205 (2007) 48–55www.elsevier.com/locate/yexnr

Experimental Neurology

Erythropoietin is both neuroprotective and neuroregenerative following opticnerve transection

Carolyn E. King a, Jennifer Rodger a,b, Carole Bartlett a, Tammy Esmaili a,Sarah A. Dunlop a,b, Lyn D. Beazley a,b,⁎

a School of Animal Biology, University of Western Australia Nedlands, 6009, Western Australiab Western Australian Institute for Medical Research, University of Western Australia Nedlands, 6009, Western Australia

Received 6 September 2006; revised 19 December 2006; accepted 12 January 2007Available online 25 January 2007

Abstract

The cytokine hormone erythropoietin (EPO) is neuroprotective in models of brain injury and disease, and protects retinal ganglion cells (RGC)from cell death after axotomy. Here, we assessed EPO's neuroprotective properties in vivo by examining RGC survival and axon regeneration at4 weeks following intraorbital optic nerve transection in adult rat. EPO was administered as a single intravitreal injection at the time of transection(5, 10, 25, 50 units, PBS control). Intravitreal EPO (5, 10 units) significantly increased RGC somata and axon survival between the eye andtransection site. Twenty five units did not improve survival of RGC somata but did increase axon survival between the eye and transection site. Inaddition, a small proportion of axons penetrated the transection site and regenerated up to 1 mm into the distal nerve. In a second series, intravitrealEPO (25 units) doubled the number of RGC axons regenerating along a length of peripheral nerve grafted onto the retrobulbar optic nerve. Our invivo evidence of both neuroregeneration and neuroprotection, taken together with the natural occurrence of EPO within the body and its ability tocross the blood–brain barrier, suggests that it offers promise as a therapeutic agent for central nerve repair.© 2007 Elsevier Inc. All rights reserved.

Keywords: Erythropoietin; Optic nerve; Transection; Peripheral nerve graft; Retina; Rat

Introduction

A therapeutic agent for repair of the damaged or diseasedmammalian central nervous system (CNS) would ideally com-bine neuroprotective and neuroregenerative properties. Severalstudies have demonstrated agents that offer neuroprotection inthe absence of neuroregeneration, an example being axotomizedretinal ganglion cells (RGCs) transduced to up-regulate extra-cellular signal-related kinases 1/2 (Erk1/2, (Pernet et al., 2005)).However, increased cell survival may not be associated withneuroregeneration, examples being axotomized RGCs treatedwith fibroblast growth factor-2 (Sapieha et al., 2003) or dibutyrlcyclic AMP, amembrane-permeable cAMP analogue (Monsul et

⁎ Corresponding author. Neurobiology Laboratory, School of Animal Biology,University of Western Australia, Nedlands, 6907, Western Australia. Fax: +61 86488 1029.

E-mail address: lynb@cyllene.uwa.edu.au (L.D. Beazley).

0014-4886/$ - see front matter © 2007 Elsevier Inc. All rights reserved.doi:10.1016/j.expneurol.2007.01.017

al., 2004). Moreover, a recent study has shown that RGCneuroprotection may be induced at the expense of neuroregen-eration. Brain-derived neurotrophic factor (BDNF) acts syner-gistically with lens injury to promote RGC survival. However,the presence of BDNFmasks the neuroregenerative properties oflens injury, leading to the suppression of axon regeneration andoptic nerve dystrophy (Pernet and Di Polo, 2006).

We here test the possibility that the cytokine hormoneerythropoietin (EPO) can confer both neuroprotection andneuroregeneration. The neuroprotective properties of EPO arewell established. In addition to its role in the propagation oferythroid cells, the neuroprotective properties of EPO have beendemonstrated in experimental CNS models as disparate as RGCaxotomy (Weishaupt et al., 2004), autoimmune-mediated in-flammation (Agnello et al., 2002), Parkinson's disease (Kanaanet al., 2006), hypoxic/ischemic insults (Sun et al., 2005), excito-toxicity (Kawakami et al., 2001; Morishita et al., 1997), oxi-dative stress (Calapai et al., 2000; Sakanaka et al., 1998),

49C.E. King et al. / Experimental Neurology 205 (2007) 48–55

chemically induced neurotoxicity (Genc et al., 2001), cerebralmalaria (Kaiser et al., 2006), retinal reperfusion injury (Liu et al.,2006), photoreceptor degeneration (Grimm et al., 2006) andtraumatic brain injury (Brines et al., 2000; Gorio et al., 2002).EPOmay also be valuable in treating stroke. It has been shown toreduce infarct size in a clinical trial of cerebral ischemia(Ehrenreich et al., 2002) with promising current clinical trialsawaiting validation on a larger scale (Hasselblatt et al., 2006).EPO also seems to stimulate neurogenesis and post-strokerecovery (Tsai et al., 2006), compatible with the finding thatEPO-R knockout mice display widespread apoptosis as well asreductions in progenitor cell numbers (Yu et al., 2002) andneurogenesis (Tsai et al., 2006).

The potential of EPO as an agent for neuroregeneration, asopposed to neuroprotection, has received far less attention.However, recent findings have suggested that EPO isneuroregenerative. In vitro studies have reported that EPOstimulates neuritic outgrowth by postnatal (Bocker-Meffert etal., 2002) and adult RGCs (Kretz et al., 2005). In addition, afterspinal cord lesion, greater functional recovery was seen in EPOtreated animals compared to controls, although the result mightbe explained by mechanisms other than axon regeneration(Celik et al., 2002; Grasso et al., 2006). In the peripheralnervous system, EPO administration reduced axon degenerationin peripheral neuropathies (Keswani et al., 2004) and inducedincreased regeneration of the cavernous nerve (Allaf et al.,2005).

To test in vivo whether exogenously administered EPO issimultaneously neuroprotective and neuroregenerative in theCNS, we have employed the model of complete intraorbitaltransection of the adult rat optic nerve: the surgery isunequivocal and neuroprotection can be readily quantifiedseparately from neuroregeneration. Normally, fewer than 10% ofRGCs survive for 1 month after axotomy induced by intraorbitaloptic nerve lesion (Berkelaar et al., 1994; Mey and Thanos,1993). Moreover, after an abortive attempt to regenerate theiraxons within the first few days, axons retract from thetransection site with the formation of a growth inhibitory glialscar (Beazley and Dunlop, 1999; DeFelipe and Jones, 1991).

Following optic nerve transection, we administered a range ofconcentrations of EPO intravitreally at the time of surgery andexamined animals after 4 weeks. From these data, wedetermined the optimal dose(s) for neuroprotection of RGCsomas and for any neuroregeneration beyond the injury site.

Furthermore, we performed two further experiments; asbefore, we examined animals at 4 weeks. We examined theeffect of EPO administration, adopting the optimal intravitrealEPO dose for neuroregeneration (25 units) in animals with aperipheral nerve graft (PNG). In this model, the normal opticpathway is replaced with a length of peripheral nerve sutured tothe retrobulbar portion of the optic nerve (David and Aguayo,1981; King et al., 2006). A proportion of RGCs regenerate theiraxons into the graft with a small proportion even formingconnections in the brain (David and Aguayo, 1981; Sauve etal., 2001; Vidal-Sanz et al., 1991). In the other series, weapplied EPO to the transection site in a gelfoam cuff todetermine whether EPO was neuroprotective and/or neuror-

egenerative via this mode of administration. The results of ourexperiments provide the first in vivo demonstration that EPOexogenously administered intravitreally is both neuroprotectiveand neuroregenerative. The findings offer the possibility thatEPO may be a valuable therapeutic agent for central nerverepair, bearing in mind that it occurs naturally in the body and isable to cross the blood–brain barrier (Juul et al., 2004;Xenocostas et al., 2005).

Materials and methods

Animals and surgery

Animals and anesthesiaFifty eight female PVG hooded rats (180–200 g bw; Animal

Resources Centre) were studied. Anesthesia for surgery andintravitreal injections was by intraperitoneal injection of Iliumxylazil-20 (Troy Laboratories, Australia, Pty. Ltd, 6 mg/kg) plusketamine (Parnell Laboratories, Australia, Pty. 50 mg/kg, i.p.);terminal anesthesia was with pentobarbitone sodium (600 mg/kg bw, i.p.). Animals were excluded if the lens or retina showedsigns of direct injury at the time of surgery or showed abnormalmicroglial activity in the retina indicated immunohistochemi-cally by ferritin labeling. Animals were maintained in standardhousing conditions and procedures were approved by theInstitutional Animal Care and Use Committee.

One experimental group underwent right optic nervetransection immediately followed by injection of 5, 10, 25 or50 units of human erythropoietin (Epoeitin alpha; EPO;Johnson Pharmaceuticals, New Jersey), in 2.5 μl sterilephosphate buffered saline (PBS) to the vitreal cavity of theright eye (n=5 for each group). Controls underwent optic nervetransection plus intravitreal injection with PBS (2.5 μl; n=7). Asecond series underwent optic nerve transection followed byperipheral nerve grafting. The animals received either intravi-treal EPO (25 units in 2.5 μl PBS) or 2.5 μl PBS as a control(n=5 per group). The dose chosen conferred, after optic nervetransection, the greatest protection against RGC axon degen-eration within the proximal nerve segment (Fig. 2). Normal(non-transected, non-injected) and normal injected (25 units ofEPO; non-transected) animals were also investigated (n=7 pergroup).

Optic nerve transectionRats were anesthetized, the right optic nerve exposed

intraorbitally and a transverse slit made in the dorsal nervesheath approximately 1 mm behind the eye. The nerve paren-chyma was then lifted and completely transected with iridec-tomy scissors and the opening in the nerve sheath closed using10-0 suture thread. The ventral aspect of the nerve sheathremained intact to avoid damaging the ophthalmic circulation.Following surgery, integrity of the retinal vasculature wasconfirmed ophthalmoscopically and rats with ischemic retinaediscarded. Completeness of transection was confirmed in testanimals (n=3) 4 days after surgery by anterograde labelingwith DiI. Rats were maintained under normal conditions for4 weeks.

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Peripheral nerve graftSurgery was performed as described previously (King et al.,

2006). The left common peroneal nerve was crushed 7–9 dayspreviously to improve the efficacy of the graft to support RGCaxon regeneration (You et al., 2002). The proximal end of the2.5 cm peroneal nerve segment was anastomosed to the opticnerve stump and the distal end inserted into the contralateralsuperior colliculus, having aspirated the overlying occipital cortex.

Intravitreal injectionEPO or PBS solutions were administered to the vitreous via a

stereotactically positioned 30-gauge needle attached to a 10 μlHamilton syringe. A final volume of 2.5 μl was administered viaperipheral nasal (1.25 μl) and temporal (1.25 μl) retinal sitestaking care to avoid contact with the lens since its injury releasesneuroregenerative factors (Fischer et al., 2000). The eye wasexamined ophthalmoscopically to check that retinal vascularwas intact.

Gelfoam cuff applicationA cuff of gelfoam infiltrated with 2.5 μl EPO (25 units) or

PBS was placed around the nerve sheath at the transection siteto encapsulate the nerve immediately post nerve transection.

Tissue preparation

Terminally anesthetized rats were perfused transcardiallywith 0.9% sodium chloride followed by freshly prepared 4%paraformaldehyde in 0.1 M phosphate buffer (pH 7.2). Righteyes and optic nerves, or peripheral nerve grafts were dissectedand post-fixed in 4% paraformaldehyde for 24 h. Retinae wereprepared as wholemounts while nerves (or peripheral nervegrafts) were cryoprotected (immersion 15% sucrose o/n),serially cryosectioned (16 μm) horizontally, collected ontoSuperfrost® Plus glass slides and stored at −80 °C.

Immunohistochemistry

Free floating retinae, permeabilized with PBS containing 1%Triton X-100, were incubated overnight in a humid chamber at4 °C with TUJ1 βIII tubulin monoclonal antibody (Covance,Berkeley CA), a specific maker for RGCs (Cui et al., 2003), andwith ferritin polyclonal antibody (Dako Corporation, Carpen-teria, CA), a marker for macrophages/microglial; dilutions were1:500 and 1:1000, respectively, in PBS+1.0% Triton X-100.Following PBS washes, antibody binding was visualized withanti-mouse (1:400, Alexa fluor 488, Molecular Probes, Eugene,OR) and anti-rabbit (1:400, Alexa fluor 546, Molecular Probes,Eugene, OR) secondary antibodies. Retinae were whole-mounted on glass slides, air dried and coverslipped usingFluoromount G (Southern Biotechnology Associates, Inc).

Sections of optic nerves and peripheral nerve grafts were re-hydrated (PBS+0.2% Triton X-100, 10 min) followed byovernight incubation with TUJ1 primary antibody (1:500 inPBS+0.2% Triton X-100) and visualized as described forretinae. Labeling was viewed by conventional fluorescencemicroscopy (Lietz Diaplan).

Analysis

Analysis was performed by two independent observers blindto the treatment group.

RetinaCounts of RGC somas were undertaken in 6 non-overlapping

rectangular sample fields of 250 μm×250 μm; 3 fields wereanalyzed in both nasal and temporal quadrants (reflectingquadrants in which EPO was administered) at eccentricities of1, 2 and 3 mm from the center of the optic disk. Cell counts weremade at 400× magnification. The area sampled corresponds to6× (250×250)=375,000 μm2 or 0.375 mm2. The naso-temporal half of the retina was sampled, corresponding to atotal area of roughly 30 mm2. The sampling rate within the halfretina is therefore (0.375×100) /30=1.25%.

RGC densities/mm2 were calculated and expressed as % ofPBS-injected controls. Retinal area, estimated using Image ProPlus, did not vary significantly between animals (EPO series:57.50 mm2±3.00; control series: 60.60 mm2±1.90; p>0.05).

Transected optic nervesAnalysis was performed at two sites in the proximal nerve

(between the retina and injury site), these beingwithin 200 μmofthe back of the eye and immediately before the transection site.The distal nerve (beyond the injury site) was also examined.Abundance of TUJ1 labeled axons was assessed by a scoringsystem (1–3).

Peripheral nerve graftsMean RGC axon densities were calculated having counted

axons across the width of both the optic nerve stump and thePNG (n=3–5 sections per animal). Counts were performedfrom the optic nerve head to a distance of 5 mm within thenerve graft at 500 μm intervals according to Gillon et al.(2003). Axon densities were averaged across the nerve at eachinterval.

Statistical analysis

Mean densities of RGC somas were analyzed using ANOVA(StatView) and significance values calculated using the Scheffepost hoc test. The number of RGC axons per mm2 was analyzedusing a non-parametric Mann–Whitney U-test.

Control animals

To confirm the completeness of nerve transection, RGCaxons leaving the eye were traced anterogradely with DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate,Molecular Probes, Eugene, OR, USA) after optic nerve tran-section; no accompanying intravitreal injections were per-formed. Four days later, animals were transcardially perfusedwith fixative as before and DiI was placed on the optic nervehead and allowed to transport at 37 °C for 4 weeks. Followingcryoprotection by immersion in 15% sucrose overnight,peripapillary retina with optic nerves attached was sectioned

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horizontally (20 μm), collected onto Superfrost® Plus glassslides and examined by fluorescence microscopy.

We also confirmed a previous demonstration (Cui et al.,2003) that TUJ1 specifically labels RGC somas and axons,leaving displaced amacrine cells (Perry, 1981) unlabeled.Labeling with TUJ1 βIII tubulin monoclonal antibody (proce-dure as before) was compared with that resulting from retrogradetracing using fluorogold (Fluorochrome Inc., Denver, CO).Fluorogold was applied to the optic nerve approximately 2 mmbehind the eye 3 days before terminal anesthesia. There was analmost 100% correlation between the two methods withcomparable labeling of RGC somas along with RGC axonlabeling. Mean RGC soma densities in wholemounted retinaewere 2145±179 cells/mm2, equivalent to a total population of114,032, a value in accord with previous reports (Cui et al.,2003; Mansour-Robaey et al., 1994).

Results

Absence of toxic effects

Following intravitreal administration of 25 units of EPO, orPBS in normal (non-nerve transected) animals, RGC somasurvival did not differ significantly (p>0.05) (EPO: 1962±70/mm2; PBS: 1966±18/mm2) from normal (2145±179/mm2);RGC distribution andmorphologywere also unchanged. Ferritinimmunohistochemistry demonstrated that EPO did not cause a

Fig. 1. (A–C) Quantitative assessment of RGC survival at 4 weeks after intra-vitreal administration of EPO at various concentrations or of PBS concomitantwith optic nerve transection. EPO at 5 and 10 units resulted in significant neuro-protection at 4 weeks. (A) Histograms represent numbers of RGC somas/mm2.Bars are standard errors; * indicates significance at p<0.05 compared to PBScontrols. (B, C) Micrographs of wholemounted retina showing RGC somas andprocesses (TUJ1 immunolabeling) at equivalent locations following administra-tion of EPO (10 units, B) or PBS (C). Scale bar=50 μm.

Fig. 2. (A–C) Qualitative assessment of RGC axons at 4 weeks after intravitrealadministration of EPO following various concentrations or of PBS concomitantwith optic nerve transection. Administration of EPO (10 and 25 units) signi-ficantly increased the distance that themajority of RGC axons projected along theproximal nerve compared to PBS controls. Axons extended beyond the tran-section site in the EPO groups but not the PBS group. (A) Histograms representRGC axon density expressed as an arbitrary scale (see text) at 2 levels in theproximal nerve, close behind the eye and immediately before the transection site(prox 1 and 2 respectively) as well as distal (dist) to the transection (arrow). Barsare standard errors; * indicates significance at p<0.05 compared to PBS controls.Cartoon depicts eye (left) and optic nerve. (B–D) Montages (retina to the left—cartoon) showing proximal nerve, transection site (white arrowheads) and distalnerve following administration of EPO (25 units). (B) Cell nuclei (Hoechst); (C)RGC axons (TUJ1 immunolabeling). Scale bars=50 μm.

significant macrophage/microglial response in either normal ornerve transected animals (not shown).

Optic nerve transection

Intravitreal administration induced dose-dependent enhance-ment of RGC soma survival with mean densities being signi-ficantly (p<0.05) raised for 5 and 10 units (333 and 344 RGCs/mm2) compared to PBS controls (253 RGCs/mm2, Figs. 1A–C)but not for 25 or 50 units (293 and 233 RGCs/mm2, Fig. 1A).

Neuroregeneration was also observed following EPOadministration. The greatest effect was observed with 10, incommon with the optimal neuroprotective dose, and 25 units(Fig. 2A) resulting in the survival of significantly more RGCaxons in the proximal nerve than for PBS controls (p<0.05;Figs. 2B, C). Whereas in controls axons had degenerated back

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towards the eye, in EPO treated rats RGC axons were moreabundant in the proximal nerve, with many abutting the tran-section site (Figs. 2B, C). Furthermore, in approximately half ofanimals examined at each EPO dose, RGC axons were presentwithin or beyond the transection site (Fig. 2C). A small numberof individual axons extended for up to 0.5 mm beyond thetransection site (Fig. 2C).

Peripheral nerve grafts

In the PNG series, intravitreal EPO administration (25 units)resulted in RGC axon densities significantly elevated comparedto PBS controls (Mann–Whitney U-test; p<0.05; Figs. 3B–E).

Fig. 3. (A–E) Retinal ganglion cell survival and RGC axon regeneration 4 weeksafter intravitreal administration of EPO (25 units) or of PBS concomitant withperipheral nerve grafting. No neuroprotection of RGC somas was seen butnumbers of RGC axons in peripheral nerve grafts were almost doubled. (A)Histograms represent numbers of RGC somas/mm2 in wholemounted retinae.Bars are standard errors; no significant differences were present (p>0.05). (B)Histograms represent mean numbers of RGC axons/mm across the width of theperipheral nerve graft. Bars are standard errors; * indicates significance atp<0.05 compared to PBS controls. (C) Graphs of numbers of RGC axons/mmacross the width of the peripheral nerve graft. Bars are standard errors. (D, E)Micrographs of horizontal sections for equivalent regions of peripheral nervegrafts (1 mm from the back of the eye) showing RGC axons (TUJ1 immuno-labeling) following administration of PBS (D) or EPO (E). Scale bar=50 μm.

When RGC axon densities were averaged, EPO treated animalsdemonstrated values approximately double those of PBScontrols (p<0.05) (Figs. 3B–C). Moreover, values wereelevated along the PNG (Fig. 3B) in contrast to other studiesthat report a peak of axon densities at the proximal end (Davidand Aguayo, 1981; Gillon et al., 2003).

As in the nerve transection animals, 25 units EPO in the PNGseries did not confer significantly greater neuroprotection ofRGC somas.Mean densities were comparable (284±79/mm2) toPBS controls (311±83.5/mm2) (p>0.05) and Fig. 3A. Theresults support other studies showing that improved neurorege-neration does not necessarily correlate with improved neuro-protection (e.g. Heiduschka et al., 2005; Monsul et al., 2004;Sapieha et al., 2003).

EPO administration to transection site

Administration of 25 units EPO via a gelfoam cuff at thetransection site did not affect RGC survival (275 RGCs/mm2) orinduce RGC axon regeneration above controls (p>0.05; data notshown).

Discussion

In summary, our studies show that EPO administeredintravitreally is both neuroprotective and neuroregenerativefor axotomized RGCs in adult rat although the dose responsecharacteristics differed for the two outcomes.

Neuroprotection

Our report of a dose-dependent RGC soma survival followingintravitreal EPO administration is compatible with a previousstudy (Weishaupt et al., 2004). Levels of RGC survival cannot bedirectly compared as we examined retinae at 4 weeks,Weishauptand colleagues at 2 weeks. Moreover, the optimal doses in ourstudy (5 and 10 units) are higher than those reported previously(1 and 2 units). However, the two studies cannot be directlycompared as we used a single administration at the time ofsurgery whereas Weishaupt and colleagues performed multipleEPO treatments at 0, 4, 7 and 10 days post surgery. We assumethat in our study, as shown by Weishaupt et al. (2004), EPOprevented caspase-3 activation in axotomized RGCs.

EPO shares its neuroprotective activity for RGCs with otherintravitreally administered agents including macrophage inhi-bitory factor (Thanos et al., 1993), the purine derivative inosine(Hou et al., 2004) and aurintricarboxylic acid, an anti-apoptoticdrug (Heiduschka et al., 2005; Heiduschka and Thanos, 2000).In addition to its action on RGCs, EPO has been shown toprotect other retinal cells. For example, it protects multiple celltypes after transient retinal ischemia (Junk et al., 2002) andphotoreceptors following light damage (Grimm et al., 2002).

Neuroregeneration

The present study is, to our knowledge, the first in vivodemonstration that exogenously administered EPO supports

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CNS neuroregeneration, extending the results of in vitro studiesof RGCs (Bocker-Meffert et al., 2002; Kretz et al., 2005). EPOshares CNS neuroregenerative potential with several othermolecules/procedures. For example, limited RGC axon regen-eration is triggered by lens injury (Fischer et al., 2000) orintravitreal insertion of the length of peripheral nerve (Berryet al., 1996). The efficacy of these procedures has recently beenascribed, at least in part, to the release of macrophage-derivedoncomodulin (Yin et al., 2003; Yin et al., 2006). Other studiesdemonstrating RGC axon regeneration have involved intravi-treal administration of agents such as cortisol in conjunctionwith aurintricarboxylic acid (Heiduschka and Thanos, 2000;Heiduschka and Thanos, 2006) or of the cAMP analoguedibutyryl cyclic AMP (Monsul et al., 2004). Alternativestrategies have been to neutralize myelin-associated inhibitorsof regeneration (Fischer et al., 2004; Weibel et al., 1994) or toimplant olfactory ensheathing cells (Li et al., 1998). Recently,nanoparticle assemblies have provided a bridge for RGC axonssevered at the optic brachium to regenerate into the superiorcolliculus (Ellis-Behnke et al., 2006).

EPO’s mode of action

The neuroprotective properties of EPO have been ascribed tonumerous actions (Milano and Collomp, 2005). For axotomizedRGCs, PI3K/Akt phosphorylation has been delineated as theprimary mediator (Weishaupt et al., 2004). In other systems,Jak2, Akt/PKB, Erk1/2 and Bcl-XL pathways have beenimplicated (Digicaylioglu et al., 2004; Siren et al., 2001; Wenet al., 2002). Other proposed mechanisms include effects oninflux of calcium ions (Koshimura et al., 1999), changes inglutathione peroxidase activity (Genc et al., 2001), IL-6 down-regulation (Agnello et al., 2002) and increased nitrous oxideproduction (Calapai et al., 2000; Digicaylioglu et al., 2004).

EPO's dual neuroprotective and neuroregenerative actionsmay share a linked mechanism. We assume that in our in vivostudy, as for the in vitro condition (Kretz et al., 2005), activationof the Jak2/Stat3 pathway is involved in the regenerativeresponse. Kretz and colleagues suggested a model of inter-related signal transduction mechanisms. These would includecascades such as PI3K/Akt and Bcl-XL to promote RGCsurvival coupled with early activation of the Jak2/Stat3pathway, necessary for cytokine-triggered axon regeneration.

Optimizing EPO’s potential for neural repair

Our finding that EPO was effective when administeredintravitreally but not to the injury site suggests that the site ofadministration should continue to be the eye rather than thenerve. However, the optimal regime(s) for RGC neuroprotec-tion and/or neuroregeneration is unclear. A key aspect toconsider is the longevity of exogenously administered EPO. Wedo not yet know its persistence after a single injection.However, we suspect that clearance time is relatively rapidsince, following intravenous injection of 200 iU, vitreal levelsfell from 137.19±66.24 miU to 54.57±16.02 miU between 4and 10 h (unpublished observations). The result is consistent

with studies showing that intravitreally injected proteins havehalf lives of between 4 and 8 h (Dias and Mitra, 2000). Never-theless we presume that some build-up took place during themultiple injection regime used by Weishaupt and colleagues(2004), explaining their reported lower optimal dose comparedto the current single injection regime. Presumably it would beadvantageous to maintain EPO levels for even more extendedperiods. Candidate procedures include viral transfection ofRGCs (Stieger et al., 2006) continuous intraperitoneal infusionas adopted for the antibacterial drug Rifampicin (Kilic et al.,2005) or containment within intravitreally injected slow releasemicrospheres, a procedure used recently to deliver regeneration-promoting oncomodulin to RGC somas (Yin et al., 2006).

We treated the retina with EPO at the time of injury. It mayprove advantageous to delay the onset of EPO administration,as was reported for lentigenically induced RGC survival(Fischer et al., 2000). Lens injury protected a greater numberof axotomized RGC somas if applied 3 or 5 days after opticnerve transection rather than on the day of injury. It may alsoprove possible to improve outcome by combining EPO withother known neuroprotective and/or neuroregenerative agents,potential candidates including growth factors such as fibroblastgrowth factor-2 (Sapieha et al., 2003) or ciliary neurotrophicfactor (Cui et al., 2003; Leaver et al., 2006). A precedent is thesynergistic effects of cortisol and aurintricarboxylic acid onaxotomized RGC survival and axon regeneration: aurintricar-boxylic acid appears to act directly on the RGCs whereas thecortisol modifies the glial environment (Heiduschka andThanos, 2006). An in vitro approach may expedite progress.

Implications for neural repair

EPO is currently a strong candidate for use in treatingconditions such as acute renal failure (Sharples and Yaqoob,2006) and chronic heart failure (Li et al., 2006). Our in vivodemonstration that EPO is not only neuroprotective but alsoneuroregenerative adds a further dimension to its therapeuticpotential. EPO has several attractive features as a putativeneuroprotective and/or neuroregenerative agent. These includeits natural occurrence in the body, the ability to cross the blood–brain barrier (Juul et al., 2004; Xenocostas et al., 2005) and theease of administration via the intranasal route, a procedurewhich has proved effective against cerebral ischemia (Yu et al.,2005). EPO may prove valuable in treating a variety ofconditions in the eye such as glaucoma and diabetic retinopathy(Osborne et al., 2004). It may also be beneficial more widely inthe CNS to treat conditions ranging from stroke and neuro-trauma to neurodegenerative diseases such as Parkinson'sdisease or amyotrophic lateral sclerosis.

Acknowledgments

Ms Sherralee Lukehurst assisted with data analysis and MrMichael Archer and Ms Marissa Penrose with figures. Theauthors are grateful for invaluable advice from Dr Liz Jazwinskaand Dr Don Birkett of Johnson and Johnson Research Pty Ltd, aswell as from Dr John Knight at Johnson and Johnson Phar-

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maceutical Research and Development LLC. Johnson andJohnson Pharmaceutical Research and Development LLCprovided the erythropoietin and Johnson and Johnson ResearchPty Ltd funded the study. SAD is an NH and MRC SeniorResearch Fellow.

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