December 2012
Volume 53, Issue 13
Free
Retina  |   December 2012
Crystallin-β-b2-Overexpressing NPCs Support the Survival of Injured Retinal Ganglion Cells and Photoreceptors in Rats
Author Affiliations & Notes
  • Michael R. R. Böhm
    From the Institute for Experimental Ophthalmology, School of Medicine, Westfalian-Wilhelms-University Münster, Münster, Germany; and the
    Interdisciplinary Centre for Clinical Research (IZKF), School of Medicine, Westfalian-Wilhelms-University Münster, Münster, Germany.
  • Sarah Pfrommer
    From the Institute for Experimental Ophthalmology, School of Medicine, Westfalian-Wilhelms-University Münster, Münster, Germany; and the
  • Carolin Chiwitt
    From the Institute for Experimental Ophthalmology, School of Medicine, Westfalian-Wilhelms-University Münster, Münster, Germany; and the
  • Matthias Brückner
    From the Institute for Experimental Ophthalmology, School of Medicine, Westfalian-Wilhelms-University Münster, Münster, Germany; and the
  • Harutyun Melkonyan
    From the Institute for Experimental Ophthalmology, School of Medicine, Westfalian-Wilhelms-University Münster, Münster, Germany; and the
  • Solon Thanos
    From the Institute for Experimental Ophthalmology, School of Medicine, Westfalian-Wilhelms-University Münster, Münster, Germany; and the
    Interdisciplinary Centre for Clinical Research (IZKF), School of Medicine, Westfalian-Wilhelms-University Münster, Münster, Germany.
  • Corresponding author: Michael R. R. Böhm, Institute of Experimental Ophthalmology, School of Medicine, Westfalian-Wilhelms-University Münster, Albert-Schweitzer-Campus 1, D15, 48149 Münster, Germany; Michael.boehm@ukmuenster.de
Investigative Ophthalmology & Visual Science December 2012, Vol.53, 8265-8279. doi:10.1167/iovs.12-10334
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      Michael R. R. Böhm, Sarah Pfrommer, Carolin Chiwitt, Matthias Brückner, Harutyun Melkonyan, Solon Thanos; Crystallin-β-b2-Overexpressing NPCs Support the Survival of Injured Retinal Ganglion Cells and Photoreceptors in Rats. Invest. Ophthalmol. Vis. Sci. 2012;53(13):8265-8279. doi: 10.1167/iovs.12-10334.

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

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Abstract

Purpose.: Crystallin β-b2 (crybb2) is known to support the regeneration of retinal ganglion cell (RGC) axons in culture. We investigated whether neuronal progenitor cells (NPCs) overexpressing crybb2 (crybb2-NPC) affect secondary retinal degeneration due to optic nerve crush in vivo.

Methods.: NPCs were produced by dissociation and propagation of rat embryonic neural tube and eye primordial cells at embryonic days 13.5 and 15. Retinal degeneration was induced by injured optic nerve crush (by suture, 20 seconds). Several groups were built: crybb2-NPC were injected into the vitreous body, while the Controls were comprised of recombinant crybb2–injected and PBS-injected groups. The eyes, in particular the retina, were analyzed by immunohistochemistry and Western blotting for different antigens at 2 and 4 weeks after surgery.

Results.: At 2 and 4 weeks post surgery, crybb2-NPC resided within the vitreoretinal compartment, and were persistently nestin-positive throughout the experimental period. The cells stained positive for various neurotrophins and acted as “living” cell factories to support the survival of injured RGCs. The crybb2-NPC migrated throughout the eye structures and sometimes became integrated within the tissue. Most of the ocular cells responded to the appearance of crybb2-NPC with marked changes of certain proteins, including Iba-1 (microglia), vimentin (glial cells), and rhodopsin (photoreceptors). Photoreceptors also displayed a better survival after crybb2-NPC injection compared to control groups.

Conclusions.: Crybb2-NPC exert beneficial effects on the vitreoretinal compartment, which suggests that modified crybb2-NPC could be used in a novel strategy for the treatment of degenerative vitreoretinal diseases. However, future studies must determine the safety of in vivo administration of crybb2-NPC.

Introduction
Retinal degenerative diseases affect millions of people worldwide 1 and, hence, methods must be developed to prevent such degeneration and the consequent visual impairment. One of these strategies could involve placing cells inside the eye that produce protective substances. 
Neural stem cells and neural progenitor cells (NPCs) have been isolated from different parts of the embryonic and adult central nervous system (CNS) of several mammalian species. 25 NPCs secrete several neurotrophic factors (NTFs) and exert protective effects on diseased neurons. 68 NPCs can be manipulated through pretreatment with growth factors or via gene transfection. 1,2,5,9 Attempts to replace lost photoreceptors so far has focused on injecting stem cells into the retina or the subretinal space. Although some of the injected cells differentiate into photoreceptors and integrate into the host tissue, the quantity of cells involved generally is low, and in most cases they fail to function. 2,1012 Alternative approaches include the modification of NPCs to rescue visual function through the engineered expression of NTFs, such as glial cell–derived NTF (GDNF)-producing human-derived NPCs. These were shown to have a positive effect after being injected into the degenerating retinas of Royal College of Surgeons (RCS) rats. 1  
In addition to classical NTFs and differentiation molecules, ocular crystallins appear to have a crucial role in the development of retinal cells, including photoreceptors. 1315 Crystallins, which also are expressed in retinal neurons, 16 have been identified in various neurodegenerative disorders, including in those affecting the retina. 17 While several studies have demonstrated that crystallin α has a role in retinal disorders, there is little information on the effects of crystallin β/γ. 1821 Adult retinal ganglion cells (RGCs) have an enhanced chance of survival when exposed to lens injury. 16,22,23 Testing of different classes of crystallins has revealed that crystallins of the β/γ superfamily possess a strong neuroprotective and regenerative potential by mimicking the effects of lens injury. 16  
Crystallins β and γ (cryg) are expressed during the development of the mouse eye. 14 Increased levels of cryg have been observed in cultured astrocytes affected by hypoxia, 23 neuronal aging processes, 24 and neuronal repair. 25 Added crystallin β-b2 (crybb2) enhances the growth of axons in retinal explants and in primary hippocampal neurons. 26 However, to our knowledge no protective effects of crybb2 on RGCs in vivo or on photoreceptors have yet been reported. 
To investigate neuroprotective features of NPCs, we used the optic nerve crush model to induce retinal degeneration and injected transfected NPCs, overexpressing crybb2 intravitreally after surgery. In controls, NPCs were replaced with intravitreal injection of recombinant crybb2 protein or injection of PBS. The aims of our study were to elucidate the survival and migration of NPCs within the eye, the intravitreal production of NTFs and crystallins, and the effects of overexpressed crybb2 on degenerating RGCs and photoreceptors. Therefore, immunohistochemistry and Western blotting (WB) were used to examine intravitreal distribution of NPCs and production of NTFs that may affect the fate of photoreceptors, glial cells, and microglia. 
Methods
Animals and Drugs
Pregnant Sprague-Dawley (SD) rats at embryonic days (E)13.5 and 15 (n = 52) were used as a source of NPCs for these studies. Adult SD rats at approximately 3 months of age (n = 64) of both sexes were used for the surgery. All experiments were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Animals were housed in standard animal rooms under a 12-hour light/dark cycle, with food and water provided ad libitum. All surgical experiments were performed under general anesthesia induced by a mixture of 10% ketamine (2 mg/kg per body weight; Ceva Sanofi, Düsseldorf, Germany) and xylazine (1 mg/kg per body weight; Ceva-Sanofi), administered intraperitoneally. After surgical intervention, gentamicin (Dr. Mann Pharma, Berlin, Germany) was applied topically to prevent infections. All surgical manipulations were unilateral, with the contralateral eye serving as a control. Experimental and control groups consisting of 16 animals are shown in Table 1
Table 1. 
 
Experimental Groups (In Vivo)
Table 1. 
 
Experimental Groups (In Vivo)
Experimental Group N Weights of SD Rats Experimental Manipulation Times, wk
Effects of crybb2 16 180 to 250 g Left ONC with intravitreal injection of crybb2 2, 4
IHC 4
WB 4
Effects of crybb2-NPC 16 180 to 250 g Left ONC with intravitreal injection of crybb2-NPC 2, 4
IHC 4
WB 4
Effects of PBS (control) 16 180 to 250 g Left ONC with intravitreal injection of PBS 2, 4
IHC 4
WB 4
Adult control 16 180 to 250 g Untreated 2, 4
IHC 4
WB 4
Preparation and Transfection of NPCs
To retrieve and culture NPCs, embryos were removed under deep anesthesia by cesarean section at days E13.5 and E15, and were stored in sterile Hank's balanced salt solution (HBSS; PAA Laboratories, Pasching, Germany) with 1% PenStrep (Sigma-Aldrich, Hamburg, Germany), on ice until dissection. The brains were removed and homogenized mechanically. The homogenate then was treated with 0.1% trypsin in HBSS to obtain single cells. The suspension was transferred into a culture medium comprising Dulbecco's modified Eagle's medium–Ham's (DMEM-F12; PAA Laboratories) supplemented with 1% PenStrep, 1% N2 Supplement (PAA Laboratories), 10 ng/μL recombinant human basic fibroblast growth factor (bFGF; RD Systems, Minneapolis, MN), and 10 μL insulin (2.5 mg/mL; PromoCell, Heidelberg, Germany). The suspension was triturated gently and 4.5 × 106 cells were plated at a density of 200,000 cells/mL on noncoated Advanced TC Petri dishes (Greiner Bio-One, Frickenhausen, Germany) to inhibit further differentiation. The cells were incubated at 37°C in a humidified atmosphere of 5% CO2. All cell harvesting steps were performed under sterile conditions. 
Rat crybb2 was cloned into pIRES-ACGFP (Clontech, Palo Alto, CA) so that rat crybb2 and green fluorescent protein (GFP) could be translated from a single bicistronic mRNA. For transfection of NPCs, cultured cells that formed neurospheres were dissociated mechanically into single cells and centrifuged for 5 minutes at 400 rpm (4°C). The culture medium was removed and approximately 5 × 106 cells were resuspended in 100 μL Amaxa Rat NSC Nucleofector Kit (Lonza Cologne, Cologne, Germany). Vector (3.5 μg DNA) was added, the cells and DNA were mixed gently, and then the cells were electroporated using the Nucleofector device (Lonza Cologne). The transfection protocol used was very similar to that described by the manufacturer, thus resulting in the highest transfection efficacies. 5,27 Cells were used for a maximum of 2 weeks after preparation. After finishing this procedure, crybb2-overexpressing NPCs (crybb2-NPC) were placed into the culture medium and cultured for up to 7 days, and the efficacy of transfection was monitored using GFP fluorescence. 
Transfected crybb2-NPC were prepared for injection by first washing the cell suspensions with HBSS. Cell numbers then were counted using the trypan-blue exclusion method, and then the cells were transferred to the transplantation medium, HBSS. Surgery and cell injection were performed within 1 hour of cell preparation. 
Unilateral Optic Nerve Crush (ONC) and Intravitreal Treatment
To generate the primary degeneration of RGCs and a photoreceptor transsynaptic response, we performed unilateral ONC to induce damage of axons followed by the intravitreal injection of crybb2-NPC. After a skin incision near the superior orbital rim, ONC was performed according to the “mini lesion” technique, which involves ligation with a suture (8.0 Ethicon; Johnson & Johnson, St-Stevens-Woluwe, Belgium) for exactly 20 seconds. 26 A 10 μl volume of crybb2-NPC was injected through the sclera via a glass capillary, with care being taken to avoid damage to the lens. To reveal the effects administered by intravitreal injected crybb2-NPC, several groups in the experimental setup were built. In the controls, 10 μL of recombinant crybb2 protein (180 μg/μL) were injected intravitreally to reveal the effects of recombinant crybb2 protein compared to crybb2-NPC due to retinal degeneration. After subcloning into pQE32 (Qiagen, Hidden, Germany), rat crybb2 was expressed and purified: bacterial cultures were centrifuged (1500g for 20 minutes) and subsequently resuspended in buffer 1 (8 M urea, 50 mM NaH2PO4, 15 mM imidazole, 10 mM Tris-HCl, and 100 mM NaCl; pH 8.0). After cloning, cell lysates were centrifuged at 20,000g for 30 minutes. The supernatant was purified using immobilized metal affinity chromatography (Clontech); elution was performed using buffer 1 at pH 6.0 supplemented with 100 mM imidazole. The proteins were dialyzed in aqua dest. For control operations (see above), 10 μL sterile PBS were injected after ONC instead of crybb2-NPC to exclude beneficial effects of the method of intravitreal injection due to retinal degeneration. The experimental protocols are listed in Table 1
Immunohistochemistry
Cultured NPCs were examined by immunohistochemistry for selected proteins: oncomodulin, brain-derived NTF (BDNF), calcineurin, and cryg and crybb2. Cultures were fixed in fresh 4% paraformaldehyde for 10 minutes at room temperature (RT), washed in PBS, and then fixed with methanol (10 minutes at −20°C). After rinsing the cells with PBS, they were incubated with blocking solution containing 10% goat serum (2 hours at RT); the primary antibodies then were applied (Table 1). After washing, cells were incubated with secondary antibodies (1 hour at RT, Table 1) and then washed with PBS. Antifade mounting medium was used to cover the cells (Mowiol; Hoechst, Frankfurt, Germany). 
The expression, intraocular localization, and effects of intravitreally injected crybb2-NPC were investigated by sacrificing operated rats using CO2 inhalation (n = 4 from each group). The eyeballs were explanted, frozen in Tissue-Tek (Sakura Finetek Europe, Alphen aan den Rijn, The Netherlands), and then stored at −80°C until further use. Sections were cut along the visual axis with a cryostat (10 μm thick, CM 1500; Leica, Bremen, Germany) and mounted onto glass slides. The same primary antibodies used to detect NTFs were used in vivo (see above). In addition, retinal cells were stained for specific antigens to identify potential changes caused by the NPCs. Photoreceptors were detected with rhodopsin, while glial cells were visualized with glial fibrillary acidic protein (GFAP) and antivimentin antibodies. The quantitative analysis of the retinal ganglion cell axons was performed as follows: retinal slices (10 μm) were labeled with antibodies against NF-200 revealing the axonal cytoskeleton of retinal ganglion cells. 28,29 Five microscope fields (each 0.2 mm2) of each sample were adjusted and NF-labeled axons were counted. The mean count of axons was calculated and expressed as a percentage of retinal ganglion cells compared to the number of NF-stained axons in the untreated contralateral retina. Because each ganglion cell is emitting one axon, the axon numbers are related to the ganglion cell numbers. 
ED-1 was used to identify microglial cells in different layers of the retina, while the ionized calcium-binding adaptor molecule 1 was used to identify activated microglia. 
The procedure used to stain cell nuclei with 4′,6-diamidino-2-phenylindole is described above. The slides were viewed with the appropriate filter on a microscope equipped with epifluorescence (Axiophot; Carl Zeiss Meditec, Oberkochen, Germany). Negative controls comprised sections processed without the addition of the primary antibodies. Control and experimental sections were stained simultaneously to avoid variations in immunohistochemical staining. Table 2 lists the antibodies used, including their working dilutions. All stained cells were counted within a region of 0.2 mm2 (five randomized regions per eye) at a magnification of ×400. 
Table 2. 
 
Antibodies Used for IHC
Table 2. 
 
Antibodies Used for IHC
Antibody Company Isotype Dilution Tissue
Primary antibodies
 BDNF Millipore (Temecula, CA) Rabbit polyclonal 1:500 NPCs in vitro/in vivo
 Calcineurin Millipore Rabbit polyclonal 1:500 NPCs in vitro/in vivo
 Crybb2 Santa Cruz Biotechnology (Santa Cruz, CA) Goat polyclonal 1:100 NPCs in vitro/in vivo
 Cryg Santa Cruz Biotechnology Rabbit polyclonal 1:100 NPCs in vitro/in vivo
 ED-1 BMA Biomedicals (Augst, Switzerland) Mouse monoclonal 1:200 Microglia
 Iba-1 Wako Pure Chemical Industries (Osaka, Japan) Rabbit 1:500 Microglia
 Nestin Millipore Rabbit polyclonal 1:200 NPCs in vitro/in vivo
 Nestin AbD Serotoec (Puchheim, Germany) Mouse monoclonal 1:100 NPCs in vitro/in vivo
 Neurofilament 200 Sigma-Aldrich (St. Louis, MO) Mouse monoclonal 1:400 Retinal ganglion cells
 Oncomodulin Swant (Marly, Switzerland) Rabbit polyclonal 1:500 NPCs in vitro/in vivo
 Rhodopsin Millipore Mouse monoclonal 1:100 Photoreceptors
 Vimentin Sigma-Aldrich Mouse monoclonal 1:200 Retinal glial cells
Secondary antibodies
 Cy-2 Jackson ImmunoResearch Europe Ltd. (Suffolk, UK) Goat antimouse 1:200
 Cy-2 Jackson ImmunoResearch Europe Ltd. Goat antirabbit 1:200
 Cy-2 Abcam (Cambridge, UK) Donkey Antigoat 1:100
 TRITC Sigma-Aldrich Goat antirabbit 1:400
 TRITC Sigma-Aldrich Goat antimouse 1:300
 TRITC Jackson ImmunoResearch Europe Ltd. Donkey antigoat 1:100
Western Blotting
WB was performed to confirm the immunohistochemical findings and evaluate retinal degeneration. Eyes taken from four animals of each group were enucleated. The extraocular tissues were dissected, the retina was discharged carefully from the sclera, and the vitreous was peeled off. All isolated samples were frozen in liquid nitrogen and kept at −80°C until further use. 
Samples were homogenized in SDS sample buffer (62.5 mM Tris-HCl, 2% wt/vol SDS, 10% glycerol, 50 mM dithiothreitol, and 0.01% wt/vol bromophenol blue). The samples were sonicated and heated, and then the protein concentration was determined using the Bradford method. 
A total of 50 μg protein from each sample was fractionated on 8%, 10%, or 12% SDS-polyacrylamide gel electrophoresis (depending on the protein) with a protein marker (BioRad, Hercules, CA). After electrophoresis, the proteins were transferred onto a nitrocellulose membrane (Schleicher & Schüll BioScience, Dassel, Germany). The blots were incubated in blocking solution (5% freshly prepared fat-free dried milk and 0.1% Tween-20 PBS) for 1 hour, followed by incubation overnight at 4°C with a variety of antibodies (Table 3). The membrane then was incubated with the secondary antibody conjugated with horseradish peroxidase in blocking solution for 1 hour at RT. Antibodies were detected with enhanced chemoluminescence (Amersham, Rockville, UK) and documented on X-ray films (Amersham Pharmacia Biotech, Amersham, UK). Loading of comparable amounts of protein was confirmed with anticalnexin (see below). Each gel was run three times. The relative densities of the protein spots were analyzed using Alpha Ease (Alpha Ease FC software 4.0; Alpha Innotech, San Leandro, CA). The protein density of a fixed area was determined for each spot by subtracting the specific background density of the particular application control. The lowest spot density for each of the different proteins was defined as the reference mark, and the relative values were determined and processed. The mean and standard deviation of the relative relationships of the proteins were generated from gels of four individual samples, each one run three times for the individual group. 
Table 3. 
 
Antibodies Used for WB
Table 3. 
 
Antibodies Used for WB
Antibody Company Isotype Dilution Molecular Mass
Primary antibodies
 BRN-3b Covance (Princeton, NJ) Rabbit polyclonal 1:2,000 45 kD
 Rhodopsin Millipore Mouse monoclonal 1:1,000 39 kD
 Vimentin Sigma-Aldrich Mouse monoclonal 1:500 58 kD
Secondary antibodies
 Secondary rhodopsin Cell Signaling Technology, Inc. (Danvers, MA) Horse antimouse 1:4,000
 Secondary vimentin Cell Signaling Technology, Inc. Horse antimouse 1:4,000
 Secondary BRN-3b Amersham Pharmacia Biotech Donkey antirabbit 1:10,000
Loading control
 Calnexin Sigma-Aldrich Rabbit 1:10,000 90 kD
Data Evaluation
The standard deviation and optical density of the WB analysis and several cell counts were analyzed with a test for two independent samples (SPPS; SSPS, Chicago, IL; Statistica ver. 7; StatSoft, Tulsa, OK) to examine the Gaussian distribution, and processed using the independent-samples t-test (Gaussian) and Kruskal-Wallis H test. Non-Gaussian values were prepared with image-processing software (Photoshop; Adobe Systems, San Jose, CA). The overall brightness and contrast were adjusted without retouching. 
Results
Morphologic Characterization and Factor Expression of crybb2-NPC In Vitro
As expected, NPCs harvested from embryonic brain and eye primordial tissue exhibited various morphologies in culture. Single cells were found along with clusters and neurospheres of NPCs (Figs. 1A, 1B). These stem cells possessed long protrusions with a shaped cell body (Fig. 1B). NPCs stained positive for several NTFs, including BDNF (Figs. 1C–E), oncomodulin (Figs. 1F–H), calcineurin (Figs. 1I–K), crybb2 (Figs. 1L–N), and cryg (Figs. 1O–Q). Since crybb2 is known to be associated with regenerative retinal functions in vitro, 26 it was selected as the first-choice protein for analysis in vivo. We observed a transfection rate of 55 to 67%, which is comparable to previous reports. 5  
Figure 1. 
 
Morphologic and immunohistochemical characterization of NPCs derived from cultured rat fetal neural tissue. (A, B) Cultured NPCs were found in cell clusters, called neurospheres (arrows). (B) Single cells with morphologic features reminiscent of stem cells also were found (arrowheads). (CQ) Immunohistochemical characterization of cultured NPCs (using nestin, green) and several other markers (red) in vitro. Labeling with antibodies raised against BDNF (CE), oncomodulin (FH), calcineurin (IK), crybb2 (LN), and cryg (OQ) revealed that these molecules were expressed in NPCs in vitro. 4′,6-Diamidino-2-phenylindole (DAPI, blue) was used to stain cell nuclei. Scale bars: 20 μm (A, B, E, H, K, N, Q).
Figure 1. 
 
Morphologic and immunohistochemical characterization of NPCs derived from cultured rat fetal neural tissue. (A, B) Cultured NPCs were found in cell clusters, called neurospheres (arrows). (B) Single cells with morphologic features reminiscent of stem cells also were found (arrowheads). (CQ) Immunohistochemical characterization of cultured NPCs (using nestin, green) and several other markers (red) in vitro. Labeling with antibodies raised against BDNF (CE), oncomodulin (FH), calcineurin (IK), crybb2 (LN), and cryg (OQ) revealed that these molecules were expressed in NPCs in vitro. 4′,6-Diamidino-2-phenylindole (DAPI, blue) was used to stain cell nuclei. Scale bars: 20 μm (A, B, E, H, K, N, Q).
NPCs within the Eye
Immunohistochemistry was used to examine whether the injected crybb2-NPC continued expression of the same NTFs within the eye. It appeared that all proteins still were produced within the vitreous body (Figs. 2A–O). Particular attention was paid to crybb2 that was stained within clusters of cells located within the vitreous (Figs. 2J–L). These data supported the view that non-transfected NPCs produce baseline levels of crybb2. Compared to crybb2-positive RGCs in the PBS group (Fig. 3A), crybb2-NPC–treated eyes exhibited increased and diffuse staining of RGCs after 2 weeks (Fig. 3C). No crybb2 staining was observed 4 weeks after ONC (Figs. 3B, 3D). When the immunohistochemistry was performed with sections from crybb2-NPC–treated eyes, most cells stained positive for crybb2 (Fig. 3E). 
Figure 2. 
 
(AO) Immunohistochemical characterization of intravitreally transplanted NPCs 2 weeks after propagation in culture. The same antibodies were used for BNDF (AC), oncomodulin (DF), calcineurin (GI), crybb2 (JL), and cryg (MO). DAPI (blue) was used to stain the cell nuclei. Scale bar: 50 μm (C, F, I, L, O).
Figure 2. 
 
(AO) Immunohistochemical characterization of intravitreally transplanted NPCs 2 weeks after propagation in culture. The same antibodies were used for BNDF (AC), oncomodulin (DF), calcineurin (GI), crybb2 (JL), and cryg (MO). DAPI (blue) was used to stain the cell nuclei. Scale bar: 50 μm (C, F, I, L, O).
Figure 3. 
 
(AF) Expression of retinal crybb2 (red) shown by immunohistochemical staining of slices (10 μm) of samples from animals treated with PBS (A, B), and crybb2 (C, D) after 2 and 4 weeks. (E) Expression of crybb2 by intravitreally implanted crybb2-NPC after 2 weeks. Cell nuclei are marked blue with DAPI. (F) The negative control is shown with cyanine (Cy-3) as a secondary antibody. Scale bar: 20 μm (AF).
Figure 3. 
 
(AF) Expression of retinal crybb2 (red) shown by immunohistochemical staining of slices (10 μm) of samples from animals treated with PBS (A, B), and crybb2 (C, D) after 2 and 4 weeks. (E) Expression of crybb2 by intravitreally implanted crybb2-NPC after 2 weeks. Cell nuclei are marked blue with DAPI. (F) The negative control is shown with cyanine (Cy-3) as a secondary antibody. Scale bar: 20 μm (AF).
Intraocular Localization of Intravitreally Injected crybb2-NPC
Nestin was used as a stem-cell marker to identify the distribution of crybb2-NPC at 2 and 4 weeks after intravitreal transplantation. NPCs were observed as follows: arrested crybb2-NPC in the vitreous cavity, arrested and migrating crybb2-NPC in the vitreoretinal compartment, and arrested and migrating crybb2-NPC in the anterior segment, including the anterior chamber, cornea, and ciliary body (Table 4). 
Table 4. 
 
Localization of Grafted NPCs after Transplantation
Table 4. 
 
Localization of Grafted NPCs after Transplantation
Localization NPC – 2 weeks NPC – 4 weeks
Cornea X
AC X X
Ciliary body X
Vitreous X X
NFL
GCL X X
OPL X
ONL X X
IPL X
INL X X
RPE
Choroidea X
Arrested nestin-positive cells were localized in the vitreal cavity, at the vitreoretinal interface (Figs. 4G–I), in most retinal layers (Figs. 4A–C), and in the subretinal space, including the choroidea (Figs. 4D–F) 2 weeks after transplantation. They also were found in the anterior chamber, associated with the corneal endothelium (Figs. 4J–L) and epithelium of the ciliary body (Figs. 4M–O) 4 weeks after transplantation. In the host retina, NPCs were detected in the ganglion cell layer (GCL), inner nuclear layer (INL), and outer nuclear layer (ONL). These observations show that NPCs are redistributed within the eye during the first 2 weeks after injection. The distribution of NPCs was similar at 4 weeks post surgery (Fig. 4), with some NPCs having reached quite distant locations, such as the cornea (Figs. 4J–L). These data supported the view that NPCs can migrate within the eye. 
Figure 4. 
 
(AO) Staining for nestin-expressing NPCs (green) in sections throughout the eye. Two weeks after transplantation, grafted NPCs were found throughout the retina (e.g., IPL) (AC), including the subretinal space (DF) and the vitreoretinal compartment (GI). After 4 weeks, nestin-positive cells were detectable in the anterior chamber and corneal endothelium (JL) and the epithelium of the ciliary body (MO). NPCs were found only in the GCL, OPL, and IPL, and not in the other retinal layers after 4 weeks (data not shown). DAPI (blue) was used to stain the cell nuclei (P, R). The negative control is shown with Cy-2 as a secondary antibody. Arrows indicate nestin-positive NPCs in all images. Scale bar: 20 μm (C, F, I, L, O, R).
Figure 4. 
 
(AO) Staining for nestin-expressing NPCs (green) in sections throughout the eye. Two weeks after transplantation, grafted NPCs were found throughout the retina (e.g., IPL) (AC), including the subretinal space (DF) and the vitreoretinal compartment (GI). After 4 weeks, nestin-positive cells were detectable in the anterior chamber and corneal endothelium (JL) and the epithelium of the ciliary body (MO). NPCs were found only in the GCL, OPL, and IPL, and not in the other retinal layers after 4 weeks (data not shown). DAPI (blue) was used to stain the cell nuclei (P, R). The negative control is shown with Cy-2 as a secondary antibody. Arrows indicate nestin-positive NPCs in all images. Scale bar: 20 μm (C, F, I, L, O, R).
Staining of Ocular Tissue after Injection of crybb2-NPC
The marker neurofilament 200 was used to examine the response of RGCs to damage and to the intravitreal injection of NPCs (Figs. 5A–D). A moderate loss of RGCs was found in the crybb2-NPC group compared to the PBS group. Compared to the adult control, the population of RGCs had reduced to 82.63 ± 5.60% in the crybb2-NPC group and to 55.56 ± 8.56% in the PBS group 2 weeks after implantation. After 4 weeks, these values were 52.78 ± 5.73% and 18.75 ± 3.99%, respectively. In the crybb2 group, the population of RGCs had reduced to 58.30 ± 2.41% after 2 weeks and 55.56 ± 3.99% after 4 weeks (Fig. 5E). After 4 weeks the population of RGCs was significantly higher in the crybb2 group than in the PBS group. These percentages were confirmed by WB analysis using antibodies detecting brain-specific homeobox/POU domain protein 3b (Fig. 5F). 30 These data showed that crybb2-producing NPCs support the survival of injured RGCs. 
Figure 5. 
 
(AF) Effects of crybb2 and crybb2-NPC on RGCs in the retina. Expression of neurofilament 200 (NF-200, red) shown by immunohistochemical staining of slices (10 μm) of samples from animals treated with PBS (A, B) and crybb2-NPC (C, D) after 2 and 4 weeks. Cell nuclei are marked blue with DAPI. Cy-3 was used for negative control as a secondary antibody (data not shown). (E) Number of persistent RGCs (per 0.2 mm2) in retinal slices (10 μm) in relation to that of adult control tissue (in %). The number of RGCs decreased in the PBS group (P < 0.001) and crybb2 group (P < 0.01) compared to crybb2-NPC–treated animals after 2 weeks. Decreased numbers of RGCs were observed in the PBS group compared to the crybb2 (P < 0.001) and the crybb2-NPC (P < 0.001) groups after 4 weeks. (F) WB analyses of retinas. Lysates of retinas treated as described were prepared and tested for brain-specific homeobox/POU domain protein 3b (BRN-3b, 45 kiloDaltons). Calnexin was used as the loading protein control. Less protein was detected in the PBS group than in the crybb2 and crybb2-NPC groups after 2 and 4 weeks. Scale bar: 20 μm (AD). Statistical differences: **P < 0.01, ***P < 0.001.
Figure 5. 
 
(AF) Effects of crybb2 and crybb2-NPC on RGCs in the retina. Expression of neurofilament 200 (NF-200, red) shown by immunohistochemical staining of slices (10 μm) of samples from animals treated with PBS (A, B) and crybb2-NPC (C, D) after 2 and 4 weeks. Cell nuclei are marked blue with DAPI. Cy-3 was used for negative control as a secondary antibody (data not shown). (E) Number of persistent RGCs (per 0.2 mm2) in retinal slices (10 μm) in relation to that of adult control tissue (in %). The number of RGCs decreased in the PBS group (P < 0.001) and crybb2 group (P < 0.01) compared to crybb2-NPC–treated animals after 2 weeks. Decreased numbers of RGCs were observed in the PBS group compared to the crybb2 (P < 0.001) and the crybb2-NPC (P < 0.001) groups after 4 weeks. (F) WB analyses of retinas. Lysates of retinas treated as described were prepared and tested for brain-specific homeobox/POU domain protein 3b (BRN-3b, 45 kiloDaltons). Calnexin was used as the loading protein control. Less protein was detected in the PBS group than in the crybb2 and crybb2-NPC groups after 2 and 4 weeks. Scale bar: 20 μm (AD). Statistical differences: **P < 0.01, ***P < 0.001.
Photoreceptor Response
The marker rhodopsin was used to examine the response of photoreceptors to axotomy due to optic nerve crush and to the intravitreal injection of NPCs. Immunohistochemical evaluation of rhodopsin revealed intense labeling of the photoreceptor outer segments (POS) in adult retinas. A slight decrease in rhodopsin was found in all treated groups after 2 weeks (Figs. 6A–D). Comparison of crybb2-NPC and crybb2 groups with the PBS group revealed less decay of stained POS after 4 weeks (Figs. 6E–H). 
Figure 6. 
 
(AD) Expression of retinal rhodopsin (green) shown by immunohistochemical staining of slices (10 μm) of samples from animals treated with PBS (A, E), crybb2 (B, F), and crybb2-NPC (C, G) after 2 and 4 weeks. The negative control is shown with Cy-2 (D, H) as the secondary antibody. (I) WB analyses of retinas were prepared and tested for rhodopsin expression. The expression of calnexin verified the amount of protein that loaded per lane. (J) Corresponding densitometric analyses of the WB results (n = 4) in PBS-, crybb2-, and crybb2-NPC–treated animals 2 and 4 weeks after ONC. The rhodopsin expression was lower in the crybb2-NPC group than in the crybb2 group after 2 weeks (P < 0.05). A significant reduction in rhodopsin expression was observed in the PBS group compared to the crybb2 (P < 0.001) and crybb2-NPC groups (P < 0.01) after 4 weeks. The rhodopsin expression was lowest in the PBS-treated group. Scale bar: 20 μm (AH). Statistical differences: *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 6. 
 
(AD) Expression of retinal rhodopsin (green) shown by immunohistochemical staining of slices (10 μm) of samples from animals treated with PBS (A, E), crybb2 (B, F), and crybb2-NPC (C, G) after 2 and 4 weeks. The negative control is shown with Cy-2 (D, H) as the secondary antibody. (I) WB analyses of retinas were prepared and tested for rhodopsin expression. The expression of calnexin verified the amount of protein that loaded per lane. (J) Corresponding densitometric analyses of the WB results (n = 4) in PBS-, crybb2-, and crybb2-NPC–treated animals 2 and 4 weeks after ONC. The rhodopsin expression was lower in the crybb2-NPC group than in the crybb2 group after 2 weeks (P < 0.05). A significant reduction in rhodopsin expression was observed in the PBS group compared to the crybb2 (P < 0.001) and crybb2-NPC groups (P < 0.01) after 4 weeks. The rhodopsin expression was lowest in the PBS-treated group. Scale bar: 20 μm (AH). Statistical differences: *P < 0.05, **P < 0.01, ***P < 0.001.
Additional WB analysis revealed a massive decay of rhodopsin in the PBS group (89.40 ± 4.16% at 2 weeks and 21.06 ± 3.53% at 4 weeks). Comparable rhodopsin was detected in the crybb2-NPC group after 2 weeks (94.95 ± 12.27%), while a smaller decrease in rhodopsin was observed after 4 weeks (66.36 ± 14.88%, P < 0.01; Figs. 6I, 6J). Significantly less rhodopsin was found in the crybb2-treated animals after 2 weeks (79.45 ± 5.27%) than in the PBS group (Figs. 6I, 6J; P < 0.05). Significantly less rhodopsin was lost in the crybb2 group (53.61 ± 2.48%) compared to the PBS group after 4 weeks (Figs. 6I, 6J; P < 0.001). Comparable staining of rhodopsin was found in the crybb2-NPC group after 2 weeks (94.95 ± 12.27%), while a reduced loss of rhodopsin was seen after 4 weeks (66.36 ± 14.88%, P < 0.01; Figs. 6I, 6J). These data showed that photoreceptors respond to the injury-induced loss of RGCs with a reduction in rhodopsin, and that this reduction can be neutralized at least partly by the addition of crybb2-producing NPCs. 
Response of Glial Cells
Antibodies detecting GFAP and vimentin were used to detect retinal glial reactivity. A distinct labeling of both proteins in the nerve fiber layer (NFL), ONL, and OPL in the crybb2-NPC group was observed after 2 weeks (Figs. 7E, 7F). The intensity of the vimentin staining in the inner retinal layers was comparable to that in the adult retinal control (data not shown). Increased labeling of vimentin (Fig. 7C) and GFAP (Fig. 7D) in all retinal layers of the crybb2 group was seen after 2 weeks. Compared to the PBS group, the crybb2-NPC animals exhibited reduced staining of vimentin and GFAP in the outer retinal layers, including the NFL, RGC layer, inner plexiform layer (IPL), INL, and the outer plexiform layer (OPL), after 4 weeks. Increased labeling of the OPL was detected. At 4 weeks, the staining profiles of both proteins were similar to those found at 2 weeks (Figs. 7A, 7B). WB analysis revealed lower levels of vimentin in the PBS group (122.48 ± 48.17%, P < 0.05) and the crybb2-NPC group (144.3 ± 10.3%, P < 0.01) compared to the crybb2 group (204.84 ± 17.42%) after 2 weeks (Figs. 7I, 7J). In contrast, significantly decreased vimentin was observed in the crybb2-NPC group (109.53 ± 55.83%) compared to the crybb2 (205.23 ± 28.46%) and PBS groups (186.66 ± 11.31%) after 4 weeks (P < 0.05; Figs. 7I, 7J). 
Figure 7. 
 
(AH) Expression of retinal vimentin and GFAP, shown by immunohistochemical staining of slices (10 μm) of samples from animals treated with PBS (A, B), crybb2 (C, D), and crybb2-NPC (E, F) 2 weeks after ONC. Glial cells are marked red with vimentin and green with GFAP. The negative control is shown with Cy-3 (G) and Cy-2 (H) as secondary antibodies. (I) WB analyses of retinas. Lysates of retinas treated as described were prepared and tested for vimentin expression. Calretinin expression verified the amount of protein that loaded per lane. (J) Corresponding densitometric analyses of the WB results (n = 4) in PBS-, crybb2-, and crybb2-NPC–treated animals 2 and 4 weeks after ONC in relation to control. The vimentin expression significantly increased in the crybb2-treated animals compared to the control and the animals treated with PBS (P < 0.05) and crybb2-NPC (P > 0.01) after 2 weeks. After 4 weeks there were no significant differences in vimentin in the PBS- and crybb2-treated groups (P > 0.05), whereas there was less vimentin expression in the crybb2-NPC–treated group (P < 0.05). Scale bar: 20 μm (AH). Statistical difference: *P < 0.05, **P < 0.01.
Figure 7. 
 
(AH) Expression of retinal vimentin and GFAP, shown by immunohistochemical staining of slices (10 μm) of samples from animals treated with PBS (A, B), crybb2 (C, D), and crybb2-NPC (E, F) 2 weeks after ONC. Glial cells are marked red with vimentin and green with GFAP. The negative control is shown with Cy-3 (G) and Cy-2 (H) as secondary antibodies. (I) WB analyses of retinas. Lysates of retinas treated as described were prepared and tested for vimentin expression. Calretinin expression verified the amount of protein that loaded per lane. (J) Corresponding densitometric analyses of the WB results (n = 4) in PBS-, crybb2-, and crybb2-NPC–treated animals 2 and 4 weeks after ONC in relation to control. The vimentin expression significantly increased in the crybb2-treated animals compared to the control and the animals treated with PBS (P < 0.05) and crybb2-NPC (P > 0.01) after 2 weeks. After 4 weeks there were no significant differences in vimentin in the PBS- and crybb2-treated groups (P > 0.05), whereas there was less vimentin expression in the crybb2-NPC–treated group (P < 0.05). Scale bar: 20 μm (AH). Statistical difference: *P < 0.05, **P < 0.01.
Retinal Microglia Responses to Intravitreal Injection of crybb2-NPC and crybb2
Immunohistochemical evaluation revealed a significant increase in the recruitment and activation of microglia in the PBS group. Microglia were found in the GCL and NFL, while in the crybb2 and crybb2-NPC groups those cells were observed in the IPL, INL, and OPL (Figs. 8A–D). Quantitatively, after 2 weeks, 22.40 ± 3.36 cells/0.2 mm2 of recruited microglia were found in the PBS group, compared to 16.5 ± 3.7 cells/0.2 mm2 in the crybb2 group (P < 0.05) and 13.86 ± 1.58 cells/0.2 mm2 in the crybb2-NPC group (P < 0.01; Fig. 8E). After 2 weeks, increased numbers of activated microglia were found in the PBS group (13.20 ± 1.79 cells/0.2 mm2) compared to the crybb2 group (7.50 ± 2.38 cells/0.2 mm2, P < 0.05) and the crybb2-NPC group (6.00 ± 2.74 cells/0.2 mm2, P < 0.01; Fig. 8F). After 4 weeks, microglia were observed in the GCL, NFL, and OPL in the PBS group, while in the crybb2 and crybb2-NPC groups they also were found in the ONL (data not shown). 
Figure 8. 
 
(AD) Expression of ED-1 and Iba-1 in retinal microglial cells, as assessed by antibody staining of slices (10 μm) from animals treated with PBS (A), crybb2 (B), and NPC (C) 2 weeks after ONC. Microglial cells are marked red for ED-1 and green for Iba-1. ED-1 and IBA-1 positive cells were found only in the NFL and GCL in the PBS-treated animals, whereas they also were found in the INL, IPL, and OPL in the crybb2- and NPC-treated groups. The negative control is shown with Cy-2 and Cy-3 (D) as the secondary antibodies. (E, F) Quantification of retinal microglial cells. (E) Count of ED-1/Iba-1-stained cells in the retinal layers. (F) Count of Iba-1 positive microglia cells. Scale bars: 20 μm (AD). Statistical differences: *P < 0.05, **P < 0.01.
Figure 8. 
 
(AD) Expression of ED-1 and Iba-1 in retinal microglial cells, as assessed by antibody staining of slices (10 μm) from animals treated with PBS (A), crybb2 (B), and NPC (C) 2 weeks after ONC. Microglial cells are marked red for ED-1 and green for Iba-1. ED-1 and IBA-1 positive cells were found only in the NFL and GCL in the PBS-treated animals, whereas they also were found in the INL, IPL, and OPL in the crybb2- and NPC-treated groups. The negative control is shown with Cy-2 and Cy-3 (D) as the secondary antibodies. (E, F) Quantification of retinal microglial cells. (E) Count of ED-1/Iba-1-stained cells in the retinal layers. (F) Count of Iba-1 positive microglia cells. Scale bars: 20 μm (AD). Statistical differences: *P < 0.05, **P < 0.01.
In summary, fewer microglia were found in the crybb2 group than in the other groups. Significantly increased numbers of microglia were found in the PBS group (18.25 ± 3.60 cells/0.2 mm2, P < 0.05) and the crybb2-NPC group (16.75 ± 1.58 cells/0.2 mm2, P < 0.01) compared to the crybb2 group (14.50 ± 0.55 cells/0.2 mm2; Fig. 8E). Increased numbers of activated cells were found in the crybb2-NPC group (12.13 ± 1.73 cells/0.2 mm2) compared to the PBS group (9.5 ± 1.0 cells/0.2 mm2, P < 0.01) and the crybb2 group (8.17 ± 1.33 cells/0.2 mm2) after 4 weeks (P < 0.05; Fig. 8F). The data showed that microglial cells appear to be sensitive indicators of activation in all of the experimental groups of this study. 
Discussion
In our study we examined the fate of intravitreally grafted NPCs within the eye and the responses of retinal cells to the grafting of either nontransfected or transfected NPCs exemplified by crybb2-NPC. There were three principal findings in this study: 
  1.  
    NPCs reside and migrate within the eye and continue to produce NTFs.
  2.  
    Crybb2 and crybb2-NPC reduce the axotomy-induced loss of RGCs and changes in photoreceptor rhodopsin. crybb2-NPC reduce glial reactivity, indicating that crybb2 is involved in retinal repair mechanisms.
  3.  
    Crybb2 reduces the recruitment and activation of retinal microglia, suggesting a reduced inflammatory response.
Intraocular Characteristics of NPCs
NPCs harvested from cultures and injected into the vitreous cavity were able to produce the same markers used to characterize them in culture. This finding suggested that the host environment does not change the profile of proteins synthesized within the vitreous body. This observation was crucial for setting the basic data for injecting transfected NPCs. We observed a migration of intravitreally applied crybb2-NPC within the entire eye for up to 4 weeks after grafting. A previous study demonstrated an overall integration of NPCs into the neonatal retina of rats after 4 weeks. 11 In agreement with our results, previous studies have detected the retina as the main donor for implanted NPCs in cases of injured, 2 ischemic, 27 or degenerating retinas. 10 The preferred location was the ONL, the subretinal debris zone, and the layer of photoreceptors. 10 Our observations revealed the detection of transplanted crybb2-NPC in the vitreoretinal compartment, subretinal space, and choroid after 2 weeks, while crybb2-NPC were found in the GCL, INL, and ONL after 4 weeks. Crybb2-NPC were associated with the anterior chamber, corneal endothelium, and ciliary body after 4 weeks. To our knowledge, this is the first demonstration of an overall migration of grafted crybb2-NPC through the entire rat eye. Migration over long distances is not surprising, since it has been shown that NPCs are able to produce extracellular matrix–degrading metalloproteases. 8  
Responses of the Ocular Tissue
Crybb2 was found to support the survival of axotomized RGCs due to compression and prevent the dissolution of rhodopsin in photoreceptors. Crybb2 usually is produced within the regenerating retina and facilitates the elongation of regenerating axons in retinal explants. 26 Therefore, it is not surprising that significantly fewer RGCs were lost in the crybb2 and crybb2-NPC groups than in the PBS group. These data confirmed the involvement of crybb2 in the repair of RGC metabolism after injury to retinal axons, although the mechanisms underlying the signal transduction remain to be elucidated. Liedtke et al. have shown that GFP-labeled crybb2 is internalized by RGCs in culture. 26 It is most likely that such uptake takes place in vivo and is responsible for the survival effects observed in our study. 
Reduction of rhodopsin and its delocalization within the POS is an early indication of degenerative onset. 31 We found that crybb2-NPC delayed rhodopsin decay. 
It has been assumed that growth-factor failure is one reason for photoreceptor death in inherited retinal photoreceptor degenerations. 32 The external supply of growth factors, such as bFGF, protects photoreceptors. 33 Higher levels of growth factors, such as bFGF, ciliary NTF (CNTF), and BNDF, 34 were found to be associated with photoreceptor death in RCS rats 35,36 and rats with light-damaged retinas. 37 It is likely that the factors produced by NPCs within the eye have a role in protecting RGCs and photoreceptors from death. To this end, we have examined closely the overexpression of one of these factors, crybb2. 
Crystallins maintain their effects on retinal proteins and are an important inhibitor of apoptosis due to toxic intermediates. 15 Increased levels of crystallins of the β/γ superfamily were found in animal models of retinal diseases, like retinitis pigmentosa, light-induced damage, and diabetic retinopathy. 3840 Compared to observations of increased crybb2 expression in regenerating retinas (e.g., GCL and INL) after ONC, no expression was found in unaffected eyes. 26 This line of evidence, together with our own findings, suggests that crybb2 exerts beneficial effects on damaged RGCs and secondarily on photoreceptors. One of the mechanisms operating through crybb2 may be an increase in the amount of neuroprotective CNTF released by astrocytes. 16 To our knowledge, no other studies have investigated the neuroprotective effects of intravitreal crybb2 in vivo. 
An early and significant increase in glial cell activity due to crybb2 administration was found along with decreased vimentin expression in the crybb2-NPC group. A complex glial network, including Müller cells and astrocytes, has neuroprotective effects, releasing NTFs, such as bFGF, CNTF, and antioxidants after retinal injury. 16,41,42  
Early upregulation of vimentin and GFAP occurs in several retinal disorders, such as retinopathia pigmentosa, macular dystrophies, or secondary retinal degenerations. 43,44 and after ONC. 45 The dynamic relationship between intermediate filaments and retinal glial cells is the key mechanism underlying the rapid modification of the Müller cell structure in response to changes in the retinal environment. 46,47 It has been shown that RGCs and astrocytes are donors of retinal crystallins in vitro. 23 Crystallins of the β/γ superfamily are released during periods of critical stress in the retina. 16,24 Increased retinal GFAP expression induces glial cells to switch into a regenerative state after the intravitreal injection of crystallin β/γ. 16 Our observations confirmed the finding of a direct association between glial reactivity and crybb2. The crybb2-NPC–mediated effects on glial cells differ considerably. We concluded that additionally secreted NTFs by crybb2-NPC may inhibit retinal glial activity. 
NPCs and NTFs
While the exact mechanism underlying the effects of NPCs within the retina are not understood fully, it is likely to be based on growth-factor production. 48 Retinal NTFs exert protective effects on neighboring host neurons. 49,50 Several NTFs delay RGC death and photoreceptor loss: fibroblast growth factors (e.g., bFGF), CNTF, brain-derived NTFs (e.g., BDNF), and insulin-like growth factors. RGCs appear to be better protected by BDNF, while photoreceptors may be better protected by bFGF and CNTF. 34 In our study, modified crybb2-NPC overexpressed crybb2 in addition to NTFs for up to 4 weeks after transplantation in vivo. 
Nestin is expressed during CNS stem-cell and progenitor-cell proliferation, and neuronal migration, 51 and protects progenitor cells from apoptosis. 33 BDNF, which is expressed by glial cells and neuronal cells, 34,52 prolongs the survival of RGCs in vitro, reduces RGC death during development, and exerts survival effects on injured adult neurons. 33,53,54 Calcineurin is expressed in the RGCs of rat retinas and exerts important cellular effects in the immune and nervous systems. 55,56 It is involved in acute and chronic disorders resulting from glaucoma. 56 Oncomodulin has a high affinity for RGCs, exerting axon-promoting effects on these cells in mature optic nerves in vivo, 57 and has a central role in inflammation-induced regeneration. 58  
Microglia
Microglial recruitment and activation are associated with retinal degeneration, as shown in the RCS rat. 59 These cells respond to several proinflammatory factors, including lipopolysaccharides and cytokines. 60,61 On the other hand, they produce and secrete NTFs, such as CNTF, GDNF, and nerve growth factor, to protect photoreceptors from cell death. 62  
The number of activated microglial cells was highest in the PBS group after 2 weeks, suggesting an increased response to dying neurons and inflammatory disorders. Inflammatory cytokines may promote the transformation of ramified to active microglia. The effect is more predictable compared to that of crybb2, which we attribute to increased cell death within the retina. An increased activation and recruitment of microglia in the crybb2-NPC group after 4 weeks was another interesting finding. An attenuated loss of RGCs and photoreceptors is indicative of a lower rate of retinal cell death in this group. Furthermore, several NTFs secreted by NPCs have inhibitory effects on the cellular immune response. We suggest a microglial reaction to the cellular response of NPCs. 
Conclusions
In conclusion, to our knowledge this the first study to show the migration of intravitreally injected NPCs through the entire rat eye. This finding implies that NPCs are able to interact with different types of ocular tissue. Crybb2-NPC prevents RGC loss and decay of rhodopsin in the injury-induced model of retinal degeneration. In addition to their survival and migration within the eye, crybb2-producing NPCs increase the chances of survival of RGCs and photoreceptors. The expression of further survival-promoting factors may be introduced in these cells, thus assuring a permanent and approximately constant delivery within the eye. This strategy of creating factor-producing “cell-factories” in the neighborhood of areas of degeneration may become a fruitful approach for the treatment of retinal disorders by providing bioavailable growth factors. 
Acknowledgments
M. Wissing and M. Langkamp-Flock provided skillful technical assistance, and M. Reis typed the manuscript. English Science Editing (ESE) provided native linguistic editing of the manuscript. 
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Footnotes
 Supported by a DFG Grant Th 386/18 (ST) and by IZKF grants (MRRB) and Grant THa3/002/09 (ST). The authors alone are responsible for the content and writing of this paper.
Footnotes
 Disclosure: M.R.R. Böhm, None; S. Pfrommer, None; C. Chiwitt, None; M. Brückner, None; H. Melkonyan, None; S. Thanos, None
Figure 1. 
 
Morphologic and immunohistochemical characterization of NPCs derived from cultured rat fetal neural tissue. (A, B) Cultured NPCs were found in cell clusters, called neurospheres (arrows). (B) Single cells with morphologic features reminiscent of stem cells also were found (arrowheads). (CQ) Immunohistochemical characterization of cultured NPCs (using nestin, green) and several other markers (red) in vitro. Labeling with antibodies raised against BDNF (CE), oncomodulin (FH), calcineurin (IK), crybb2 (LN), and cryg (OQ) revealed that these molecules were expressed in NPCs in vitro. 4′,6-Diamidino-2-phenylindole (DAPI, blue) was used to stain cell nuclei. Scale bars: 20 μm (A, B, E, H, K, N, Q).
Figure 1. 
 
Morphologic and immunohistochemical characterization of NPCs derived from cultured rat fetal neural tissue. (A, B) Cultured NPCs were found in cell clusters, called neurospheres (arrows). (B) Single cells with morphologic features reminiscent of stem cells also were found (arrowheads). (CQ) Immunohistochemical characterization of cultured NPCs (using nestin, green) and several other markers (red) in vitro. Labeling with antibodies raised against BDNF (CE), oncomodulin (FH), calcineurin (IK), crybb2 (LN), and cryg (OQ) revealed that these molecules were expressed in NPCs in vitro. 4′,6-Diamidino-2-phenylindole (DAPI, blue) was used to stain cell nuclei. Scale bars: 20 μm (A, B, E, H, K, N, Q).
Figure 2. 
 
(AO) Immunohistochemical characterization of intravitreally transplanted NPCs 2 weeks after propagation in culture. The same antibodies were used for BNDF (AC), oncomodulin (DF), calcineurin (GI), crybb2 (JL), and cryg (MO). DAPI (blue) was used to stain the cell nuclei. Scale bar: 50 μm (C, F, I, L, O).
Figure 2. 
 
(AO) Immunohistochemical characterization of intravitreally transplanted NPCs 2 weeks after propagation in culture. The same antibodies were used for BNDF (AC), oncomodulin (DF), calcineurin (GI), crybb2 (JL), and cryg (MO). DAPI (blue) was used to stain the cell nuclei. Scale bar: 50 μm (C, F, I, L, O).
Figure 3. 
 
(AF) Expression of retinal crybb2 (red) shown by immunohistochemical staining of slices (10 μm) of samples from animals treated with PBS (A, B), and crybb2 (C, D) after 2 and 4 weeks. (E) Expression of crybb2 by intravitreally implanted crybb2-NPC after 2 weeks. Cell nuclei are marked blue with DAPI. (F) The negative control is shown with cyanine (Cy-3) as a secondary antibody. Scale bar: 20 μm (AF).
Figure 3. 
 
(AF) Expression of retinal crybb2 (red) shown by immunohistochemical staining of slices (10 μm) of samples from animals treated with PBS (A, B), and crybb2 (C, D) after 2 and 4 weeks. (E) Expression of crybb2 by intravitreally implanted crybb2-NPC after 2 weeks. Cell nuclei are marked blue with DAPI. (F) The negative control is shown with cyanine (Cy-3) as a secondary antibody. Scale bar: 20 μm (AF).
Figure 4. 
 
(AO) Staining for nestin-expressing NPCs (green) in sections throughout the eye. Two weeks after transplantation, grafted NPCs were found throughout the retina (e.g., IPL) (AC), including the subretinal space (DF) and the vitreoretinal compartment (GI). After 4 weeks, nestin-positive cells were detectable in the anterior chamber and corneal endothelium (JL) and the epithelium of the ciliary body (MO). NPCs were found only in the GCL, OPL, and IPL, and not in the other retinal layers after 4 weeks (data not shown). DAPI (blue) was used to stain the cell nuclei (P, R). The negative control is shown with Cy-2 as a secondary antibody. Arrows indicate nestin-positive NPCs in all images. Scale bar: 20 μm (C, F, I, L, O, R).
Figure 4. 
 
(AO) Staining for nestin-expressing NPCs (green) in sections throughout the eye. Two weeks after transplantation, grafted NPCs were found throughout the retina (e.g., IPL) (AC), including the subretinal space (DF) and the vitreoretinal compartment (GI). After 4 weeks, nestin-positive cells were detectable in the anterior chamber and corneal endothelium (JL) and the epithelium of the ciliary body (MO). NPCs were found only in the GCL, OPL, and IPL, and not in the other retinal layers after 4 weeks (data not shown). DAPI (blue) was used to stain the cell nuclei (P, R). The negative control is shown with Cy-2 as a secondary antibody. Arrows indicate nestin-positive NPCs in all images. Scale bar: 20 μm (C, F, I, L, O, R).
Figure 5. 
 
(AF) Effects of crybb2 and crybb2-NPC on RGCs in the retina. Expression of neurofilament 200 (NF-200, red) shown by immunohistochemical staining of slices (10 μm) of samples from animals treated with PBS (A, B) and crybb2-NPC (C, D) after 2 and 4 weeks. Cell nuclei are marked blue with DAPI. Cy-3 was used for negative control as a secondary antibody (data not shown). (E) Number of persistent RGCs (per 0.2 mm2) in retinal slices (10 μm) in relation to that of adult control tissue (in %). The number of RGCs decreased in the PBS group (P < 0.001) and crybb2 group (P < 0.01) compared to crybb2-NPC–treated animals after 2 weeks. Decreased numbers of RGCs were observed in the PBS group compared to the crybb2 (P < 0.001) and the crybb2-NPC (P < 0.001) groups after 4 weeks. (F) WB analyses of retinas. Lysates of retinas treated as described were prepared and tested for brain-specific homeobox/POU domain protein 3b (BRN-3b, 45 kiloDaltons). Calnexin was used as the loading protein control. Less protein was detected in the PBS group than in the crybb2 and crybb2-NPC groups after 2 and 4 weeks. Scale bar: 20 μm (AD). Statistical differences: **P < 0.01, ***P < 0.001.
Figure 5. 
 
(AF) Effects of crybb2 and crybb2-NPC on RGCs in the retina. Expression of neurofilament 200 (NF-200, red) shown by immunohistochemical staining of slices (10 μm) of samples from animals treated with PBS (A, B) and crybb2-NPC (C, D) after 2 and 4 weeks. Cell nuclei are marked blue with DAPI. Cy-3 was used for negative control as a secondary antibody (data not shown). (E) Number of persistent RGCs (per 0.2 mm2) in retinal slices (10 μm) in relation to that of adult control tissue (in %). The number of RGCs decreased in the PBS group (P < 0.001) and crybb2 group (P < 0.01) compared to crybb2-NPC–treated animals after 2 weeks. Decreased numbers of RGCs were observed in the PBS group compared to the crybb2 (P < 0.001) and the crybb2-NPC (P < 0.001) groups after 4 weeks. (F) WB analyses of retinas. Lysates of retinas treated as described were prepared and tested for brain-specific homeobox/POU domain protein 3b (BRN-3b, 45 kiloDaltons). Calnexin was used as the loading protein control. Less protein was detected in the PBS group than in the crybb2 and crybb2-NPC groups after 2 and 4 weeks. Scale bar: 20 μm (AD). Statistical differences: **P < 0.01, ***P < 0.001.
Figure 6. 
 
(AD) Expression of retinal rhodopsin (green) shown by immunohistochemical staining of slices (10 μm) of samples from animals treated with PBS (A, E), crybb2 (B, F), and crybb2-NPC (C, G) after 2 and 4 weeks. The negative control is shown with Cy-2 (D, H) as the secondary antibody. (I) WB analyses of retinas were prepared and tested for rhodopsin expression. The expression of calnexin verified the amount of protein that loaded per lane. (J) Corresponding densitometric analyses of the WB results (n = 4) in PBS-, crybb2-, and crybb2-NPC–treated animals 2 and 4 weeks after ONC. The rhodopsin expression was lower in the crybb2-NPC group than in the crybb2 group after 2 weeks (P < 0.05). A significant reduction in rhodopsin expression was observed in the PBS group compared to the crybb2 (P < 0.001) and crybb2-NPC groups (P < 0.01) after 4 weeks. The rhodopsin expression was lowest in the PBS-treated group. Scale bar: 20 μm (AH). Statistical differences: *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 6. 
 
(AD) Expression of retinal rhodopsin (green) shown by immunohistochemical staining of slices (10 μm) of samples from animals treated with PBS (A, E), crybb2 (B, F), and crybb2-NPC (C, G) after 2 and 4 weeks. The negative control is shown with Cy-2 (D, H) as the secondary antibody. (I) WB analyses of retinas were prepared and tested for rhodopsin expression. The expression of calnexin verified the amount of protein that loaded per lane. (J) Corresponding densitometric analyses of the WB results (n = 4) in PBS-, crybb2-, and crybb2-NPC–treated animals 2 and 4 weeks after ONC. The rhodopsin expression was lower in the crybb2-NPC group than in the crybb2 group after 2 weeks (P < 0.05). A significant reduction in rhodopsin expression was observed in the PBS group compared to the crybb2 (P < 0.001) and crybb2-NPC groups (P < 0.01) after 4 weeks. The rhodopsin expression was lowest in the PBS-treated group. Scale bar: 20 μm (AH). Statistical differences: *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 7. 
 
(AH) Expression of retinal vimentin and GFAP, shown by immunohistochemical staining of slices (10 μm) of samples from animals treated with PBS (A, B), crybb2 (C, D), and crybb2-NPC (E, F) 2 weeks after ONC. Glial cells are marked red with vimentin and green with GFAP. The negative control is shown with Cy-3 (G) and Cy-2 (H) as secondary antibodies. (I) WB analyses of retinas. Lysates of retinas treated as described were prepared and tested for vimentin expression. Calretinin expression verified the amount of protein that loaded per lane. (J) Corresponding densitometric analyses of the WB results (n = 4) in PBS-, crybb2-, and crybb2-NPC–treated animals 2 and 4 weeks after ONC in relation to control. The vimentin expression significantly increased in the crybb2-treated animals compared to the control and the animals treated with PBS (P < 0.05) and crybb2-NPC (P > 0.01) after 2 weeks. After 4 weeks there were no significant differences in vimentin in the PBS- and crybb2-treated groups (P > 0.05), whereas there was less vimentin expression in the crybb2-NPC–treated group (P < 0.05). Scale bar: 20 μm (AH). Statistical difference: *P < 0.05, **P < 0.01.
Figure 7. 
 
(AH) Expression of retinal vimentin and GFAP, shown by immunohistochemical staining of slices (10 μm) of samples from animals treated with PBS (A, B), crybb2 (C, D), and crybb2-NPC (E, F) 2 weeks after ONC. Glial cells are marked red with vimentin and green with GFAP. The negative control is shown with Cy-3 (G) and Cy-2 (H) as secondary antibodies. (I) WB analyses of retinas. Lysates of retinas treated as described were prepared and tested for vimentin expression. Calretinin expression verified the amount of protein that loaded per lane. (J) Corresponding densitometric analyses of the WB results (n = 4) in PBS-, crybb2-, and crybb2-NPC–treated animals 2 and 4 weeks after ONC in relation to control. The vimentin expression significantly increased in the crybb2-treated animals compared to the control and the animals treated with PBS (P < 0.05) and crybb2-NPC (P > 0.01) after 2 weeks. After 4 weeks there were no significant differences in vimentin in the PBS- and crybb2-treated groups (P > 0.05), whereas there was less vimentin expression in the crybb2-NPC–treated group (P < 0.05). Scale bar: 20 μm (AH). Statistical difference: *P < 0.05, **P < 0.01.
Figure 8. 
 
(AD) Expression of ED-1 and Iba-1 in retinal microglial cells, as assessed by antibody staining of slices (10 μm) from animals treated with PBS (A), crybb2 (B), and NPC (C) 2 weeks after ONC. Microglial cells are marked red for ED-1 and green for Iba-1. ED-1 and IBA-1 positive cells were found only in the NFL and GCL in the PBS-treated animals, whereas they also were found in the INL, IPL, and OPL in the crybb2- and NPC-treated groups. The negative control is shown with Cy-2 and Cy-3 (D) as the secondary antibodies. (E, F) Quantification of retinal microglial cells. (E) Count of ED-1/Iba-1-stained cells in the retinal layers. (F) Count of Iba-1 positive microglia cells. Scale bars: 20 μm (AD). Statistical differences: *P < 0.05, **P < 0.01.
Figure 8. 
 
(AD) Expression of ED-1 and Iba-1 in retinal microglial cells, as assessed by antibody staining of slices (10 μm) from animals treated with PBS (A), crybb2 (B), and NPC (C) 2 weeks after ONC. Microglial cells are marked red for ED-1 and green for Iba-1. ED-1 and IBA-1 positive cells were found only in the NFL and GCL in the PBS-treated animals, whereas they also were found in the INL, IPL, and OPL in the crybb2- and NPC-treated groups. The negative control is shown with Cy-2 and Cy-3 (D) as the secondary antibodies. (E, F) Quantification of retinal microglial cells. (E) Count of ED-1/Iba-1-stained cells in the retinal layers. (F) Count of Iba-1 positive microglia cells. Scale bars: 20 μm (AD). Statistical differences: *P < 0.05, **P < 0.01.
Table 1. 
 
Experimental Groups (In Vivo)
Table 1. 
 
Experimental Groups (In Vivo)
Experimental Group N Weights of SD Rats Experimental Manipulation Times, wk
Effects of crybb2 16 180 to 250 g Left ONC with intravitreal injection of crybb2 2, 4
IHC 4
WB 4
Effects of crybb2-NPC 16 180 to 250 g Left ONC with intravitreal injection of crybb2-NPC 2, 4
IHC 4
WB 4
Effects of PBS (control) 16 180 to 250 g Left ONC with intravitreal injection of PBS 2, 4
IHC 4
WB 4
Adult control 16 180 to 250 g Untreated 2, 4
IHC 4
WB 4
Table 2. 
 
Antibodies Used for IHC
Table 2. 
 
Antibodies Used for IHC
Antibody Company Isotype Dilution Tissue
Primary antibodies
 BDNF Millipore (Temecula, CA) Rabbit polyclonal 1:500 NPCs in vitro/in vivo
 Calcineurin Millipore Rabbit polyclonal 1:500 NPCs in vitro/in vivo
 Crybb2 Santa Cruz Biotechnology (Santa Cruz, CA) Goat polyclonal 1:100 NPCs in vitro/in vivo
 Cryg Santa Cruz Biotechnology Rabbit polyclonal 1:100 NPCs in vitro/in vivo
 ED-1 BMA Biomedicals (Augst, Switzerland) Mouse monoclonal 1:200 Microglia
 Iba-1 Wako Pure Chemical Industries (Osaka, Japan) Rabbit 1:500 Microglia
 Nestin Millipore Rabbit polyclonal 1:200 NPCs in vitro/in vivo
 Nestin AbD Serotoec (Puchheim, Germany) Mouse monoclonal 1:100 NPCs in vitro/in vivo
 Neurofilament 200 Sigma-Aldrich (St. Louis, MO) Mouse monoclonal 1:400 Retinal ganglion cells
 Oncomodulin Swant (Marly, Switzerland) Rabbit polyclonal 1:500 NPCs in vitro/in vivo
 Rhodopsin Millipore Mouse monoclonal 1:100 Photoreceptors
 Vimentin Sigma-Aldrich Mouse monoclonal 1:200 Retinal glial cells
Secondary antibodies
 Cy-2 Jackson ImmunoResearch Europe Ltd. (Suffolk, UK) Goat antimouse 1:200
 Cy-2 Jackson ImmunoResearch Europe Ltd. Goat antirabbit 1:200
 Cy-2 Abcam (Cambridge, UK) Donkey Antigoat 1:100
 TRITC Sigma-Aldrich Goat antirabbit 1:400
 TRITC Sigma-Aldrich Goat antimouse 1:300
 TRITC Jackson ImmunoResearch Europe Ltd. Donkey antigoat 1:100
Table 3. 
 
Antibodies Used for WB
Table 3. 
 
Antibodies Used for WB
Antibody Company Isotype Dilution Molecular Mass
Primary antibodies
 BRN-3b Covance (Princeton, NJ) Rabbit polyclonal 1:2,000 45 kD
 Rhodopsin Millipore Mouse monoclonal 1:1,000 39 kD
 Vimentin Sigma-Aldrich Mouse monoclonal 1:500 58 kD
Secondary antibodies
 Secondary rhodopsin Cell Signaling Technology, Inc. (Danvers, MA) Horse antimouse 1:4,000
 Secondary vimentin Cell Signaling Technology, Inc. Horse antimouse 1:4,000
 Secondary BRN-3b Amersham Pharmacia Biotech Donkey antirabbit 1:10,000
Loading control
 Calnexin Sigma-Aldrich Rabbit 1:10,000 90 kD
Table 4. 
 
Localization of Grafted NPCs after Transplantation
Table 4. 
 
Localization of Grafted NPCs after Transplantation
Localization NPC – 2 weeks NPC – 4 weeks
Cornea X
AC X X
Ciliary body X
Vitreous X X
NFL
GCL X X
OPL X
ONL X X
IPL X
INL X X
RPE
Choroidea X
×
×

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