FULL PAPER Anatomy

Expression of Small Leucine-Rich in the Developing Retina and Kainic Acid-Induced Retinopathy in ICR Mice

Safwat Ali Mohamed ALI1,2,3), Yoshinao Z. HOSAKA1,2)* and Masato UEHARA1,2)

1)Laboratory of Basic Veterinary Science, United Graduate School of Veterinary Science, Yamaguchi University, Yamaguchi 753–8515, 2)Department of Veterinary Anatomy, Faculty of Agriculture, Tottori University, Tottori 680–8553, Japan and 3)Department of Anatomy and Embryology, Faculty of Veterinary Medicine, Menoufia University, Sadat City, Egypt

(Received 18 October 2010/Accepted 10 November 2010/Published online in J-STAGE 24 November 2010)

ABSTRACT. The aim of this study was to determine the developmental changes of small leucine-rich proteoglycans (PGs), , and , in ICR mouse retinas and to elucidate their role in the adult retina using kainic acid (KA)-induced retinal degeneration model. Retinas of prenatal, postnatal and adult mice were collected for histological and immunohistochemical staining to investigate the changes in distribution of these PGs. Decorin-and fibromodulin-immunostainings were diffusely distributed at prenatal and early post- natal stages and were stronger in the adult retina. However, biglycan was moderately distributed in the prenatal and early postnatal stages and was faint in the adult retina. Retinas were collected at 1, 3 and 7 days after intravitreal injection of KA. Retinas of KA injected eyes underwent shrinkage accompanied by serious damage in the inner layers. Decorin and fibromodulin were upregulated in the inner retinal layers of KA-injected eyes compared to the normal ones. Our results suggest that decorin and fibromodulin play key roles in retinal differentiation, and contribute to the retinal damage and repair process. However, biglycan may have no or only a limited role in the mouse retinal development or repair process. KEY WORDS: biglycan, decorin, fibromodulin, kainic acid, retinal development and damage. J. Vet. Med. Sci. 73(4): 439–445, 2011

Proteoglycans (PGs) play key roles in all of the funda- ber of the leucine-rich repeat family, has been shown mental biological processes and behave as potent effectors to bind to type I and to regulate fibrogenesis [8], of cellular pathways. PGs consist of a core protein to which and it has been also shown to be a constituent of , (GAG) side chains are bound. GAG , and sclera [33, 35]. However, there are no reports chains attached to the core protein vary markedly in length on the localization and developmental changes of fibromod- and number. These variations in GAG chain number and ulin in the retina. degree of sulfation determine the unique functions of differ- PGs specifically bind to many cell surface molecules and ent PGs within and between PG classes [7]. extracellular matrix molecules that are involved in various The families of small leucine-rich PGs (SLRPs) comprise developmental events in the brain [17, 19, 20]. These devel- at least nine members that, though structurally related, have opmental events include proliferation and migration of neu- evolved from different , have acquired unique func- roblasts, neurite outgrowth, and formation of specific tions, and have undergone a significant degree of structural synapses. However, there were a few studies on an involve- sophistication. They can be synthesized as either glycopro- ment of PG in the retinal development. Additionally, in the teins containing N-linked oligosaccharides or as PGs con- neural retina, it has been inferred that alterations in the taining chondroitin/ or expression of PGs are involved in a number of pathologic chains [14]. The core of decorin, biglycan and conditions as retinal degeneration [16, 27]. Although the fibromodulin are similar in size (36–42 kDa). These core relationships between PGs and retinal diseases have been proteins have a central domain containing leucine-rich studied, molecular biological studies on the expressional repeats and terminal domains with cysteine residues in con- regulation of SLRP core proteins in normal and pathologic served positions. Fibromodulin is a keratan sulfate PG, retinas are limited, and there have been only a few reports on while decorin and biglycan are chondroitin/dermatan sulfate the identification of PG core proteins expressed in the retina PGs [39]. [1, 12]. Decorin and biglycan have been isolated from mamma- Glutamate is the principal excitatory neurotransmitter in lian connective tissues [28], and it has been suggested that central nervous system and eye [22, 40]. Endogenous decorin and biglycan are small PGs related to cell prolifera- glutamate may contribute to the brain damage occurring tion and extracellular matrix assembly [15, 29] and play a acutely after status epileptics, cerebral ischemia or traumatic role in the regeneration of nervous tissues in central nervous brain injury [22]. The increase of glutamate level was found system injury [34]. Fibromodulin, the most abundant mem- in the animal model of retinal ischemia and optic nerve crush [40]. It has been shown that increased glutamate in *CORRESPONDENCE TO: HOSAKA, Y. Z., Department of Veterinary Anatomy, Faculty of Agriculture, Tottori University, 4–101 neuro-degenerative disease in the retina was similar to that Koyama-Minami, Tottori 680–8553, Japan. occurred after the intravitreal injection of kainic acid (KA), e-mail: [email protected] a glutamate receptor agonist [11, 18]. In retinal degenera- 440 S. A. M. ALI, Y. Z. HOSAKA AND M. UEHARA tion, decorin is also found to be upregulated in a retinal of normal and KA-injected eyes, some sections were stained ischemic model [12], but an involvement of other SLRPs in with 1 μg/ml 4’,6-diamidino-2-phenylindole (DAPI; Molec- retinal degeneration has not been clarified yet. ular Probes, Eugene, OR, U.S.A.) in PBS for 10 min. Then In this study, relationships among three SLRPs, decorin, the slides were washed 3 times for 5 min each time in PBS. biglycan and fibromodulin, to development and pathogene- To reduce photobleaching, the sections were mounted with sis in the retina were investigated through their immunohis- Vectashield mounting medium (Vector Laboratories, Burl- tochemical distributions in the developing and mature retina ingame, CA, U.S.A.) and examined under a fluorescence and in the retina with KA-induced injury of ICR mice. Fur- microscope (IX71, Olympus, Tokyo, Japan). thermore, the expression of these SLRPs was exam- Antibodies: The antibodies used in this study were pur- ined in the degenerated retina. chased from different companies; polyclonal anti-biglycan and polyclonal anti-fibromodulin antibodies from Santa MATERIALS AND METHODS Cruz Biotechnology (Santa Cruz, CA, U.S.A.), polyclonal anti-decorin from R&D Systems (Abingdon, U.K.) and All animal care and experimental procedures were polyclonal anti-Pax6 antibody from Covance (San Diego, approved by the Animal Research Committee, Tottori Uni- CA, U.S.A.). Pax6 antibody was used to identify the cells in versity, Japan (Approval number: 09-T-19). the ganglion cell layer (GCL), amacrine cells and horizontal Animals and tissue processing for developmental studies: cells in the inner nuclear layer (INL) of normal retina and ICR mice were purchased from CLEA Japan, Inc. (Tokyo, compared to that of KA-injected retinas. Japan). The animals were maintained in a 12:12 hr light- Immunohistochemistry: Paraffin sections of mouse retina dark cycle with free access to food and water throughout the were deparaffinized, rehydrated, and rinsed with PBS. Indi- experiment. One male mouse was placed to mate with rect immunostaining was performed using a Histofine SAB- female mice overnight and pregnant females were identified PO (G) (for goat primary antibodies; fibromodulin and by the presence of a vaginal plug the next morning. Thus, decorin) or SAB-PO (R) (for rabbit primary antibodies; big- embryonic day zero (E0) was set on the first day the vaginal lycan and Pax6) immunohistochemical staining kit plug was observed. Mouse embryos at embryonic age of 18 (Nichirei, Tokyo, Japan). Endogenous peroxidase activity days (E18, n=8) obtained by cesarean section from these was eliminated with 3% hydrogen peroxide in methanol for pregnant mice were enucleated. Eyes of mice at postnatal 5 min. After washing with PBS, the retinal sections were ages of 7 (P7, n=4), 14 (P14, n=4), and 42 (P42, n=6) days sequentially blocked with normal blocking serum for 25 min were also used in this experiment. The mice were eutha- at room temperature. The sections were then incubated at nized by cervical dislocation, and both eyes in each mouse 4°C overnight with specific primary antibodies (1:50 in PBS were collected and fixed with 4% paraformaldehyde in for fibromodulin and biglycan, 1:300 for Pax6 and 0.15 μg/ phosphate buffered saline (PBS, pH 7.4) overnight at 4°C ml for decorin). Control sections were incubated with a with gentle shaking. Serial sagittal sections of the central buffer solution after treatment with the blocking serum. retina were cut at 4 μm for histological and immunohis- After washing with PBS, biotinylated anti-goat or rabbit tochemical studies. IgG was applied for 30 min at room temperature. After Animal model of KA-induced retinal degeneration: Three washing with PBS, peroxidase-conjugated streptavidin groups of five ICR mice at P14, P42 and P120 were used. solution was applied for 30 min and peroxidase labeling was The mice were anesthetized by intraperitoneal injection of visualized using 0.05% 3-3’diaminobenzidine (Sigma, 1.25% Avertin (2, 2, 2-tribromoethanol in tert-amyl alcohol; Tokyo, Japan). Micrographs of the sections were taken with 17 μl/g body weight; Aldrich, Milwaukee, WI, U.S.A.). a digital camera for a microscope (DP-71, Olympus, Tokyo, Intravitreal injection of KA was performed as previously Japan). described [18]. Throughout this study, intravitreal injec- RNA isolation and reverse transferase-polymerase chain tions were performed in a final volume of 2 μl using a heat- reaction (RT-PCR): Retinas were removed and kept at pulled glass pipette connected to a microsyringe (Microdis- –80°C until use. Total RNA was isolated from the retina by penser; Drummond Scientific Company, Broomall, PA, using Trizol (Invitrogen, Tokyo, Japan). For RT-PCR anal- U.S.A.). In this experiment, right eyes of each stage were ysis, complementary DNA (cDNA) was prepared by Rever- injected with 2 μl of 10 mM corresponding to 20 nmol of TraAce (Toyobo, Osaka, Japan,). The cDNA samples were KA (Tocris Bioscience, Ellisville, MO, U.S.A.) prepared in then used for RT-PCR of decorin, biglycan, fibromodulin PBS. The left eyes were injected with 2 μl of PBS and used and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as controls. Animals were euthanized by cervical disloca- as an internal control. The cDNA was amplified by using tion at 1, 3 and 7 days after injection for morphological and Quick Taq (Toyobo) for 35 cycles in a thermal cycler molecular studies. (Takara, Osaka Japan) according to the manufacturer’s pro- Morphological analysis: Retinal sections were stained tocol. Each cycle consisted of denaturation for 15 sec at with hematoxylin and eosin (HE) at a distance of 1 mm from 94°C, primer annealing for 30 sec at 59°C and extension for the optic disc. HE-stained sections were observed and 1 min at 68°C. The amplified products were electrophore- thickness of the retina was measured using Image J soft- sed in 2.0% (w/v) agarose gels at 100 V for 30 min in TAE ware. To distinguish changes in the nuclei of retinal layers buffer (40 mM Tris–acetate, 1 mM EDTA, pH 8.0). SMALL PROTEOGLYCANS IN MOUSE EYE 441

Table 1. Primers used for RT-PCR Gene Size Primer Sequence (Genbank) (bp) Decorin 5’-GCC TTC CAG GGA CTG AAG AGT-3’ 149 (BC132521) 5’-AGT CCT TTC AGG CTG GGT GC-3’ Biglycan 5’-TGT CAC ACC TAC CTT CAG TG-3’ 171 (L20276) 5’-TTG AAG TCA TCC TTG CGA -3’ Fibromodulin 5’-GCT ACC AAC ACC TTC AAC TC-3’ 149 (BC064779) 5’-GTG CAG AAG CTG CTG ATG-3’ GAPDH 5’-GAG AGG CCC TAT CCC AAC TC-3’ 193 (NM_008084) 5’-GTG GGT GCA GCG AAC TTT AT-3’

Sequences for primers (Sigma Genosys, Hokkaido, Japan) the INL (3–7 days after injection), whereas the outer layers used in this study are listed in Table 1. of the retina remained almost intact for 7 days (Fig. 2). In Statistical analysis: The data were analyzed by ANOVA, the normal retina, Pax6 was distributed diffusely from the followed by Fisher’s protected least significant difference GCL to ONL, and there was prominent staining of cells con- (LSD) test. All values are presented as means ± SD. P<0.05 sistent with ganglion cells located in the GCL, and cells in was considered statistically significant. INL, supposed to be amacrine cells and horizontal cells. With damage of the GCL 1 day and 3 days after KA injec- RESULTS tion, Pax6-immunopostive cells were confined to the INL. At 7 days Pax6 was distributed diffusely throughout the ret- Distributional changes in SLRPs during retinal develop- ina. DAPI staining result showed that progressive loss of ment: At E18 and P7, the GCL and inner plexiform layer cells occurred in the inner retinal layer of KA-injected eyes. (IPL) were distinguishable from other neuroblasts cell mass. Distribution and expression of SLRPs of KA-induced ret- Decorin and fibromodulin were diffusely distributed inal degeneration model: One day after KA injection, the throughout the retina (Fig. 1). The photoreceptor layer distributions of decorin and fibromodulin showed the same (PRL) had formed at P14, and the retina was in a mature pattern as that in the control (normal) retina but with stron- state at P42. Decorin was detected diffusely in the retina ger immunostaining levels, especially in the inner retinal with higher affinity confined to cell consistent with ganglion layers (GCL, IPL and INL). Three days after KA injection, cells in the GCL. Fibromodulin was moderately distributed when progressive loss of inner retinal cells was observed, in the retina with higher affinity confined to astrocytes and strong immunoreactivity for decorin and fibromodulin was ganglion cells in the GCL and cells in the inner side of INL; detected in inner retinal layers, and decorin was also supposed to be amacrine cells, in addition to nerve fiber rich detected diffusely in outer retinal layers. However, in reti- layers; nerve fiber layer and IPL. Biglycan showed a mod- nas at the late stage (7 days after injection), when significant erate distribution throughout the retina at E18 and P7 and loss of inner retinal cells was observed, decorin and fibro- with weak staining at P14 and P42. modulin immunostaining was distributed throughout the ret- Morphological analysis of KA-induced retinal degenera- ina including the PRL (Fig. 3A). Immunostaining for tion model: Intravitreal injection of KA at three stages of ret- biglycan was weak in both the normal retina and the retina inal development (P14, P42 and P120) resulted in after KA injection. In addition to the immunohistochemical progressive damage and shrinkage of the retinas comparing results, all retinas of normal and KA-injected eyes at 1, 3 to normal ones. The damage was confined to the inner ret- and 7 days after injection expressed decorin and fibromodu- ina. Moreover, these retinas were detached from the retinal lin mRNA throughout the experimental periods. However, pigment epithelium and there were deformities in retinal the expression level of biglycan mRNA was very low in ret- shape in many parts resulting in a tortuous shape. The inas of both normal and KA-injected eyes (Fig. 3B). results obtained from morphological studies of HE samples from these stages revealed that the effects of KA on retinas DISCUSSION at P14, P42 and P120 were almost similar (Table 2). Hence, we used the P42 mouse as a model of KA-induced injury to PGs play pivotal roles in various cellular processes such the retina and we evaluated the effects on the expression and as proliferation, migration, cell adhesion, and survival [10]. distribution of SLRPs. Their influence on neurite extension [25, 31, 38] makes The inner retinal layers were seriously damaged in the them potential candidates for interactions with growing experimental eyes after KA injection. The time course of axons during development and after injury of the mamma- morphological changes in the retinas showed that ganglion lian central nervous system. PGs have been shown to play cell damage occurred at an early stage (1 day after injection) an important role in neurite outgrowth of retinal ganglion followed by shrinkage of the IPL and loss of retinal cells in cells [3, 32]. Moreover, altered expression of PGs has been 442 S. A. M. ALI, Y. Z. HOSAKA AND M. UEHARA reported in retinal degenerative diseases [9, 24, 27]. Thus, tions in an attempt to elucidate their roles. in the present study, we investigated the expression of PGs, Our results showed that decorin and fibromodulin are dis- especially SLRPs, in developmental and pathological situa- tributed diffusely in the retina at the prenatal stage. The dis- tribution of decorin and fibromodulin becomes more obvious in early postnatal to adult stages. Astrocytes main- tain the extracellular matrix in the lamina cribrosa consist- ing of , elastic fibers and such as laminin and PGs. Astrocytes express a wide variety of growth factors and receptors, many of which serve as trophic and survival factors [37]. It is suggested that astro- cytes serve the same function in the retina. A previous study showed that PGs can be neurotrophic factors for retinal ganglion cells [30], suggesting that deco- rin, a chondroitin/dermatan sulfate PG, may be one of the neurotrophic factors surrounding retinal ganglion cells and their neurite. Therefore, the distribution pattern of decorin and fibromodulin in the retina suggests that these PGs are related to the differentiation of retinal cells in addition to their survival. It was reported that SLRP-deficient mice have a compensatory deposition of other SLRPs from the respective SLRP class, i.e., more biglycan in cartilage and in decorin-deficient mice [41]. In this study, biglycan was weakly distributed in the mouse retina compared with the distribution of decorin. This finding suggests that a decorin-biglycan compensation system is also present in the retina. The adult retina of KA-injected mice underwent shrink- age confined mainly to the inner retina, GCL and INL. It has been suggested that KA-induced shrinkage in the retina is accompanied by loss of the GCL and IPL [11]. Previous studies showed that cultured retinal ganglion cells (RGCs) were highly sensitive to glutamate-induced excitotoxicity. However, in the presence of neurotrophic factors, RCG were resistant to glutamate excitotoxicity, whereas retinal amacrine cells were highly sensitive to elevated glutamate levels [36, 42]. These reports suggested that the decrease in the number of cells observed in the GCL after intravitreal glutamate injections might have resulted from a loss of ama- crine cells and from a lack of trophic support for RGCs. KA induces activation of retinal astrocytes and Müller cells, which reflects a metabolic change of the cells in response to Fig. 1. HE staining and immunostaining of decorin, biglycan and degenerative changes of their neighboring neurons, which fibromodulin in the retinas of mice at E18, P7, P14, and P42. HE, hematoxylin and eosin; Dcn, decorin; Bgn, biglycan; Fmod, may give evidence of repair process in KA-induced damage fibromodulin; GCL&NFL, ganglion cell layer and nerve fiber [5]. Previous studies have shown that Pax6 mRNA was layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, expressed during development of the mouse retina and in outer plexiform layer; ONL, outer nuclear layer; NB, neuroblast the adult mouse retina [21] and was upregulated during reti- cell mass; PRL, photoreceptor layer. Scale bar=50 m. nal regeneration [13]. In the present study, Pax6 protein in

Table 2. Retinal thickness after KA injection (m) Day 0 Day 1 Day 3 Day 7

P14 871.3  8.2a) 708.0  18.5b) 645.3  5.9c) 517.3  31.2d) P42 884.7  34.5a) 646.0  21.9b) 590.7  9.8b) 483.3  14.5c)

P120 805.7  6.2a) 560.7  5.2b) 504.3  17.1c) 413.3  10.7d) Values represent mean SD. Different letters (a–d) indicate significant difference among the same age groups: P<0.05. SMALL PROTEOGLYCANS IN MOUSE EYE 443

Fig. 2. HE staining, Pax6-immunostaining and DAPI staining of P42 mice retinas in the kainic acid (KA)- injured model. HE, hematoxylin and eosin; N, control; Fig. 3. Immunostaining (A) and RT-PCR (B) results for decorin 1D, 3D and 7D: 1, 3 and 7 days after KA injection, (Dcn), biglycan (Bgn) and fibromodulin (Fmod) in the P42 mice respectively; L: lens; arrows: interface between vitreous retinas of kainic acid (KA)-injected model. N, control; 1D, 3D body and retina. Scale bar=50 m. and 7D: 1, 3 and 7 days after KA injection, respectively; L: lens. Scale bar=50 m. the retinas of KA-injected eyes showed lower density than that in retinas of normal eyes, suggesting weak or no regen- decreased in response to ischemia in a rat model of oxygen- eration up to 7 days post-injection. induced retinopathy [26]. Moreover, in transient retinal Decorin is known to be involved in the development of ischemia, expression level of decorin mRNA decreased in certain eye structures, namely the cornea and sclera, by the early stage (6–28 hr) and recovered to near-normal lev- modulating collagen fibrogenesis. Decorin expression els in the late stage at 4 days to 1 week [12]. Taken together, 444 S. A. M. ALI, Y. Z. HOSAKA AND M. UEHARA these findings suggest that decreased decorin reflects cellu- microvilli. Mol. Cell Proteomics 3: 1119–1127. lar damage and death and that recovery of the level of deco- 3. Brittis, P. A. and Silver, J. 1995. Multiple factors govern intr- rin may occur in response to a regenerative process and/or aretinal axon guidance: a time-lapse study. Mol. Cell Neurosci. the secretion of numerous cytokines by inflammatory cells 6: 413–432. and activated retinal glial cells. 4. Chakravarti, S., Paul, J., Roberts, L., Chervoneva, I., Oldberg, A. and Birk, D. E. 2003. 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