Crystallin-β-b2-Overexpressing NPCs Support the Survival of Injured Retinal Ganglion Cells and Photoreceptors in Rats

Retina

Crystallin-b-b2-Overexpressing NPCs Support the Survivalof Injured Retinal Ganglion Cells and Photoreceptors inRats

Michael R. R. Bohm,1,2 Sarah Pfrommer,1 Carolin Chiwitt,1 Matthias Bruckner,1

Harutyun Melkonyan,1 and Solon Thanos1,2

PURPOSE. Crystallin b-b2 (crybb2) is known to support theregeneration of retinal ganglion cell (RGC) axons in culture.We investigated whether neuronal progenitor cells (NPCs)overexpressing crybb2 (crybb2-NPC) affect secondary retinaldegeneration due to optic nerve crush in vivo.

METHODS. NPCs were produced by dissociation and propaga-tion of rat embryonic neural tube and eye primordial cells atembryonic days 13.5 and 15. Retinal degeneration was inducedby injured optic nerve crush (by suture, 20 seconds). Severalgroups were built: crybb2-NPC were injected into the vitreousbody, while the Controls were comprised of recombinantcrybb2–injected and PBS-injected groups. The eyes, inparticular the retina, were analyzed by immunohistochemistryand Western blotting for different antigens at 2 and 4 weeksafter surgery.

RESULTS. At 2 and 4 weeks post surgery, crybb2-NPC residedwithin the vitreoretinal compartment, and were persistentlynestin-positive throughout the experimental period. The cellsstained positive for various neurotrophins and acted as ‘‘living’’cell factories to support the survival of injured RGCs. Thecrybb2-NPC migrated throughout the eye structures andsometimes became integrated within the tissue. Most of theocular cells responded to the appearance of crybb2-NPC withmarked changes of certain proteins, including Iba-1 (microg-lia), 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 thevitreoretinal compartment, which suggests that modifiedcrybb2-NPC could be used in a novel strategy for the treatmentof degenerative vitreoretinal diseases. However, future studies

must determine the safety of in vivo administration of crybb2-NPC. (Invest Ophthalmol Vis Sci. 2012;53:8265–8279) DOI:10.1167/iovs.12-10334

Retinal degenerative diseases affect millions of peopleworldwide1 and, hence, methods must be developed to

prevent such degeneration and the consequent visual impair-ment. One of these strategies could involve placing cells insidethe eye that produce protective substances.

Neural stem cells and neural progenitor cells (NPCs) havebeen isolated from different parts of the embryonic and adultcentral nervous system (CNS) of several mammalian species.2–5

NPCs secrete several neurotrophic factors (NTFs) and exertprotective effects on diseased neurons.6–8 NPCs can bemanipulated through pretreatment with growth factors or viagene transfection.1,2,5,9 Attempts to replace lost photorecep-tors so far has focused on injecting stem cells into the retina orthe subretinal space. Although some of the injected cellsdifferentiate into photoreceptors and integrate into the hosttissue, the quantity of cells involved generally is low, and inmost cases they fail to function.2,10–12 Alternative approachesinclude the modification of NPCs to rescue visual functionthrough the engineered expression of NTFs, such as glial cell–derived NTF (GDNF)-producing human-derived NPCs. Thesewere shown to have a positive effect after being injected intothe 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 thedevelopment of retinal cells, including photoreceptors.13–15

Crystallins, which also are expressed in retinal neurons,16 havebeen identified in various neurodegenerative disorders, includ-ing in those affecting the retina.17 While several studies havedemonstrated that crystallin a has a role in retinal disorders,there is little information on the effects of crystallin b/c.18–21

Adult retinal ganglion cells (RGCs) have an enhanced chance ofsurvival when exposed to lens injury.16,22,23 Testing of differentclasses of crystallins has revealed that crystallins of the b/csuperfamily possess a strong neuroprotective and regenerativepotential by mimicking the effects of lens injury.16

Crystallins b and c (cryg) are expressed during thedevelopment of the mouse eye.14 Increased levels of cryghave been observed in cultured astrocytes affected byhypoxia,23 neuronal aging processes,24 and neuronal repair.25

Added crystallin b-b2 (crybb2) enhances the growth of axonsin retinal explants and in primary hippocampal neurons.26

However, to our knowledge no protective effects of crybb2 onRGCs in vivo or on photoreceptors have yet been reported.

To investigate neuroprotective features of NPCs, we usedthe optic nerve crush model to induce retinal degeneration andinjected transfected NPCs, overexpressing crybb2 intravitreally

From the 1Institute for Experimental Ophthalmology, School ofMedicine, Westfalian-Wilhelms-University Munster, Munster, Ger-many; and the 2Interdisciplinary Centre for Clinical Research (IZKF),School of Medicine, Westfalian-Wilhelms-University Munster,Munster, Germany.

Supported by a DFG Grant Th 386/18 (ST) and by IZKF grants(MRRB) and Grant THa3/002/09 (ST). The authors alone areresponsible for the content and writing of this paper.

Submitted for publication June 5, 2012; revised October 30,2012; accepted November 1, 2012.

Disclosure: M.R.R. Bohm, None; S. Pfrommer, None; C.Chiwitt, None; M. Bruckner, None; H. Melkonyan, None; S.Thanos, None

Corresponding author: Michael R. R. Bohm, Institute ofExperimental Ophthalmology, School of Medicine, Westfalian-Wilhelms-University Munster, Albert-Schweitzer-Campus 1, D15,48149 Munster, Germany; [email protected].

Investigative Ophthalmology & Visual Science, December 2012, Vol. 53, No. 13

Copyright 2012 The Association for Research in Vision and Ophthalmology, Inc. 8265

after surgery. In controls, NPCs were replaced with intravitrealinjection of recombinant crybb2 protein or injection of PBS.The aims of our study were to elucidate the survival andmigration of NPCs within the eye, the intravitreal productionof NTFs and crystallins, and the effects of overexpressedcrybb2 on degenerating RGCs and photoreceptors. Therefore,immunohistochemistry and Western blotting (WB) were usedto examine intravitreal distribution of NPCs and production ofNTFs that may affect the fate of photoreceptors, glial cells, andmicroglia.

METHODS

Animals and Drugs

Pregnant Sprague-Dawley (SD) rats at embryonic days (E)13.5 and 15

(n¼ 52) were used as a source of NPCs for these studies. Adult SD rats

at approximately 3 months of age (n¼ 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, Dusseldorf, 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.

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/lL

recombinant human basic fibroblast growth factor (bFGF; RD Systems,

Minneapolis, MN), and 10 lL insulin (2.5 mg/mL; PromoCell,

Heidelberg, Germany). The suspension was triturated gently and 4.5

3 106 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 378C 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 (48C). The

culture medium was removed and approximately 5 3 106 cells were

resuspended in 100 lL Amaxa Rat NSC Nucleofector Kit (Lonza

Cologne, Cologne, Germany). Vector (3.5 lg 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) andIntravitreal 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 ll 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 lL of recombinant

crybb2 protein (180 lg/lL) 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 resus-

pended 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

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

IHC, immunohistochemistry; WB, Western blotting; wk, weeks.

8266 Bohm et al. IOVS, December 2012, Vol. 53, No. 13

operations (see above), 10 lL 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�208C). 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 ¼ 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�808C until further use.

Sections were cut along the visual axis with a cryostat (10 lm 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 lm) 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 40,6-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 3400.

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 �808C 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 lg 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 & Schull 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 48C 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 (Amer-

sham, 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

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

TRITC, tetramethylrhodamine isothiocyanate; Cy, cyanine.

IOVS, December 2012, Vol. 53, No. 13 Crystallin-b-b2-Overexpressing NPCs 8267

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.

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 indepen-

dent-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 FactorExpression of crybb2-NPC In Vitro

As expected, NPCs harvested from embryonic brain and eyeprimordial tissue exhibited various morphologies in culture.Single cells were found along with clusters and neurospheresof NPCs (Figs. 1A, 1B). These stem cells possessed longprotrusions with a shaped cell body (Fig. 1B). NPCs stainedpositive 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 tobe associated with regenerative retinal functions in vitro,26 itwas selected as the first-choice protein for analysis in vivo. Weobserved a transfection rate of 55 to 67%, which is comparableto previous reports.5

NPCs within the Eye

Immunohistochemistry was used to examine whether theinjected crybb2-NPC continued expression of the same NTFswithin the eye. It appeared that all proteins still were producedwithin the vitreous body (Figs. 2A–O). Particular attention waspaid to crybb2 that was stained within clusters of cells locatedwithin the vitreous (Figs. 2J–L). These data supported the viewthat 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 diffusestaining of RGCs after 2 weeks (Fig. 3C). No crybb2 staining

was observed 4 weeks after ONC (Figs. 3B, 3D). When theimmunohistochemistry was performed with sections fromcrybb2-NPC–treated eyes, most cells stained positive forcrybb2 (Fig. 3E).

Intraocular Localization of Intravitreally Injected

crybb2-NPC

Nestin was used as a stem-cell marker to identify thedistribution of crybb2-NPC at 2 and 4 weeks after intravitrealtransplantation. NPCs were observed as follows: arrestedcrybb2-NPC in the vitreous cavity, arrested and migratingcrybb2-NPC in the vitreoretinal compartment, and arrested andmigrating crybb2-NPC in the anterior segment, including theanterior chamber, cornea, and ciliary body (Table 4).

Arrested nestin-positive cells were localized in the vitrealcavity, at the vitreoretinal interface (Figs. 4G–I), in most retinallayers (Figs. 4A–C), and in the subretinal space, including thechoroidea (Figs. 4D–F) 2 weeks after transplantation. They alsowere found in the anterior chamber, associated with thecorneal endothelium (Figs. 4J–L) and epithelium of the ciliarybody (Figs. 4M–O) 4 weeks after transplantation. In the hostretina, NPCs were detected in the ganglion cell layer (GCL),inner nuclear layer (INL), and outer nuclear layer (ONL). Theseobservations show that NPCs are redistributed within the eyeduring the first 2 weeks after injection. The distribution ofNPCs was similar at 4 weeks post surgery (Fig. 4), with someNPCs having reached quite distant locations, such as thecornea (Figs. 4J–L). These data supported the view that NPCscan migrate within the eye.

Staining of Ocular Tissue after Injection of crybb2-

NPC

The marker neurofilament 200 was used to examine theresponse of RGCs to damage and to the intravitreal injection ofNPCs (Figs. 5A–D). A moderate loss of RGCs was found in thecrybb2-NPC group compared to the PBS group. Compared tothe adult control, the population of RGCs had reduced to 82.636 5.60% in the crybb2-NPC group and to 55.56 6 8.56% in thePBS group 2 weeks after implantation. After 4 weeks, thesevalues were 52.78 6 5.73% and 18.75 6 3.99%, respectively.In the crybb2 group, the population of RGCs had reduced to58.30 6 2.41% after 2 weeks and 55.56 6 3.99% after 4 weeks(Fig. 5E). After 4 weeks the population of RGCs wassignificantly higher in the crybb2 group than in the PBS group.These percentages were confirmed by WB analysis usingantibodies detecting brain-specific homeobox/POU domain

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

BRN-3b, brain-specific homeobox/POU domain protein 3b.


FIGURE 1. Morphologic and immunohistochemical characterization of NPCs derived from cultured rat fetal neural tissue. (A, B) Cultured NPCswere found in cell clusters, called neurospheres (arrows). (B) Single cells with morphologic features reminiscent of stem cells also were found(arrowheads). (C–Q) Immunohistochemical characterization of cultured NPCs (using nestin, green) and several other markers (red) in vitro.Labeling with antibodies raised against BDNF (C–E), oncomodulin (F–H), calcineurin (I–K), crybb2 (L–N), and cryg (O–Q) revealed that thesemolecules were expressed in NPCs in vitro. 40,6-Diamidino-2-phenylindole (DAPI, blue) was used to stain cell nuclei. Scale bars: 20 lm (A, B, E, H,K, N, Q).


protein 3b (Fig. 5F).30 These data showed that crybb2-producing NPCs support the survival of injured RGCs.

Photoreceptor Response

The marker rhodopsin was used to examine the response ofphotoreceptors to axotomy due to optic nerve crush and to theintravitreal injection of NPCs. Immunohistochemical evalua-tion of rhodopsin revealed intense labeling of the photorecep-tor outer segments (POS) in adult retinas. A slight decrease inrhodopsin was found in all treated groups after 2 weeks (Figs.6A–D). Comparison of crybb2-NPC and crybb2 groups withthe PBS group revealed less decay of stained POS after 4 weeks(Figs. 6E–H).

Additional WB analysis revealed a massive decay ofrhodopsin in the PBS group (89.40 6 4.16% at 2 weeks and21.06 6 3.53% at 4 weeks). Comparable rhodopsin wasdetected in the crybb2-NPC group after 2 weeks (94.95 6

12.27%), while a smaller decrease in rhodopsin was observedafter 4 weeks (66.36 6 14.88%, P < 0.01; Figs. 6I, 6J).Significantly less rhodopsin was found in the crybb2-treatedanimals after 2 weeks (79.45 6 5.27%) than in the PBS group(Figs. 6I, 6J; P < 0.05). Significantly less rhodopsin was lost inthe crybb2 group (53.61 6 2.48%) compared to the PBS groupafter 4 weeks (Figs. 6I, 6J; P < 0.001). Comparable staining of

rhodopsin was found in the crybb2-NPC group after 2 weeks(94.95 6 12.27%), while a reduced loss of rhodopsin was seenafter 4 weeks (66.36 6 14.88%, P < 0.01; Figs. 6I, 6J). Thesedata showed that photoreceptors respond to the injury-induced loss of RGCs with a reduction in rhodopsin, and thatthis reduction can be neutralized at least partly by the additionof crybb2-producing NPCs.

Response of Glial Cells

Antibodies detecting GFAP and vimentin were used to detectretinal glial reactivity. A distinct labeling of both proteins in thenerve fiber layer (NFL), ONL, and OPL in the crybb2-NPCgroup was observed after 2 weeks (Figs. 7E, 7F). The intensityof the vimentin staining in the inner retinal layers wascomparable to that in the adult retinal control (data notshown). Increased labeling of vimentin (Fig. 7C) and GFAP(Fig. 7D) in all retinal layers of the crybb2 group was seen after2 weeks. Compared to the PBS group, the crybb2-NPC animalsexhibited reduced staining of vimentin and GFAP in the outerretinal layers, including the NFL, RGC layer, inner plexiformlayer (IPL), INL, and the outer plexiform layer (OPL), after 4weeks. Increased labeling of the OPL was detected. At 4 weeks,the staining profiles of both proteins were similar to thosefound at 2 weeks (Figs. 7A, 7B). WB analysis revealed lower

FIGURE 2. (A–O) Immunohistochemical characterization of intravitreally transplanted NPCs 2 weeks after propagation in culture. The sameantibodies were used for BNDF (A–C), oncomodulin (D–F), calcineurin (G–I), crybb2 (J–L), and cryg (M–O). DAPI (blue) was used to stain the cellnuclei. Scale bar: 50 lm (C, F, I, L, O).


levels of vimentin in the PBS group (122.48 6 48.17%, P <0.05) and the crybb2-NPC group (144.3 6 10.3%, P < 0.01)compared to the crybb2 group (204.84 6 17.42%) after 2weeks (Figs. 7I, 7J). In contrast, significantly decreasedvimentin was observed in the crybb2-NPC group (109.53 6

55.83%) compared to the crybb2 (205.23 6 28.46%) and PBSgroups (186.66 6 11.31%) after 4 weeks (P < 0.05; Figs. 7I,7J).

Retinal Microglia Responses to Intravitreal

Injection of crybb2-NPC and crybb2

Immunohistochemical evaluation revealed a significant in-crease in the recruitment and activation of microglia in thePBS group. Microglia were found in the GCL and NFL, while inthe crybb2 and crybb2-NPC groups those cells were observedin the IPL, INL, and OPL (Figs. 8A–D). Quantitatively, after 2weeks, 22.40 6 3.36 cells/0.2 mm2 of recruited microglia werefound in the PBS group, compared to 16.5 6 3.7 cells/0.2 mm2

in the crybb2 group (P < 0.05) and 13.86 6 1.58 cells/0.2mm2 in the crybb2-NPC group (P < 0.01; Fig. 8E). After 2weeks, increased numbers of activated microglia were found inthe PBS group (13.20 6 1.79 cells/0.2 mm2) compared to thecrybb2 group (7.50 6 2.38 cells/0.2 mm2, P < 0.05) and thecrybb2-NPC group (6.00 6 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).

In summary, fewer microglia were found in the crybb2group than in the other groups. Significantly increasednumbers of microglia were found in the PBS group (18.25 6

3.60 cells/0.2 mm2, P < 0.05) and the crybb2-NPC group(16.75 6 1.58 cells/0.2 mm2, P < 0.01) compared to thecrybb2 group (14.50 6 0.55 cells/0.2 mm2; Fig. 8E). Increasednumbers of activated cells were found in the crybb2-NPCgroup (12.13 6 1.73 cells/0.2 mm2) compared to the PBS

FIGURE 3. (A–F) Expression of retinal crybb2 (red) shown by immunohistochemical staining of slices (10 lm) of samples from animals treated withPBS (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 aremarked blue with DAPI. (F) The negative control is shown with cyanine (Cy-3) as a secondary antibody. Scale bar: 20 lm (A–F).

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

Grafted crybb2-NPC were found throughout the retinal layers, andin extraretinal tissue 2 and 4 weeks after grafting. AC, anteriorchamber.


group (9.5 6 1.0 cells/0.2 mm2, P < 0.01) and the crybb2group (8.17 6 1.33 cells/0.2 mm2) after 4 weeks (P < 0.05;Fig. 8F). The data showed that microglial cells appear to besensitive indicators of activation in all of the experimentalgroups of this study.

DISCUSSION

In our study we examined the fate of intravitreally graftedNPCs within the eye and the responses of retinal cells to thegrafting of either nontransfected or transfected NPCs exempli-fied by crybb2-NPC. There were three principal findings in thisstudy:

1. NPCs reside and migrate within the eye and continue toproduce NTFs.

2. Crybb2 and crybb2-NPC reduce the axotomy-inducedloss of RGCs and changes in photoreceptor rhodopsin.crybb2-NPC reduce glial reactivity, indicating thatcrybb2 is involved in retinal repair mechanisms.

3. Crybb2 reduces the recruitment and activation of retinalmicroglia, suggesting a reduced inflammatory response.

Intraocular Characteristics of NPCs

NPCs harvested from cultures and injected into the vitreouscavity were able to produce the same markers used tocharacterize them in culture. This finding suggested that the

host environment does not change the profile of proteinssynthesized within the vitreous body. This observation wascrucial for setting the basic data for injecting transfectedNPCs. We observed a migration of intravitreally appliedcrybb2-NPC within the entire eye for up to 4 weeks aftergrafting. A previous study demonstrated an overall integra-tion of NPCs into the neonatal retina of rats after 4 weeks.11

In agreement with our results, previous studies havedetected the retina as the main donor for implanted NPCsin cases of injured,2 ischemic,27 or degenerating retinas.10

The preferred location was the ONL, the subretinal debriszone, and the layer of photoreceptors.10 Our observationsrevealed the detection of transplanted crybb2-NPC in thevitreoretinal compartment, subretinal space, and choroidafter 2 weeks, while crybb2-NPC were found in the GCL,INL, and ONL after 4 weeks. Crybb2-NPC were associatedwith the anterior chamber, corneal endothelium, and ciliarybody after 4 weeks. To our knowledge, this is the firstdemonstration of an overall migration of grafted crybb2-NPCthrough the entire rat eye. Migration over long distances isnot surprising, since it has been shown that NPCs are ableto produce extracellular matrix–degrading metallopro-teases.8

Responses of the Ocular Tissue

Crybb2 was found to support the survival of axotomized RGCsdue to compression and prevent the dissolution of rhodopsin

FIGURE 4. (A–O) Staining for nestin-expressing NPCs (green) in sections throughout the eye. Two weeks after transplantation, grafted NPCs werefound throughout the retina (e.g., IPL) (A–C), including the subretinal space (D–F) and the vitreoretinal compartment (G–I). After 4 weeks, nestin-positive cells were detectable in the anterior chamber and corneal endothelium (J–L) and the epithelium of the ciliary body (M–O). NPCs werefound 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 lm (C, F, I,L, O, R).


in photoreceptors. Crybb2 usually is produced within the

regenerating retina and facilitates the elongation of regenerat-

ing 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 survivaleffects observed in our study.

Reduction of rhodopsin and its delocalization within thePOS is an early indication of degenerative onset.31 We foundthat crybb2-NPC delayed rhodopsin decay.

It has been assumed that growth-factor failure is one reasonfor photoreceptor death in inherited retinal photoreceptordegenerations.32 The external supply of growth factors, such asbFGF, protects photoreceptors.33 Higher levels of growthfactors, such as bFGF, ciliary NTF (CNTF), and BNDF,34 werefound to be associated with photoreceptor death in RCS

FIGURE 5. (A–F) Effects of crybb2 and crybb2-NPC on RGCs in the retina. Expression of neurofilament 200 (NF-200, red) shown byimmunohistochemical staining of slices (10 lm) of samples from animals treated with PBS (A, B) and crybb2-NPC (C, D) after 2 and 4 weeks. Cellnuclei 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 lm) 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 groupcompared 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 asdescribed were prepared and tested for brain-specific homeobox/POU domain protein 3b (BRN-3b, 45 kiloDaltons). Calnexin was used as theloading 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: 20lm (A–D). Statistical differences: **P < 0.01, ***P < 0.001.


FIGURE 6. (A–D) Expression of retinal rhodopsin (green) shown by immunohistochemical staining of slices (10 lm) of samples from animals treatedwith 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 loadedper lane. (J) Corresponding densitometric analyses of the WB results (n¼ 4) in PBS-, crybb2-, and crybb2-NPC–treated animals 2 and 4 weeks afterONC. The rhodopsin expression was lower in the crybb2-NPC group than in the crybb2 group after 2 weeks (P < 0.05). A significant reduction inrhodopsin expression was observed in the PBS group compared to the crybb2 (P < 0.001) and crybb2-NPC groups (P < 0.01) after 4 weeks. Therhodopsin expression was lowest in the PBS-treated group. Scale bar: 20 lm (A–H). Statistical differences: *P < 0.05, **P < 0.01, ***P < 0.001.


FIGURE 7. (A–H) Expression of retinal vimentin and GFAP, shown by immunohistochemical staining of slices (10 lm) of samples from animalstreated 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 describedwere prepared and tested for vimentin expression. Calretinin expression verified the amount of protein that loaded per lane. (J) Correspondingdensitometric analyses of the WB results (n¼ 4) in PBS-, crybb2-, and crybb2-NPC–treated animals 2 and 4 weeks after ONC in relation to control.


FIGURE 8. (A–D) Expression of ED-1 and Iba-1 in retinal microglial cells, as assessed by antibody staining of slices (10 lm) from animals treated withPBS (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 werefound 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-treatedgroups. The negative control is shown with Cy-2 and Cy-3 (D) as the secondary antibodies. (E, F) Quantification of retinal microglial cells. (E) Countof ED-1/Iba-1-stained cells in the retinal layers. (F) Count of Iba-1 positive microglia cells. Scale bars: 20 lm (A–D). Statistical differences: *P < 0.05,**P < 0.01.

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 lm (A–H). Statistical difference: *P< 0.05, **P < 0.01.


rats35,36 and rats with light-damaged retinas.37 It is likely thatthe factors produced by NPCs within the eye have a role inprotecting RGCs and photoreceptors from death. To this end,we have examined closely the overexpression of one of thesefactors, crybb2.

Crystallins maintain their effects on retinal proteins and arean important inhibitor of apoptosis due to toxic intermedi-ates.15 Increased levels of crystallins of the b/c superfamilywere found in animal models of retinal diseases, like retinitispigmentosa, light-induced damage, and diabetic retinopa-thy.38–40 Compared to observations of increased crybb2expression in regenerating retinas (e.g., GCL and INL) afterONC, no expression was found in unaffected eyes.26 This lineof evidence, together with our own findings, suggests thatcrybb2 exerts beneficial effects on damaged RGCs andsecondarily on photoreceptors. One of the mechanismsoperating through crybb2 may be an increase in the amountof neuroprotective CNTF released by astrocytes.16 To ourknowledge, no other studies have investigated the neuropro-tective effects of intravitreal crybb2 in vivo.

An early and significant increase in glial cell activity due tocrybb2 administration was found along with decreasedvimentin expression in the crybb2-NPC group. A complexglial network, including Muller cells and astrocytes, hasneuroprotective effects, releasing NTFs, such as bFGF, CNTF,and antioxidants after retinal injury.16,41,42

Early upregulation of vimentin and GFAP occurs in severalretinal disorders, such as retinopathia pigmentosa, maculardystrophies, or secondary retinal degenerations.43,44 and afterONC.45 The dynamic relationship between intermediatefilaments and retinal glial cells is the key mechanismunderlying the rapid modification of the Muller cell structurein response to changes in the retinal environment.46,47 It hasbeen shown that RGCs and astrocytes are donors of retinalcrystallins in vitro.23 Crystallins of the b/c superfamily arereleased during periods of critical stress in the retina.16,24

Increased retinal GFAP expression induces glial cells to switchinto a regenerative state after the intravitreal injection ofcrystallin b/c.16 Our observations confirmed the finding of adirect association between glial reactivity and crybb2. Thecrybb2-NPC–mediated effects on glial cells differ considerably.We concluded that additionally secreted NTFs by crybb2-NPCmay inhibit retinal glial activity.

NPCs and NTFs

While the exact mechanism underlying the effects of NPCswithin the retina are not understood fully, it is likely to bebased on growth-factor production.48 Retinal NTFs exertprotective effects on neighboring host neurons.49,50 SeveralNTFs delay RGC death and photoreceptor loss: fibroblastgrowth factors (e.g., bFGF), CNTF, brain-derived NTFs (e.g.,BDNF), and insulin-like growth factors. RGCs appear to bebetter protected by BDNF, while photoreceptors may be betterprotected by bFGF and CNTF.34 In our study, modified crybb2-NPC overexpressed crybb2 in addition to NTFs for up to 4weeks after transplantation in vivo.

Nestin is expressed during CNS stem-cell and progenitor-cell proliferation, and neuronal migration,51 and protectsprogenitor cells from apoptosis.33 BDNF, which is expressedby glial cells and neuronal cells,34,52 prolongs the survival ofRGCs in vitro, reduces RGC death during development, andexerts survival effects on injured adult neurons.33,53,54

Calcineurin is expressed in the RGCs of rat retinas and exertsimportant cellular effects in the immune and nervoussystems.55,56 It is involved in acute and chronic disordersresulting from glaucoma.56 Oncomodulin has a high affinity forRGCs, 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 withretinal degeneration, as shown in the RCS rat.59 These cellsrespond to several proinflammatory factors, including lipo-polysaccharides and cytokines.60,61 On the other hand, theyproduce and secrete NTFs, such as CNTF, GDNF, and nervegrowth factor, to protect photoreceptors from cell death.62

The number of activated microglial cells was highest inthe PBS group after 2 weeks, suggesting an increasedresponse to dying neurons and inflammatory disorders.Inflammatory cytokines may promote the transformation oframified to active microglia. The effect is more predictablecompared to that of crybb2, which we attribute to increasedcell death within the retina. An increased activation andrecruitment of microglia in the crybb2-NPC group after 4weeks was another interesting finding. An attenuated loss ofRGCs and photoreceptors is indicative of a lower rate ofretinal cell death in this group. Furthermore, several NTFssecreted by NPCs have inhibitory effects on the cellularimmune response. We suggest a microglial reaction to thecellular response of NPCs.

CONCLUSIONS

In conclusion, to our knowledge this the first study to showthe migration of intravitreally injected NPCs through the entirerat eye. This finding implies that NPCs are able to interact withdifferent types of ocular tissue. Crybb2-NPC prevents RGC lossand decay of rhodopsin in the injury-induced model of retinaldegeneration. In addition to their survival and migration withinthe eye, crybb2-producing NPCs increase the chances ofsurvival of RGCs and photoreceptors. The expression offurther survival-promoting factors may be introduced in thesecells, thus assuring a permanent and approximately constantdelivery within the eye. This strategy of creating factor-producing ‘‘cell-factories’’ in the neighborhood of areas ofdegeneration may become a fruitful approach for thetreatment of retinal disorders by providing bioavailable growthfactors.

Acknowledgments

M. Wissing and M. Langkamp-Flock provided skillful technicalassistance, and M. Reis typed the manuscript. English ScienceEditing (ESE) provided native linguistic editing of the manuscript.

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Crystallin-β-b2-Overexpressing NPCs Support the Survival of Injured Retinal Ganglion Cells and Photoreceptors in Rats