Glial Cell Line–Derived Neurotrophic Factor (GDNF)Promotes the Survival of Axotomized RetinalGanglion Cells in Adult Rats: Comparison to andCombination with Brain-Derived NeurotrophicFactor (BDNF)

Qiao Yan, Jue Wang, Christine R. Matheson, Janal L. Urich

Department of Neuroscience, Amgen, Inc., One Amgen Center Drive, Thousand Oaks,California 91320

Received 9 December 1997; accepted 2 September 1998

ABSTRACT: Adult rat retinal ganglion cells(RGC) undergo degeneration after optic nerve transec-tion. Studies have shown that exogenously applied neu-rotrophic factors such as brain-derived neurotrophicfactor (BDNF) can attenuate axotomy-induced as well asdevelopmental RGC death. Here, we examined whetherglial cell line–derived neurotrophic factor (GDNF), aknown neurotrophic factor for dopaminergic neuronsand motor neurons, could provide neurotrophic supportto RGC in adult rats. We determined whether RGCcould retrogradely transport GDNF from their targettissue. After injection into the superior colliculus ofadult rats, 125I-GDNF was retrogradely transported tocontralateral eyes but not to ipsilateral eyes. The trans-port of 125I-GDNF could be blocked by coinjection ofexcess unlabeled GDNF, indicating that it was receptormediated. We tested whether intravitreally appliedGDNF could prevent axotomy-induced RGC degenera-

tion. The RGC were prelabeled with Fluorogold (FG)and axotomized by intraorbital optic nerve transection.GDNF, BDNF (positive control), cytochrome c (negativecontrol), or a GDNF/BDNF combination was injectedintravitreally on days 0 and 7. On day 14, FG-labeledRGC were counted from whole-mount retinas. Wefound that, similar to BDNF, GDNF could significantlyattenuate the degeneration of RGC in a dose-dependentfashion. Furthermore, the combination treatment ofGDNF and BDNF showed better protection than eitherfactor used individually. Our data indicate that GDNF isa neurotrophic factor for the adult rat RGC. GDNF, likeBDNF, may be useful for the treatment of human RGCdegenerative diseases.© 1999 John Wiley & Sons, Inc. J Neuro-

biol 38: 382–390, 1999

Keywords:RGC; GDNF receptor; retrograde transport;axotomy; cell death

In adult rats, intraorbital optic nerve transectioncauses most of the retinal ganglion cells (RGC) todegenerate within 2 weeks (Villegas-Perez et al.,1993; Berkelaar et al., 1994). The axotomy separatesthe neuronal cell bodies from their targets and sup-porting tissues along their axons. Some axons of RGCare able to regenerate by sciatic nerve grafts, whichprovide a permissive environment for RGC axon

growth (Aguayo, 1985) and neurotrophic factors(Thanos et al., 1989). These neurons can survive,sprout axons, and re-form synapses in the superiorcolliculus under these conditions (Vidal-Sanz et al.,1987). The reestablishment of such connections and aconstant supply of neurotrophic support derived fromtheir targets or tissue along their axons appears toprevent further RGC loss.

Many lines of evidence indicate brain-derived neu-rotrophic factor (BDNF) is an endogenous neurotro-phic factor for RGC. BDNF mRNA is present in theretina (Perez and Canimos, 1995), optic nerve, and

Correspondence to:Q. Yan© 1999 John Wiley & Sons, Inc. CCC 0022-3034/99/030382-09

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superior colliculus (Conner et al., 1997). TrkB, thespecific tyrosine kinase receptor for BDNF, is ex-pressed by RGC (Jelsma et al., 1993; Perez and Cani-mos, 1995). Administration of exogenous BDNF tothe superior colliculus of neonatal hamster reducesdevelopmental RGC death (Ma et al., 1998). Further-more, intravitreal administration of BDNF (Mey andThanos, 1993; Mansour-Robaey et al., 1994; Peinado-Ramon et al., 1996) can rescue these neurons fromaxotomy-induced cell death. In addition to BDNF,other growth factors such as nerve growth factor(NGF) (Carmignoto et al., 1989) and NT-4/5 (Cui andHarvey, 1994; Cohen et al., 1994; Peinado-Ramon etal., 1996) have also been shown to prevent axotomy-induced RGC degeneration after intravitreal adminis-tration. Whether these protein factors act on RGC asendogenous neurotrophic factors has not been deter-mined.

Glial cell line–derived neurotrophic factor(GDNF) is a member of the transforming growthfactor-b (TGF-b) superfamily (Lin et al., 1993).GDNF has been shown to be a potent neurotrophicfactor for dopaminergic neurons (Beck et al., 1995;Tomac et al., 1995), motoneurons (Henderson et al.,1994; Oppenheim et al., 1995; Yan et al., 1995), anddorsal root ganglion neurons (Matheson et al., 1997).GDNF interacts with a specific cell-surface receptor,GDNFR-a and its biological effects are mediatedthrough the interaction of GDNF, GDNFR-a, and atyrosine kinase receptor, Ret (Jing et al., 1996;Treanor et al., 1996). In this study, we examinedwhether exogenous GDNF was retrogradely trans-ported by RGC from the superior colliculus, the targetof RGC. We then compared the rescue effect ofGDNF with BDNF on axotomized RGC. We foundthat exogenous GDNF was transported by RGC andprotected RGC from axotomy-induced cell death. Ourdata establish that GDNF is an important neurotrophicfactor for RGC. The preliminary results of this studywere published elsewhere in abstract form (Wang andYan, 1997).

MATERIALS AND METHODS

Materials

Recombinant human GDNF and BDNF were produced inEscherichia coli transfected with expression vectors andpurified by the Amgen Protein Chemistry group. GDNF wasiodinated by the lactoperoxidase technique (Marchalonis,1969) using Na125I (NEN/DuPont, Wilmington, DE).125I-GDNF was separated from free125I by a G-25 SephadexQuick Spin column (Boehringer Mannheim, Indianapolis,IN). The specific activity of125I-GDNF used in this ex-periment was 3700 cpm/fmol. Lactoperoxidase and other

chemicals were purchased from Sigma Chemical Co. (St.Louis, MO). NTB-2 autoradiographic emulsion was pur-chased from Kodak (Rochester, NY). Adult female (2–4months) Sprague–Dawley rats were purchased from CharlesRiver (Hollister, CA).

Retrograde Transport Study

A total of six adult female rats were anesthetized with acocktail containing 43 mg/mL of ketamine hydrochloride,8.6 mg/mL of xylazine, and 1.43 mg/mL of acepromazine ata dose of 0.7 mL/kg body weight. One microliter of125I-GDNF (2.423 106 cpm) with or without a 110-fold excessof nonlabeled GDNF was injected over 1 min into the centerof the right superior colliculus (interaural 2.2 mm, lateral1.5 mm, 3.6 mm below the skull) (Paxinos and Watson,1986) using a 5-mL Hamilton syringe. About 20 h later,animals were sacrificed by inhalation of CO2. Eyes wereremoved and immersion fixed in 4% paraformaldehyde,2.5% glutaraldehyde, 0.1M sodium phosphate buffer, pH7.2, for 1 h. The radioactivity of each eye was measured bya g-scintillation counter. The mean number of counts accu-mulated in the eyes was calculated for each treatment groupand means were compared using analysis of variance(ANOVA). The eyes were then cryoprotected with 20%sucrose in phosphate-buffered saline (PBS) and sectionedthrough the optic disc in the vertical axis at 10mm using acryostat microtome. Sections were mounted on glass slidesand dipped in Kodak NTB-2 emulsion, exposed for 15 days,and counterstained with Toluidine blue.

RGC Labeling, Optic Nerve Transection,and Intravitreal Injection

To evaluate thein vivo neuroprotective effect of GDNF onRGC, we used an animal model of optic nerve axotomydescribed by Mansour-Robaey et al. (1994). Adult femalerats were anesthetized with ketamine cocktail as describedabove. RGC were retrogradely labeled by applying Gel-foam-soaked Fluorogold (FG) (2% FG in 0.9% NaCl, 10%dimethyl sulfoxide; Florochrome, Englewood, CO) to thesurface of both superior colliculi. Seven days after the FGapplication, the right optic nerve was transected intraorbit-ally at about 0.5 mm from the eye. Immediately followingthe optic nerve transection, 1mL of test sample was injectedinto the vitreous space using a 10-mL Hamilton syringe witha 30-gauge needle through the pars plana at the temporalside of the eye. The test samples were GDNF, BDNF,cytochrome c (1 or 5mg), or a GDNF/BDNF combination(1 mg 1 1 mg or 5 mg 1 5 mg) in PBS. The injection wasrepeated at day 7 postoptic nerve transection. The injectionprocedure was monitored by stereo microscopy, and ani-mals with eye bleeding or lens injury due to the injectionprocedure were excluded. Optic nerve transection resultedin activation of microglia/macrophages which took in Flu-orogold released by injured RGC. To positively identifythese cells from RGC, we made cross sections by eithervibratome or cryostat microtome from axotomized retinaswith or without the treatment of BDNF, GDNF, or cyto-

Effect of GDNF on RGC 383

chrome c. We stained the sections with either OX42 anti-body (anti-CD11b/c; PharMingen, CA) or fluorescein-la-beled griffonia simplicifolia lectin 1 (Vector Labs, CA).Both probes are known to label microglia/macrophage incentral nervous system (CNS) tissue. We saw strong Fluo-rogold labeling of cells in the retinal ganglion cell layer, butno fluorescent staining with OX42 antibody or the fluores-cein isothiocyanate (FITC)-lectin. We tried many times andwith several different protocols, but were unable to doublelabel the Fluorogold-labeled spindle-shaped cells. Althoughwe could not explain why these reactivated microglia cellsdid not stain with microglia markers in this study, based oncriteria in the published literature (Mansour-Robaey et al.,1994; Huxlin et al., 1995; Peinado-Ramon et al., 1996), webelieved that these brightly labeled, spindle-shaped cellswere microglia or macrophages.

Analysis of RGC Survival

Animals were sacrificed at day 14 postoptic nerve transec-tion by inhalation of CO2, and the orientation of each eyewas marked. Eyes were removed and immersion fixed in 4%paraformaldehyde in 0.1M sodium phosphate, pH 7.2, for1 h at room temperature. The retinas were dissected, orien-tation marked, and whole mounted on glass slides. Eachretina was divided into superior, inferior, nasal, and tempo-ral quadrants. The retina was examined and photographedunder a fluorescent microscope. Three pictures were takenat325 magnification with Kodak Ektachrome ISO 400 filmfrom each retinal quadrant centered at 1, 2, and 3 mm off theoptic disc. A total of 12 pictures were taken from eachretina. The number of FG-labeled RGC was counted asrelatively round somata with dendritic processes from aslide-projected image with 0.373 0.53 mm2 area of retina.The spindle-shaped, FG-positive microglia could be easilyrecognized from these photographs and were not counted.The person performing the counting was blinded to thetreatment groups.

RESULTS

Retrograde Transport of GDNF fromSuperior Colliculus to RGC

Long-distance–projecting neurons derive some oftheir neurotrophic support from their target throughretrograde transport. As an initial step in determiningwhether adult RGC may express GDNF receptor andrespond to GDNF biologically, we tested whetherRGC could retrogradely transport GDNF from theirtarget in a receptor-mediated fashion. About 20 h afterinjection of 125I-GDNF into the superior colliculus,radioactivity accumulated in the contralateral but notthe ipsilateral eyes, as measured by ag-counter (Fig.1). Since the majority of RGC project to the contralat-eral superior colliculus in the rat, the selective accu-mulation in the contralateral eyes indicated the125I-

GDNF was transported by axons rather than bydiffusion or leakage into the circulation. Coinjectionof excess nonlabeled GDNF could block this accumu-lation, indicating the retrograde transport was specificand receptor mediated.

Emulsion autoradiography of frozen sections ofeyes was examined to confirm that radioactivity wasassociated with RGC soma and axons (Fig. 2). Bothdark-field and bright-field microscopy confirmed thatsilver grains overlaid the RGC cell bodies and theoptic nerve composed of RGC axons.

GDNF Rescue RGC from Axotomy-Induced Cell Death

The retrograde transport of GDNF by RGC promptedus to look for potential neuroprotective effects ofGDNF on RGC. A reproducible cell death occursafter intraorbital optic nerve axotomy in adult rats,making such a model ideal for assessing the survival-promoting activity of factors on RGC. It has beenshown previously that although axotomy-inducedRGC degeneration is an apoptotic process (Berkelaaret al., 1994) and that RGC become committed todegeneration early, the majority of RGC degenerationoccurs within 2 weeks (Villegas-Perez et al., 1993;Berkelaar et al., 1994). Consistent with the literature,we found only about 50% of the RGC degenerated 7

Figure 1 Quantification of the retrograde transport of125I-GDNF from the right superior colliculus to eyes. Theamounts of retrogradely transported125I-GDNF are ex-pressed as the mean counts per min (6S.E.M.) in the eye (n5 3). 125I-GDNF was significantly accumulated in the eyescontralateral to the injection side compared to the ipsilateraleyes (paired Studentt test, two-tailed, *p , .0001). Theretrograde transport was blocked by coinjection of excess ofcold GDNF.

384 Yan et al.

days after axotomy in a preliminary study (notshown), but over 90% of the RGC degenerated by 2weeks. In this study, we therefore examined RGCsurvival 2 weeks after optic nerve axotomy.

The effects of axotomy and drug treatments areshown in Figure 3. Optic nerve axotomy caused dra-matic RGC degeneration [Fig. 3(A) vs. Fig. 3(B)]. Inthe axotomized eyes with no treatment [Fig. 3(B)] orcytochrome c treatment [Fig. 3(F)], the majority offluorescent cells were microglia which were highlyfluorescent and spindle shaped. Retinas treated withGDNF and BDNF showed many more surviving RGCafter axotomy than cytochrome c–treated controls.

The effects of neurotrophic factor treatment onRGC were quantified by counting FG-labeled RGC.

The density of RGC is high in the center and low inthe peripheral retina [Fig. 4(A)]. Axotomy causedrelatively more RGC degeneration in the central retinathan in the peripheral retina, and the surviving RGCwere relatively evenly distribution throughout the ret-ina. Proportionally, both GDNF and BDNF hadgreater rescue effects on the peripheral RGC than thecentral RGC [Fig. 4(A)]. There was no significantdifference in the RGC numbers derived from thesimilar region (same distance from the optic disc) ofeach quadrant of retina (not shown), indicating theaxotomy and neurotrophic factor treatment affectedfour retinal quadrants equally. To make the quantita-tive comparison easier, we pooled cell counts from 12areas of each retina [Fig. 4(B)] (see Materials and

Figure 2 Dark-field autoradiograms of the retrograde transport of125I-GDNF to the eye. (A)shows the accumulation of125I-GDNF in the eye contralateral to the injection side. The silver grainsare concentrated over the retinal ganglion cell bodies in the retinal ganglion cell layer (RGC) andover retinal ganglion cell dendrites in the inner plexiform layer (IPL). The silver grains also heavilyaccumulated over the optic nerve composed of the axons of retinal ganglion cells (D). Few or nosilver grains are seen in the ipsilateral eye (B), which is consistent with the topographic projectionof retinal ganglion cells in rats. The accumulation of125I-GDNF was blocked by coinjection ofexcess of nonlabeled GDNF (C), confirming that the retrograde transport of125I-GDNF is receptormediated. INL5 inner nuclear layer; LENS5 lens; ON5 optic nerve; ONL5 outer nuclear layer.Scale bar [in (D)]: 125mm for (A–C), 250mm for (D).

Effect of GDNF on RGC 385

386 Yan et al.

Methods). Axotomy caused over 90% RGC degener-ation in retinas without treatment. GDNF treatmentresulted in a dose-dependent significant rescue ofaxotomized RGC: 43.8% survival with 1mg treatmentand 47.8% with 5mg treatment. BDNF treatmentshowed greater effects than GDNF at similar doses:48.9% with 1 mg treatment and 61.1% with 5mgtreatment. Retinas treated with cytochrome c showeda small increase (17.4%) in RGC survival comparedto nontreated eyes (8.8%), suggesting that the injec-tion itself might induce the release of some endoge-nous neurotrophic activity in the eye. Since the neu-rotrophic effects of GDNF and BDNF are mediatedthrough different receptor systems and neither mole-cule at high doses can rescue all the RGC, we exam-ined the effect of a combination of GDNF and BDNFtreatment. The combination of these two moleculesresulted in better rescue of RGC than either moleculeused alone: 63.8% with 1mg combination treatmentand 75.2% with 5mg combination treatment. At the1-mg dose level, a combination of BDNF and GDNFresulted in a statistically significant enhancement ofRGC survival, while at the 5-mg dose level, the com-bination was better than GDNF alone but not statis-tically significant better than BDNF alone. It shouldbe pointed out that the combination of 1mg of BDNF1 1 mg of GDNF did not differ significantly from thebest treatment with 5mg BDNF 1 5 mg GDNF or 5mg of BDNF alone. Since the rescue effect of com-bination treatment was less than the sum of individualrescue effects, there must be some overlap betweenthe subpopulations of RGC rescued by GDNF and byBDNF.

DISCUSSION

We have shown here that adult rat RGC could uptakeand retrogradely transport125I-GDNF from their tar-get, the superior colliculus, to their cell bodies. Sincethis transport was blockable by coinjection with ex-cess of nonradioactive GDNF, indicative of limitedbinding sites on the nerve terminals, our data demon-strated that adult RGC express functional GDNF re-

Figure 4 Effects of GDNF and BDNF treatment on RGCsurvival after optic nerve axotomy. (A) The density of RGCin normal rat eyes (Norm.) shows a central to peripheralgradient of distribution. Optic nerve axotomy caused RGCdegeneration throughout the retina in uninjected eyes (Un-inj.). Cytochrome c injection (Cyto c) resulted in slightlymore RGC survival compared to uninjected eyes. BDNF orGDNF (5 mg) treatment resulted in a clear protection ofRGC. 1 mm, 2 mm, and 3 mm5 distance between thecenter of the optic nerve head and the area of the retinaexamined. Data are expressed as mean numbers of RGC perretinal area6 S.E.M. (error bar in the Uninj. group is therange) and the number of animals used are indicated. (B)Cell counts from 12 areas of each retina are pooled (seeMaterials and Methods) and data presented are mean6 S.E.M. (error bar in the Uninj. group is the range) of allanimals used for each treatment. The data were analyzed byANOVA followed by Dunnettt tests. *p , .001 comparedto cytochrome c–treated group.#p , .05 compared to 1-mginjection of BDNF or GDNF.¶p , .01 compared to 5mg ofGDNF treatment, but not significant in comparison to 5mgBDNF treatment.

Figure 3 Fluorogold-labeled RGC in whole mounted retinas (2 mm away from the optic nervehead): (A) control retina with no axotomy and no treatment. (B) Axotomy with no treatment. (C)Axotomy with 5mg of BDNF treatment. (D) Axotomy with 5mg of GDNF treatment. (E) Axotomywith 5 mg of BDNF and 5mg of GDNF combination treatment. (F) Axotomy with 5mg ofcytochrome c treatment. Axotomy with no treatment resulted the death of the majority of RGC (B);the intensely fluorescent microglial cells with their characteristic spindle shape can be easilyidentified. Some representative microglial cells in (B,F) are marked by arrowheads. Some repre-sentative RGC in (A,C–E) are marked by arrows. Scale bar [in (F)]5 50 mm for all panels.

Effect of GDNF on RGC 387

ceptor. Recent studies show that GDNF interacts witha GPI-linked cell-surface receptor called GDNFR-a(Jing et al., 1996; Treanor et al., 1996). In turn,GDNFR-a together with bound GDNF interacts withthe tyrosine kinase receptor Ret. Binding of GDNF tothe cell-surface receptor activates the Ret tyrosinekinase (Jing et al., 1996; Treanor et al., 1996). Dataavailable so far suggest that GDNF needs to interactwith GDNFR-a first; then it may interact with Retdirectly. In the developing eye of 13.5-day-old mouseembryos, Ret mRNA expression was localized toRGC, amacrine, and horizontal cells (Pachnis et al.,1993). The expression of GDNFR-a and Ret in theeye of adult animals remains to be studied.

If GDNF acts as a target-derived neurotrophic fac-tor for RGC, as the results of our retrograde transportand optic nerve axotomy studies suggested, one willexpect the expression of GDNF in the target region ofRGC. The expression of GDNF in the CNS has beenshown to be relatively limited (Pochon et al., 1997;Trupp et al., 1997). In developing animals, GDNF isreadily detected in the striatum, the target of dopami-nergic neurons of the substantia nigra, but not in thehippocampus and the cortex (Stromberg et al., 1993).The expression of GDNF in the striatum of adultanimals is down-regulated and below the level ofdetection (Stromberg et al., 1993). The expression ofGDNF can be up-regulated with epilepsy-inducingagents such as kainate or pilocarpine (Humpel et al.,1994). Under those conditions, GDNF mRNA isfound in the striatum, hippocampus, and cortex ofadult animals. So far, no study has reported the ex-pression of GDNF in the superior colliculus and lat-eral geniculate, two targets of RGC or the optic nerve(Trupp et al., 1997). It is entirely possible that theGDNF expression level is below the limits of currentdetection methods. Whether GDNF expression in thetargets of RGC can be up-regulated after injury or byepilepsy-inducing agents remains to be demonstrated.RGC may also have access to GDNF secreted fromthe tissue along the optic nerve or from tissue withinthe eye, although there are not yet reports of GDNFexpression in these tissues. More recently, two newmembers of the GDNF family, neurturin and perse-phin, have been identified (Kotzbauer et al., 1996;Milbrandt et al., 1998). GDNF, neurturin, and perse-phin share about 40% protein homology with eachother. It has been demonstrated that neurturin can alsointeract with GDNFR-a and Ret and has similar bio-logical activities as GDNF (Kotzbauer et al., 1996).On the other hand, persephin interacts with Ret butnot GDNFR-a, and has overlap with as well as dif-ferent biological activities from GDNF and neurturin(Milbrandt et al., 1998). If future studies show thatneurturin or persephin is expressed in the target areas

of RGC, it may function as an endogenous target-derived neurotrophic factor for RGC.

The rescue effect of intravitreally administratedGDNF on axotomized adult RGC is dose dependent.With 5 mg GDNF treatment, the highest dose used inthis study,,50% of the RGC were rescued. Quanti-tatively, BDNF showed a better rescue effect thanGDNF. More than 50% of RGC were rescued by 5mgBDNF treatment. Since we only used two doses ofGDNF and two injections owing to the relativelyinvasive nature of intravitreal injection in this study, itpossible that we did not reach the optimal dosage orfrequency of dosing with GDNF. It is interesting tonote that neurotrophic factor treatment had a biggerpositive effect on the peripheral RGC than the centralRGC. Since optic nerve lesion resulted in a closeraxotomy for the central RGC than peripheral ones, itis likely that neurotrophic factor treatment was lesseffective on more severely damaged neurons. It is alsopossible that the length of remaining axon after theaxotomy may influence the uptake of exogenous aswell as endogenous neurotrophic factors.

Brain-derived neurotrophic factor and GDNF usedifferent receptor systems. The fact that neither ofthem used individually can rescue much more than50% of axotomized RGC prompted us to examine theadditive or synergistic effects of these two moleculesapplied simultaneously. The combination treatmentresulted in better than either single molecule treatedalone (at the 1-mg level). The effect was partial addi-tive but certainly not synergistic. It is unlikely that thelack of substantially additive or synergistic effect isdue to overlapping expression of BDNF receptor andGDNF receptor in a subpopulation of RGC, since ithas been shown that almost all RGC express TrkBmRNA (Jelsma et al., 1993) and the majority of RGCexpress Ret (Pachnis et al., 1993) and transport125I-GDNF. It is possible that the effects of BDNF andGDNF on RGC can converge by saturation of anintracellular signal pathway which resulted in a par-tially additive effect, since both TrkB and Ret aretyrosine kinase receptors and both can activate a mi-togen-activated protein kinase signal transductionpathway (Marsh et al., 1993; Worby et al., 1996). Onthe other hand, the RGC population is heterogeneous,with many different sizes of cell bodies which sub-serve many different physiological functions. It ispossible that each different RGC subpopulation hasdistinct survival factor requirements. BDNF andGDNF may act on an overlapping RGC subpopulationwith similar neurotrophic factor requirements. Futureexperiments are required to verify these possibilities.

This study has added GDNF to the list of neuro-trophic factors such as BDNF (Johnson et al., 1986;Mansour-Robaey et al., 1994; Peinado-Ramon et al.,

388 Yan et al.

1996; Ma et al., 1998), NT-4/5 (Cui and Harvey,1994; Cohen et al., 1994; Peinado-Ramon et al.,1996), NGF (Carmignoto et al., 1989), ciliary neuro-trophic factor (Mey and Thanos, 1993), and fibroblastgrowth factor (Sievers et al., 1987), capable of pro-moting the survival of RGC. Most of these studieswere conducted with cultured embryonic or earlypostnatal RGC, naturally occurring RGC death inneonatal animals or with optic nerve axotomy to in-duce RGC death in young or adult animals to evaluateneurotrophic activity on these neurons. Thesein vitroand in vivo models permit a rapid analysis of RGCdeath and provide valuable information on the poten-tial candidates for future clinical use to prevent ortreat RGC degeneration in human diseases. However,the long-term effect on RGC and therapeutic useful-ness of these molecules need to be studied further inmore clinically relevant animal models.

In conclusion, this study demonstrated receptor-mediated uptake and retrograde transport of GDNFfrom the target field of RGC to their cell bodies.Intravitreally applied GDNF was effective in prevent-ing the axotomy-induced RGC degeneration. GDNFmay be potentially useful for the treatment of humandiseases such as glaucoma, which involve RGC de-generation.

The authors thank the Protein Chemistry Group at Am-gen for the recombinant GDNF and BDNF, and Dr. AndyWelcher for proof reading the manuscript.

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