retinal cell numbers (B), in the numbers of BrdU-labeled cells (C), and in ganglion cell and photoreceptor counts (D). Heparin injection led to a 28% reduction in eye size (A; Hep), but retinal cell numbers (B; Hep) and BrdU incorporation (C; Hep) were unchanged.
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