neurotrophin-3 promotes cell death induced in cerebral ischemia, oxygen-glucose deprivation, and...

Neurobiology of Disease 9, 24–37 (2002)

doi:10.1006/nbdi.2001.0458, available online at http://www.idealibrary.com on

Neurotrophin-3 Promotes Cell Death Inducedin Cerebral Ischemia, Oxygen-GlucoseDeprivation, and Oxidative Stress: PossibleInvolvement of Oxygen Free Radicals

Brian Bates,* ,1,2 Lorenz Hirt,†,1,3 Sunu S. Thomas,†

Schahram Akbarian,* ,§ Dean Le,† Sepideh Amin-Hanjani,†

Michael Whalen,† Rudolf Jaenisch,* ,‡

and Michael A. Moskowitz†

*Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge,Massachusetts 02142; †Stroke and Neurovascular Regulation Laboratory, MassachusettsGeneral Hospital, Harvard Medical School, 149 13th Street, Charlestown,Massachusetts 02129; ‡Biology Department, Massachusetts Institute of Technology,Cambridge, Massachusetts; and §Department of Psychiatry, MassachusettsGeneral Hospital, Boston, Massachusetts 02114

Received April 6, 2001; revised August 17, 2001; accepted for publication October 23, 2001

To explore the role of neurotrophin-3 (NT-3) during cerebral ischemia, NT-3-deficient brains were sub-jected to transient focal ischemia. Conditional mutant brains produced undetectable amounts of NT-3mRNA, whereas the expression of the neurotrophin, BDNF, the NT-3 receptor, TrkC, and the nonselective,low-affinity neurotrophin receptor p75NTR, were comparable to wild-type. Baseline absolute blood flow,vascular and neuroanatomical features, as well as physiological measurements were also indistinguish-able from wild-type. Interestingly, the absence of NT-3 led to a significantly decreased infarct volume 23 hafter middle cerebral artery occlusion. Consistent with this, the addition of NT-3 to primary cortical cellcultures exacerbated neuronal death caused by oxygen-glucose deprivation. Coincubation with theoxygen free radical chelator, trolox, diminished potentiation of neuronal death. NT-3 also enhancedneuronal cell death and the production of reactive oxygen species caused by oxidative damage inducingagents. We conclude that endogenous NT-3 enhanced neuronal injury during acute stroke, possible by
increasing oxygen-radical mediated cell death. © 2002 Elsevier Science (USA)
INTRODUCTION

The neurotrophin family of growth factors, includ-ing nerve growth factor (NGF), brain-derived neuro-trophic factor (BDNF), neurotrophin-3 (NT-3), andneurotrophin-4/5 (NT-4/5), have a wide spectrum of

1 These two authors contributed equally to this work.2 To whom correspondence and reprint requests should be ad-

dressed at present address: Genetics Institute Inc., 35 CambridgePark Drive, Cambridge, MA, 02140. Fax:(617) 665-8350. E-mail:[email protected].

3 Present address: Laboratoire de Recherche Neurologique,
CHUV, BH 19 208, CH 1011 Lausanne, Switzerland.
24

effects on developing and mature neurons of the cen-tral and peripheral nervous systems. Classically, neu-rotrophins are thought of as neuronal survival factorsthat prevent the developmentally programmed deathof peripheral neurons or the experimentally induceddeath of central neurons (Lewin and Barde, 1996).

Given their role as neuronal survival factors, muchattention has focused on neurotrophin involvement inmodels of central nervous system injury, like cerebralischemia. After an ischemic event, BDNF and NGFmRNA were found to increase, whereas NT-3 mRNAdecreased in dentate gyrus, a region of the brain rel-
atively resistant to ischemic cell death (Lindvall et al.,
0969-9961/02 $35.00© 2002 Elsevier Science (USA)

All rights reserved.

25Neurotrophin-3 and Neuronal Cell Death

1992; Takeda et al., 1993; Boris-Moller et al., 1998). Incontrast, NT-3 mRNA was up-regulated in areas ofneuronal degeneration after intrahippocampal injec-tion of the excitotoxin, quinolinic acid (Rocamora et al.,1993). Taken together, these observations suggest thatchanges in neurotrophin synthesis, including de-creased NT-3 expression, may be part of a normalphysiological neuroprotective response after excito-toxic or ischemic damage.

Other studies have reported conflicting findings onthe survival of cultured neurons treated with neuro-trophins and exposed to excitotoxic or ischemic insults(Cheng and Mattson, 1994; Gwag et al., 1995; Koh et al.,1995; Nakao et al., 1995; Pringle et al., 1996; Samdani etal., 1997). Results from in vivo studies, however, havebeen more consistent. Application of BDNF, NT-3, andNT-4/5 have all decreased infarct volume in rodentmodels of cerebral ischemia (Beck et al., 1994; Chan etal., 1996; Chang et al., 1997; Schabitz et al., 1997; Zhanget al, 1999). However, in these studies, large amountsof neurotrophins were transiently supplied to circum-scribed areas and, therefore, did not address the roleof endogenous neurotrophin synthesis after ischemia.

In order to explore the role of endogenous NT-3 syn-thesis after cerebral ischemia, we examined the responseof an NT-3-deficient brain to transient focal ischemiainduced by middle cerebral artery occlusion (MCAO).Previously described NT-3 null mutant mice were notsuitable for this task due to early postnatal mortality(Ernfors et al., 1994; Farinas et al., 1994; Tessarollo et al.,1994). Therefore, we made use of a recently character-ized NT-3 conditional mutant that was viable yet lackedNT-3 expression in brain (Bates et al., 1999). Although theconditional mutant brain was grossly normal by mor-phological and molecular criteria, infarct volume wasdecreased following transient ischemia. Moreover, theaddition of NT-3 in vitro potentiated neuronal deathcaused by oxygen-glucose deprivation and oxidativedamage. We suggest that one mechanism by which NT-3enhanced neuronal death during acute injury was byincreasing oxygen free radical production.

MATERIALS AND METHODS

Generation of NT-3 Conditional Mutant Mice

NT-3 conditional mutant mice were generated aspreviously described using the cre/lox system (Bateset al., 1999). Briefly, loxP sites were inserted around theNT-3 coding exon (exon II) in embryonic stem cells
using a homologous targeting vector. Mice bearing the
NT-3 allele with loxP sites surrounding exon II (re-ferred to as NT-32lox) were crossed with transgenicmice that expressed cre recombinase driven by the ratnestin promoter/enhancer (Trumpp et al., 1999). Ex-pression of cre recombinase in the brain led to excisionof the NT-32lox allele resulting in viable animals thatlacked NT-3 in the brain. Control and conditional mu-tant mice used in this study were obtained from thesame litters but were of a mixed genetic background(for further details see Bates et al., 1999; Trumpp et al.,1999).

Northern Blot

Total RNA was obtained from brain subregions ofadult (3–6 months old) conditional mutant or wild-type littermates using RNAzol (Tel-Test). Ten micro-grams per sample were run on a 1.1% formaldehyde/agarose gel and transferred to a nylon membrane.Blots were probed with mouse cDNA fragments forthe indicated genes.

Histology

Histological studies were conducted in mice tran-scardially perfused with 10% phosphate-buffered for-malin. Isolated tissue was postfixed overnight in thesame fixative, cryoprotected in 30% sucrose, frozen indry ice, cut into 25-mm sections, collected into sterilePBS, and either stained with cresyl violet or thionine,or further processed as described below. For NMDAR1 in situ hybridization, parasaggital sections werehybridized with a 33P-labeled cRNA probe of the en-tire coding region of NMDA-R1 splice variant NR1-2a(NMDAR1C) as described (Sucher et al., 1993, 1995).For cytochrome oxidase staining, sections were incu-bated for 2–12 h at 37°C in 0.05% DAB, 0.025% cyto-chrome C type III (Sigma C2506), 4% sucrose in 0.1 Mphosphate buffer, then rinsed, slide-mounted, dehy-drated, and coverslipped. For NADPH-d histochem-istry, sections were first rinsed multiple times in 0.1 MTris–HCl (pH 8.0) then incubated at 37°C in 0.1 M Tris(pH 8.0), 1.2 mM NADPH (Sigma N-1630), 30 mMl-malic acid (Sigma M 1125), 0.3 mM nitroblue-tetra-zolium, 2.5% DMSO, 0.8% Triton X-100 until a lightblue background became apparent, then rinsed in PBSbefore being slide-mounted, dehydrated, and cover-slipped.

Mouse Model of Cerebral Ischemia

All animal experiments were conducted in accor-
dance with National Institute of Health and institu-
© 2002 Elsevier Science (USA)All rights reserved.

26 Bates et al.

tional guidelines. Age and gender-matched condi-tional mutant and control mice of 3–6 months of age(15–30 g) were anesthetized with 2.5% halothane andmaintained under 1% halothane in 30% oxygen and70% nitrous oxide with a face mask. In all animals,regional cerebral blood flow (rCBF) was measured bylaser-Doppler flowmetry with a flexible probe fixed onthe skull (1 mm posterior and 6 mm lateral frombregma) until 10 min after onset of ischemia. In aseparate group of animals, the right femoral arterywas cannulated for arterial blood pressure and bloodgas determination as described (Huang et al., 1994;Endres et al., 1997; Hara et al., 1997). pH, arterialoxygen pressure (pO2), and partial pressure of carbondioxide (pCO2) of arterial blood samples were ana-lyzed in a Ciba-Corning Diagnostics 248 pH/bloodgas analyzer (Ciba-Corning Diagnostics). In thisgroup, rCBF was monitored throughout the ischemiauntil 10 min after reperfusion. Focal cerebral ischemiawas induced by introducing a silicone-coated 8-0 fila-ment from the external carotid artery into the internalcarotid artery and advancing it (Huang et al., 1994;Endres et al., 1997; Hara et al., 1997). By doing so, themiddle cerebral artery was occluded. The filamentwas removed after 1 h to allow reperfusion. Rectaltemperature was controlled and maintained at 37 60.5°C with a temperature control unit (Frederick Haerand Co.) and a heating lamp during the anesthesiaperiod. No differences in rectal temperatures wereobserved between groups during the monitoring pe-riod and at sacrifice.

Infarct Measurements

The animals were sacrificed 23 h after reperfusion.The brains were frozen in liquid nitrogen vapor forcryostat sectioning or directly divided into five coro-nal 2 mm sections using a brain matrix (RBM-2000C;Activational Systems). Infarction volumes were quan-tified using MCID M4 image analysis software (Imag-ing Research Inc.) on 2% 2,3,5 triphenyl-tetrazolium-chloride stained 2-mm slices. Infarction volume wascalculated by summing the volumes of each sectiondirectly (Huang et al., 1994) or indirectly using thefollowing formula: (contralateral hemisphere (mm3)-undamaged ipsilateral hemisphere (mm3)) divided bycontralateral hemisphere (Swanson et al., 1990; Endreset al., 1997). The difference between direct and indirectinfarct volumes is likely to be accounted for by brain
swelling.

Neurological Deficits

Mice were tested for neurological deficits beforesacrifice and rated on a scale from 0 (no observabledeficit) to 3 (severe) (Huang et al., 1994; Endres et al.,1997; Hara et al., 1997). Briefly, failure to extend theforepaw when suspended vertically was graded asmild injury (1), circling to the contralateral side asmoderate (2), and loss of circling or righting reflex assevere (3). Animals were graded by an observerblinded to genotype.

Carbon Black

Plasticity of the posterior communicating arteries(PcomAs) was evaluated as described by Kawase et al.(1999) with minor modifications. Animals were deeplyanesthetized (3% halothane) and perfused transcardi-ally successively with 20 ml heparinized (10 U/ml)saline, 10 ml of 3.7% formaldehyde in PBS, and 3 ml ofa solution of 50% carbon black ink in gelatin. Animalswere then decapitated and the heads were stored in3.7% formaldehyde for 3 h at 4°C before careful dis-section. Vascular anatomy was evaluated and patencyof the PcomAs were graded by two observers blindedto the genotype. Left and right PcomAs were gradedindependently. Grade 0, no connection between pos-terior and anterior circulation; Grade 1, capillary con-nection; Grade 2, small truncal connection; Grade 3,truncal connection; scores 2 and 3 were considerednormal, and scores 0 and 1 were considered hypo-plastic.

Absolute Cerebral Blood Flow

Absolute cerebral blood flow (CBF) was determinedas described by Fuji et al. (1997) using an indicator frac-tionation. Animals (n 5 4 per group) were anesthetizedwith 1% halothane as above. The right femoral arteryand jugular vein were cannulated with PE-10 polyethyl-ene tubing. After determining mean arterial blood pres-sure and blood gases, arterial blood was withdrawncontinuously from the femoral artery at a rate of 0.3ml/min (Stoelting). One microcurie of N-isopropyl-[methyl 1,3-14C]-p-iodoamphetamine (American Radio-labeled Chemicals Inc.) dissolved in 0.1 ml saline wasinjected into the jugular vein as a bolus (,1 s). Twentyseconds after injection, the animal was decapitated andthe blood withdrawal terminated simultaneously. Thebrain was removed quickly, frozen in isopentane solu-tion, chilled with dry ice, and then dissected into right
and left hemispheres. After adding scintigest (Fisher Sci-


entific) and incubating at 50°C for 6 h, scintillation fluidand H2O2 were added. Twelve hours after shaking, ra-dioactivity in brain and blood were measured by liquidscintillation spectrometry. Absolute CBF was calculatedaccording to the method of Van Uitert and Levy (1978),and Betz and Iannotti (1983).

Mixed Neuronal/Glial Cultures

Mixed neuronal/glial cultures were established es-sentially as described in Goldberg and Choi (1993) andin Rose et al. (1993). Briefly, cortices were isolated fromBalb/c mouse embryos at day 14.5 days of gestation,treated briefly with trypsin, dissociated, and platedonto polyornithine/laminin-coated 24-well plates at adensity of 4 corticies per 24 wells. Cells were plated inMinimal Essential Medium (MEM; Gibco 330-1430)supplemented with sodium bicarbonate and glucoseto 26.2 and 25 mM final concentration, respectively(medium referred to as MS). Plating medium alsocontained 10% (v/v) defined horse serum (Hyclone),10% (v/v) fetal calf serum (Hyclone), 2 mM glu-tamine, and pen/strep (Gibco). Cells were maintainedwithout change in this medium for 5–7 days, at whichtime half the medium was removed and replaced withMEM supplemented as above but lacking FCS andcontaining 20 mM cytosine arabinoside (Ara-C, finalconcentration in wells 10 mM) to halt the growth ofglial cells. After 2–3 days cells were refed every 2–3days with the same medium lacking Ara-C until cul-tures were 14–17 days old. This procedure led tocultures in which cortical neurons sat upon a conflu-ent monolayer of glial cells.

Neuronal Injury

Combined oxygen-glucose deprivation (OGD) wasperformed essentially as described (Goldberg andChoi, 1993). Briefly, cells were extensively rinsed inserum-free MS and then incubated overnight in serumfree MS with or without NT-3 (Upstate Biotechnology,100ng/ml). The next day cells were extensively rinsedwith a deoxygenated, glucose free, balanced salt solu-tion (Rose et al., 1993) and placed in a sealed, deoxy-genated chamber (95% N2, 5% CO2). The chamber washumidified and maintained at 37°C for 50 min. Cul-tures were then removed and rinsed with oxygenated(95% O2, 5% CO2), serum-free MS. Cells were main-tained overnight in serum-free MS. Those culturespreincubated with NT-3 were maintained in its pres-ence (100 ng/ml) following the OGD period. In some
instances the vitamin E analog, trolox (Aldrich), was
added during and after the OGD period to a finalconcentration of 100 mM.

For direct induction of oxidative damage, cultureswere rinsed as described above and incubated over-night in serum free MS with or without NT-3. The nextday the medium was exchanged for MS containingFe21 (30mM) or Buthionine Sulfoximine (BSO; 1 mM;as in Gwag et al., 1995). Those cultures treated over-night with NT-3 were maintained with NT-3 (100 ng/ml) for an additional 24 h with BSO or Fe21. In somecases trolox (100 mM) was added simultaneously withBSO or Fe21.

Neuronal cell death was quantitated 24 hours afterthe termination of OGD or 24 h after the addition ofBSO or Fe21 by measuring the concentration of thecytosolic enzyme, lactate dehydrogenase (LDH), re-leased into the culture medium using a spectrophoto-metric assay (Sigma TOX-7, manufacturer’s protocolfollowed). Values obtained were subtracted by theaveraged value obtained from sister cultures exposedto sham injury and expressed as a percentage of max-imal LDH release (complete neuronal death) inducedby incubation of sister cultures overnight with 500 mMN-methyl, d-aspartic acid (NMDA).

Measurement of Superoxide Production

The intracellular superoxide production assay wasperformed as described by Bindokas et al. (1996) withminor modifications. Dihydroethidine (HEt) was ob-tained from Molecular Probes (Eugene, OR) and pre-pared as a 1 mg/ml stock in DMSO and stored undernitrogen at 280°C. The experiments were performedin 24-well Falcon plates containing 14-day-old mixedneuronal/glial cultures. Cultures were rinsed in se-rum free MS and incubated overnight in serum freeMS with or without NT-3 (100 ng/ml). The next daymedium was exchanged for working solution consist-ing of serum free MS and a 400-fold dilution of HEtstock solution. In different treatment paradigms, theworking solution also contained NT-3 and/or Fe21,where indicated. At the onset of the experiments, im-ages of cells with neuronal morphology were obtainedat 10-min intervals using a Leica DMRB/Bio-RadMRC 1024 confocal microscope equipped with kryp-ton-argon laser (488/585 nm excitation/emissionwavelengths). Captured images were transformedfrom Biorad software to Photoshop JPEG format andconverted to grey scale spectrum. NIH image 1.6 soft-ware was used to quantitate intensity of each individ-ual neuron under investigation (background sub-
tracted). Values obtained were expressed as a percent-

28 Bates et al.

age of the maximum fluorescence intensity. Maximumfluorescence intensity was determined arbitrarilybased on the average intensity of the brightest neuronsat the 50-min time-point (preincubated with NT-3:NT-3 and Fe21 present during image capture). Theseneurons were presumed to represent maximum inten-sity because their intensity was unchanged betweenthe 40 and 50-min time points.

Statistical Evaluation

Kruskal–Wallis nonparametric ANOVA followedby Dunn’s multiple comparison test was used to com-pare neurological scores and cell death in cell cultureexperiments. Culture data was also assessed usingMann–Whitney two-tailed comparison test. For oxy-gen-glucose deprivation (OGD), values obtained withOGD alone were compared to other OGD conditions.Similarly, BSO alone was compared to other BSO con-ditions and Fe21 to other Fe21 conditions. Unpairedstudent’s two-tailed T test was used for comparison ofunpaired data (infarct volumes, physiological param-eters, and HEt oxidation). P values inferior to 0.05were considered statistically significant.

RESULTS

Conditional Mutant Lacks NT-3 But Has NormalLevels of BDNF, TrkC, and p75NTR mRNAin Brain

An NT-3 conditional mutant mouse was createdusing the cre/loxP system as previously described(Bates et al., 1999). Cre recombinase, expressed underthe control of the rat nestin promoter/enhancer(Trumpp et al., 1999), catalyzed incomplete deletion ofthe NT-32lox allele in numerous tissues, but in the CNSrecombination was nearly 100%. In contrast to thepreviously published NT-3 null mutants (Ernfors et al.,1994; Farinas et al., 1994; Tesarollo et al., 1994), theNT-3 conditional mutant was viable and fertilethereby permitting the detailed study of adult ani-mals.

NT-3 conditional mutant mice lacked detectableamounts of NT-3 mRNA in the brain. Figure 1 showsa Northern blot in which NT-3 mRNA was detected inthe hippocampus and cerebellum of wild-type micebut was below detection in the conditional mutant(Fig. 1A). The absence of NT-3 expression did not leadto changes in expression of the neurotrophin family
member, BDNF (Fig. 1B). The level of expression of the

high affinity NT-3 receptor, TrkC, was also unchanged(Fig. 1C), suggesting that NT-3 was not required as asurvival factor for the majority of cells expressing TrkCin the brain. We also examined expression of the non-selective, low affinity neurotrophin receptor, p75NTR.This receptor can function as a proapoptotic factorunder certain conditions to induce neuronal cell death(reviewed in Casaccia-Bonnefil et al., 1999; Kaplan andMiller, 2000). We found expression levels of p75NTRto be unaffected by the absence of NT-3 in the condi-tional mutant (Fig. 1D).

Conditional Mutant Brain in Ischemia-SensitiveRegions Is Morphologically Normal

Whereas deletion of the NT-32lox allele was initi-ated during embryogenesis, the gross morphologyof the adult conditional mutant brain was normal,with the exception of cerebellar foliation (Bates etal., 1999). Enzyme-histochemical staining for cyto-chrome oxidase, a marker of metabolic activity(Wong-Riley, 1979), and nicotinamide-dinucleotidediaphorase (NADPH-d), which indicates expressionof nitric oxide synthase (NOS) (Bredt et al., 1991;Vincent and Kimura, 1992) revealed labeling pat-terns in the cerebral cortex (including the barrelcortex) and the striatum that were indistinguishablebetween conditional mutant and control (Fig. 2).Distribution and numbers of NADPH-d containingneurons in the striatum and all other areas of theforebrain, midbrain, and hindbrain were qualita-tively similar between conditional mutant and con-trol. In situ hybridization revealed that the NR1subunit of the NMDA ionotropic glutamate receptorwas similarly expressed in all brain regions of theconditional mutant and control, with heaviest ex-pression in the CA1 field of the hippocampus andthe retrosplenial cortex (Fig. 2). Likewise, Nisslstaining demonstrated a normal layered arrange-ment of the cortex as well as normal architectureand cellularity of the hippocampus and striatum(Fig. 2 and data not shown).

Baseline measurements of absolute cerebral bloodflow were also unaffected in the conditional mutantmice. Mean absolute cerebral blood flow was deter-mined in four animals of conditional mutant and con-trol genotypes. Values obtained were 209 6 39 ml/min/100 g tissue in conditional mutants and 212 6 24ml/min/100 g tissue in wild-type controls. There wasno difference either in mean arterial blood pressure,pCO2, or pO2 between the groups (Table 1) during
absolute CBF determination. Likewise, there was no


difference between the groups in regional cerebralblood flow measured during and after the induction ofischemia (Table 1).

Variability between mouse strains in the develop-ment of the posterior communicating artery (PcomA)has previously been described (Fujii et al., 1997). ThePcomA connects the vertebrobasilar territory of bloodsupply to the brain with the carotid artery territoryand its patency (see Materials and Methods), there-fore, influences infarct size in the suture MCAOmodel. To verify that any observed effects on infarctsize did not result from differences in cerebral vascu-lature or PcomA development, carbon black evalua-tion of the circle of Willis was performed on threeanimals per group. There were no general differencesobserved in overall cerebral vasculature nor werethere differences found between wild-type and condi-tional mutant with regard to the patency of the PcomA

FIG. 1. NT-3 mRNA was below detection in the conditional mu-tant brain but BDNF, TrkC, and p75NTR were unaffected. Northernblots of total RNA (10 mg/lane) (A) Blot probed for NT-3. NT-3mRNA detected in the hippocampus (HIP) and cerebellum (CB) ofadult wild-type (W) mice but not in conditional mutant (M). Blotprobed for BDNF (B), TrkC (C), or p75NTR (D). Similar amounts ofeach mRNA in HIP, CB, cortex (CTX), and striatum (STR) betweenconditional mutant and wild-type. (E) Loading control for A-C. Blotprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH).Equal loading in D confirmed by ethidium bromide staining ofrRNA (not shown).

(Fig. 3).

Conditional Mutant Has Smaller Infarct afterTransient Focal Ischemia

Mice were subjected to 1 h MCAO followed by 23 hof reperfusion. After 23 h, the conditional mutantsshowed significantly smaller infarcts (74 6 23 mm3,n 5 9) than wild-type controls (104 6 21 mm3, n 5 10;P 5 0.01) (Fig. 4A). However, since cerebral hemi-spheres were slightly smaller in the conditional mu-tants (16269 versus 17066 mm3, P , 0.05, calculationbased on the areas of serial 2-mm-thick coronal slices)we also analyzed infarct size in relation to the unaf-fected hemisphere. We found that infarcts were alsosmaller in the conditional mutant when expressed as apercentage of the contralateral, intact hemisphere(46 6 16% conditional mutant versus 61 6 13% wild-type, P , 0.05). Furthermore, the indirect infarcts,calculated as the ratio of the contralateral hemisphereminus the ipsilateral unaffected hemisphere dividedby the contralateral hemisphere, were also signifi-cantly smaller in the conditional mutants (38 6 10%conditional mutant versus 48 6 9% wild-type, P 50.04) (Fig. 4B).

We found no difference between conditional mu-tants and wild-type mice with regard to neurologicaldeficit evaluated before sacrifice. With 1 h ischemiaand 23 h reperfusion median score was 2 (minimum 2,maximum 2) for wild-type, and 2 for conditional mu-tants (minimum 2, maximum 3). According to theanalysis of variance by the Kruskall–Wallis nonpara-metric ANOVA, followed by Dunn’s multiple com-parison test, the variation between medians is nogreater than expected by chance. However, the condi-tional mutant mice did have a preexisting gait abnor-mality (Bates et al., 1999) that may have effected thisevaluation.

Physiological Parameters

Arterial blood pressure, pO2, pCO2, and pH weremeasured before, during, and after ischemia, andthere was no difference between conditional mutantand controls. In both groups there was an equivalentdrop in rCBF at filament insertion and an equivalentrise in rCBF at filament withdrawal (Table 1).

Primary Cortical Neuron Culture

In order to investigate possible mechanisms of NT-3-potentiated neuronal death at the cellular level, wedesigned a primary cortical culture system using pre-
viously published protocols (Goldberg and Choi, 1993;

30 Bates et al.

Rose et al., 1993; MM Behrens, personal communica-tion). Mixed neuronal/glial cultures were establishedfrom E14.5 wild-type (Balb/c) mouse embryos. After14 days, cultures were placed in serum free medium

FIG. 2. Normal cyto- and chemoarchitecture in NT-3 conditionalknock-out mice. (A, C, E, G) Wild-type controls; (B, D, F, H) condi-tional mutants. Enzyme-histochemical stain for (A, B) cytochromeoxidase and (C, D) nicotinamide-adenine-dinucleotide-phosphatediaphorase (NADPH-d) in striatum. (E, F) Film autoradiogram ofsections hybridized with 33P-labeled NMDA R1 subunit antisensecRNA. (G, H) Nissl-stained sections of somatosensory cortex. I, IV,VI refer to cortical layers.

(MS, see Materials and Methods) overnight and then


subjected to combined oxygen and glucose depriva-tion (OGD) for 50 min. Previous work demonstratedthat such treatment causes neuronal cell death, pri-marily via a necrotic mechanism, while glial cells are

FIG. 6. Preincubation with NT-3 caused increased ROS produc-tion in presumptive cortical neurons in culture. Cortical neuronalcultures were incubated overnight in the presence (A, C) or absence(B, D) of 100 ng/ml NT-3. Medium was exchanged for mediumcontaining 2.5 mg/ml dihydroethidine and 30 mM Fe21 with (A, B)or without (C, D) added NT-3 (100ng/ml). Images were captured at10-min intervals with a confocal microscope. The forty minute time-point is shown here. Note the increased flourescence in culturesincubated overnight with NT-3 indicating enhanced ROS produc-tion. (E) Time course of HEt oxidation expressed as a percentage ofmaximum. Conditions A, B, C, and D are as described above. Notegreater and more rapid oxididation of HEt in cultures preincubatedwith NT-3 (*,#P . 0.001 compared to individual controls, unpaired
two-tailed Student’s t test)


unaffected (Goldberg and Choi, 1993; Koh et al., 1995).Neuronal cell death was assessed 24 h after OGD byquantifying the amount of lactate dehydrogenase(LDH) released into the culture medium, a methodpreviously shown to reliably measure neuronal celldeath (Koh and Choi, 1987; Goldberg and Choi, 1993;Koh et al., 1995). We found that 50 min of OGD re-sulted in the death of approximately 7% of the neu-rons in our cultures (Fig. 5A). In agreement with pre-vious reports (Koh et al., 1995) we found that whencultures were incubated overnight in MS containing100 ng/ml NT-3 (human recombinant, Upstate Bio-technology) and then subjected to the same treatment,there was a dramatic enhancement in neuronal celldeath (Fig. 5A). Also similar to previous reports(Goldberg and Choi, 1993; Koh et al., 1995), we foundthat the same treatment with or without NT-3 had noeffect on the survival of control cultures containingonly glia (data not shown).

We next sought to determine if the production orhandling of reactive oxygen species (ROS) was in-volved in the NT-3-potentiation of OGD-mediatedneuronal cell death. ROS are induced upon transientischemia in vivo (Nelson et al., 1992; Chan et al., 1995;Murakami et al., 1998). In culture, increased produc-tion of ROS occurs upon exposure to the excitotoxin,

TABLE 1

Physiological Parameters Before, During, and After MCAO

Conditional mutant Wild-Type

Before 74 6 6 78 6 8 (ns)BP During 81 6 6 75 6 12 (ns)

After 81 6 9 74 6 16 (ns)Before 7.32 6 0.08 7.30 6 0.04 (ns)

pH During 7.26 6 0.03 7.23 6 0.06 (ns)After 7.26 6 0.05 7.27 6 0.04 (ns)Before 149 6 20 159 6 10 (ns)

pO2 During 113 6 25 120 6 38 (ns)After 109 6 24 109 6 27 (ns)Before 40 6 7 43 6 7 (ns)

pCO2 During 51 6 5 55 6 14 (ns)After 53 6 6 51 6 8 (ns)

rCBF During 0.14 6 0.06 0.15 6 0.01 (ns)After 0.94 6 0.29 0.98 6 0.11 (ns)

Note. Physiological parameters. Blood pressure (BP), arterial oxy-gen pressure (pO2), and arterial carbon dioxide pressure (pCO2, allin mm of Hg) of arterial blood samples, as well as arterial blood pH,were analyzed before, during, and after MCAO. Regional cerebralblood flow (rCBF) was measured by laser-Doppler flowmetry dur-ing ischemia until 10 min after reperfusion and expressed as apercentage of the baseline readings. (ns) not statistically significant.

N-methyl-d-aspartic acid (NMDA), and excitotoxicity

is thought to be a major component of neuronal deathinduced by OGD (Choi, 1988; Dugan et al., 1995; Bin-dokas et al., 1996). Direct ROS mediated neuronaldeath in culture is potentiated by neurotrophins(Gwag et al., 1995; Park et al., 1998) and BDNF, inparticular, has been found to directly stimulate ROSproduction in cultured neurons (MM Behrens and DWChoi, personal communication). The vitamin E analogand ROS scavenger, trolox (Chow et al., 1994), fullyinhibited the potentiation of OGD-induced death byNT-3, suggesting that the enhancement of death oc-curred by a mechanism involving ROS. However,trolox did not modify cell death induced by OGDalone (Fig. 5A), suggesting a limited role for ROS inneuronal killing at this level of injury.

We next examined whether NT-3 could enhanceneuronal cell death directly caused by the oxidativedamage inducing agents, Fe21, or buthionine sulfoxi-mine (BSO). Fe21 ions enhance Fenton chemistry lead-ing to increased production of the ROS, hydroxyl free

FIG. 3. Carbon black examination of the arterial circle of Willis.Brain vasculature revealed by carbon black perfusion as describedin methods. No differences were observed in the development of thePcomA (highlighted by dashed line), posterior cerebral artery(PCA), or basilar artery (BA) between the conditional mutant (A)
and wild-type (B).

32 Bates et al.

radical (Gutteridge and Halliwell, 2000). BSO irrevers-ibly inhibits g-glutamylcystine synthetase resulting indecreased cellular glutathionine levels and conse-quently, increased susceptibility to oxidative damage(Gwag et al., 1995). Fe21 (30 mM) induced the death ofapproximately 14% of the neurons without causingglial death (Fig. 5B and data not shown). Simultaneousincubation with trolox almost completely inhibitedcell killing, suggesting that neuronal death was medi-ated by ROS in this system. Preincubation of Fe21-treated cultures with NT-3 greatly enhanced neuronalcell death. NT-3-enhanced cell death was also inhib-ited by trolox. Similarly, BSO induced the trolox in-hibitable death of approximately 33% of the neurons.As seen in Fe21-treated cultures, pre-incubation withNT-3 caused nearly complete neuronal death thatagain was inhibited by trolox (Fig. 5B). These results

FIG. 4. Direct and indirect infarct measurements are decreased inthe conditional mutant. Indicated genotypes (WT, wild-type; CM,conditional mutant) were subjected to one hour MCAO and 23 hreperfusion. Infarct areas were measured on 2 mm TTC-stainedsections as described under Materials and Methods. Circles indicateindividual values used to calculate the mean. (A) Direct infarctvolumes (mean 6 SD) were significantly (P . 0.01) smaller inconditional mutant compared to wild-type littermates. (B) The cal-culated indirect infarct volumes (mean 6 SD) were also decreasedin the conditional mutant (P . 0.05).

clearly indicate that NT-3 enhanced oxidative neuro-


nal cell death. Several possibilities exist to explain thisresult such as altered synthesis of ROS detoxifyingenzymes like superoxide dismutase and catalase (Jack-son et al., 1990; Spina et al., 1992; Pan and Perez-Polo,1993; Mattson et al., 1995) or NT-3-mediated enhance-ment of ROS production by unknown mechanisms.

We next explored the ability of NT-3 to directlyinfluence ROS production by visualizing the ROS-mediated oxidation of dihydroethidine (HEt) toethidium by cultured neurons under oxidative stress.Within 40 min of adding Fe21 a fluorescent signal,indicative of HEt oxidation, was detected in cells ofneuronal morphology (Figs. 6B and 6D). The timecourse and extent of HEt oxidation did not differbetween cultures with or without NT-3 added simul-taneously with Fe21 (Fig. 6B). Overnight preincubationwith NT-3, however, caused a more rapid accumula-tion and higher absolute levels of oxidized HEt (Figs.6A and 6C). Enhanced accumulation of oxidized HEtdid not depend upon the continued presence of NT-3(Fig. 6C), but did require overnight pre-incubation(compare Figs. 6C and 6D). Therefore, the enhance-ment of ROS production was not an immediate effectof NT-3, but more likely occurred due to NT-3-medi-ated affects on downstream factors that influencedROS production or handling.

DISCUSSION

A major goal of neurotrophin research has been tounderstand neurotrophin function in adult animals.Progress toward this goal has been hampered by thelack of an animal model in which neurotrophins areabsent in the adult. The conditional knockout strategy,utilizing the cre/lox system (Rajewsky et al., 1996), haspermitted the establishment of such a model. TheNT-3 conditional mutant described in this work didnot succumb to early postnatal mortality as did thepreviously characterized NT-3 null mutant (Ernfors etal., 1994; Farinas et al., 1994, Tessarollo et al., 1994) and,therefore, allowed the detailed analysis of an NT-3-deficient adult brain.

To verify that NT-3 was not expressed in the mutantbrain and to determine if any changes in the expres-sion of neurotrophin receptors, or the neurotrophin,BDNF could account for any observed phenotypes weperformed Northern blotting experiments. Previouswork demonstrated that recombination of the NT-32lox

allele was nearly complete in the mutant brain (Bateset al., 1999) making any significant expression of
NT-3 unlikely. Furthermore, we report here that NT-3


mRNA levels were below our detection limit in con-ditional mutant hippocampus and cerebellum. Al-

FIG. 5. NT-3 enhanced OGD and ROS mediated neuronal cell death.Primary cortical cultures incubated overnight in serum free MS with orwithout NT-3 (NT, 100 ng/ml) where indicated. (A) Cultures subjectedto OGD for 50 min in the presence of trolox (T), where indicated.Culture media assayed for LDH concentration and expressed as apercentage of the average LDH concentration in sister cultures treatedwith 500 mM NMDA (total neuronal death). Values for each conditionwere averaged and reported 6 SD. Note that preincubation with NT-3(NT) enhanced cell death 3.7-fold and that this enhancement wasinhibited by trolox. Total cultures assayed: OGD, n 5 24; OGD 1 T,n 5 18; OGD 1 NT, n 5 24; OGD1NT1T, n 5 16. (B) After overnightincubation in serum free MS with or without NT-3 the indicatedadditions were made and the cultures incubated a further 24 h. Culturemedia were collected, assayed, and reported as in A. Note that prein-cubation with NT-3 enhanced Fe21 (Fe) mediated death 6.3-fold andBSO-mediated death 3.6-fold. Total cultures assayed: Fe, n 5 32; Fe 1NT, n 5 26; Fe 1 T, n 5 20; Fe 1 NT 1 T, n 5 16; BSO, n 5 15; BSO 1NT, n 5 15; BSO 1 T, n 5 12; BSO 1 NT 1 T, n 5 15. #P . 0.01 usingKruskal–Wallis nonparametric ANOVA: *P . 0.01 using Mann–Whit-ney two-tailed comparison test. ns, not significant by either test. In Acomparisons were made to OGD alone. In B comparisons were madeto either Fe21 alone or BSO alone for these respective experiments.

though not highly expressed, NT-3 mRNA is synthe-

sized in the cortex of wild-type rodents (Friedman etal., 1991; Lauterborn et al., 1994; Pitts and Miller, 2000;Vigers et al., 2000). As this expression was not detectedin wild-type mice, while unlikely, it is possible thatsome very low level of NT-3 synthesis may occur inthe conditional mutant. Expression of the NT-3 recep-tor, TrkC, and the neurotrophin family member,BDNF, were unchanged by the absence of NT-3. Also,the low-affinity neurotrophin receptor p75NTR wasexpressed at wild-type levels in the conditional mu-tant. This receptor has been implicated in neuronalapoptosis (Rabizadeh et al., 1993; Frade et al., 1996;Casaccia-Bonnefil et al., 1999; Miller and Kaplan, 2000)and its expression is increased in cerebral ischemia(Kokaia et al., 1998; Park et al., 2000; Andsberg et al.,2001). We did not determine expression levels ofp75NTR after MCAO. Therefore, it is possible that,although present at wild-type levels prior to injury, aninability to up-regulate expression of this receptorcontributes to decreased infarct size in the conditionalmutant. However, animals that lack p75NTR throughgenetic deletion (Lee et al., 1992) do not show de-creased cell death after MCAO (Andsberg et al., 2001),suggesting a limited role for this receptor in this sys-tem.

In the conditional mutant, NT-3 expression waseliminated due to recombination mediated by cre re-combinase that was expressed from the nestin pro-moter/enhancer (Bates et al., 1999; Trumpp et al.,1999). The nestin promoter is normally active duringembryogenesis in neural and myogenic precursors be-ginning as early as embryonic day 7.75 (E7.75) (Zim-merman et al., 1994; Dahlstrand et al., 1995). We havefound expression of cre and recombination of the NT-32lox allele throughout the developing central nervoussystem beginning as early as E9.5 in our mice (Bates etal., 1999, and data not shown). This early eliminationof NT-3 results in abnormal cerebellar foliation (Bateset al., 1999) and, as reported here, slightly reducedcerebral hemisphere size. Several possibilities exist toexplain decreased hemisphere size such as, decreasedneuronal number or dendritic arborization (McAllisteret al., 1997). As these parameters were not examinedhere, they cannot be ruled out as possible contributorsto our findings. However, although such factorswould likely affect direct infarct measurements, theirimpact on indirect infarcts, which were also reducedin the conditional mutant, would be diminished.

In contrast, we found that the cyto- and chemoar-chitecture of the forebrain territory affected by MCAOwas normal in the conditional mutant. The overall
morphology of the brain including the layering and

34 Bates et al.

cellularity of the cortex, hippocampus, and striatumwas not discernibly different from controls. We alsodid not find evidence for decreased synthesis of mol-ecules promoting neuronal death and injury in acutestroke. Expression of neuronal nitric oxide synthaseand an essential NMDA receptor gene, at least, ap-peared to be normal. Furthermore, absolute cerebralblood flow under basal conditions, the anatomy of thecircle of Willis, physiological parameters (BP, pH, pO2,pCO2), and regional cerebral blood flow during isch-emia and reperfusion were unaffected by the absenceof NT-3. In summary, we found no obvious differencebetween the conditional mutant and wild-type brainprior to injury that would account for our finding ofdecreased cell death upon MCAO. Therefore, we con-clude that decreased cell death is most likely a pri-mary effect of the absence of NT-3 and not a secondaryeffect brought about by some structural or develop-mental abnormality.

We observed smaller infarct volumes in conditionalmutant mice after 1 h MCAO and 23 h reperfusion,suggesting that endogenous NT-3 promotes larger in-farcts in this paradigm. Numerous other studies havealso implicated NT-3 and neurotrophin signaling asimportant factors in neuronal cell death (Rabizadeh etal., 1993; Gwag et al., 1995; Koh et al., 1995; Frade et al.,1996; Park et al., 2000; Giehl et al., 2001). For example,Xu et al. (1997) reported that the pan-Trk tyrosinekinase inhibitor, K252a, decreased ischemic damageafter MCAO. Endogenous NT-3 has also been shownto promote cell death in other neuronal injury para-digms. For example, inactivation of endogenous NT-3was found to improve the survival of axotomizedcorticospinal neurons and that survival was also im-proved in mutant mice lacking a single copy of theNT-3 gene (Giehl et al., 2001). However, given that asingle post-injury time point was examined in thepresent report, it will be important to conduct furtherstudies to determine if cell death is decreased through-out the recovery period or if this observation reflectsdelayed cell death in the absence of NT-3.

In contrast to our findings, a recent report by Zhangand coworkers (1999) demonstrated that cortical ap-plication of NT-3 after MCAO in rat had a neuropro-tective effect, with a significant reduction in infarctsize. These results rule out the possibility suggestedby this work that exogenous NT-3 may exacerbate anischemic lesion. While this observation is relevant forneuroprotective approaches, it is far from the physio-logical situation and from the role of endogenousNT-3. What has yet to be demonstrated is how this
neuroprotective effect is mediated and if it involves

TrkC, the physiological NT-3 receptor, since high con-centrations of NT-3 have been shown to activate boththe TrkA and TrkB receptors (Wyatt et al., 1997; Fari-nas et al., 1998). Interestingly, recent reports demon-strated that cortical infusion of NT-3 led to the induc-tion of BDNF expression, down-regulation of TrkC,and phosphorylation of the BDNF receptor, TrkB(Shutte et al., 2000; Giehl et al., 2001), suggesting thatthe neuroprotective action of exogenous NT-3 mayindeed be mediated by nonphysiological signalingthrough the BDNF pathway. This is also consistentwith the observation that deletion of endogenousNT-4 and BDNF (TrkB ligands) resulted in increasedinfarct volumes in MCAO (Endres et al., 2000).

We attempted to investigate possible mechanismsfor our in vivo observation using a previously charac-terized mixed neuronal/glial culture system. In thissystem, OGD and oxidative damage have been shownto induce the necrotic death of neurons while glialcells were unaffected (Goldberg and Choi, 1993; Gwaget al., 1995). Consistent with our in vivo results, wefound that NT-3 greatly increased OGD-induced neu-ronal cell death and that this was prevented by theoxygen free radical chelator, trolox, suggesting ROSinvolvement in NT-3 enhanced neuronal death.

Numerous studies have implicated ROS as impor-tant mediators of cell death after transient ischemia invivo (Nelson et al., 1992; Zini et al., 1992; Chen et al.,1995; Yamamoto et al., 1997; Murakami et al., 1998;Eliasson et al., 1999; Kawase et al., 1999). Therefore,given that the absence of NT-3 led to decreased celldeath after MCAO, and that a ROS chelator preventedNT-3-enhanced neuronal cell death in culture, the in-volvement of ROS in NT-3-potentiated death was ex-amined in more detail. We found that preincubationwith NT-3 greatly enhanced neuronal cell deathcaused by oxidative damage. Furthermore, by directlyvisualizing ROS-mediated HEt oxidation, we demon-strated that NT-3 enhanced ROS production in pre-sumptive neurons treated with Fe21. Our findings areconsistent with previous reports showing that the neu-rotrophin, BDNF, enhanced while K252a prevented,necrotic neuronal death induced by oxidative damage(Goodman and Mattson, 1994; Gwag et al., 1995).When added immediately prior to Fe21, however,NT-3 did not enhance ROS production. These resultssuggest a mechanism whereby NT-3 induced the up-regulation of genes involved in ROS production or thedown-regulation of genes necessary for ROS elimina-tion. Interestingly, neurotrophins have been shown toeffect the synthesis of ROS detoxifying enzymes, like
superoxide dismutase and catalase in other systems


(Jackson et al., 1990; Spina et al., 1992; Pan and Perez-Polo, 1993; Mattson et al., 1995). Consistent with this,Gwag et al. (1995) showed that the potentiation ofFe21-mediated death by BDNF could be blocked bythe addition of cycloheximide. While these experi-ments are informative, it will be important to deter-mine if these observations made on cultured cells arealso seen in vivo, and if the NT-3 conditional mutanthas reduced levels of ROS after MCAO.

In total, our findings support the notion that endog-enous NT-3 is detrimental to neuronal survival afteran ischemic event. Furthermore, it supports the ideathat decreases in NT-3 synthesis that normally followcerebral ischemia are not necessarily maladaptive butmay be part of a normal neuroprotective response.Finally, it would seem apparent that these resultspoint to the potential therapeutic benefits of regulat-ing neurotrophin signaling in general, and NT-3 sig-naling in particular, during early times after an isch-emic episode.

ACKNOWLEDGMENTS

The authors thank Margarita Behrens for help in setting up thecell culture system, Jessica Daussmann and Ruth Flannery for ani-mal support, Jeanne Reis for histology, and Nick Plesnila and CindyChen for technical help. The authors also thank Margarita Behrensand Dennis Choi for communicating unpublished results and KuoFen Lee for providing reagents. We also thank Margarita Behrens,Maribel Rios, Paul Yaworsky, and Seung Kwak for critical readingof the manuscript. This work was supported by SchweizerischeStiftung fuer Medizinisch Biologische Stipendien and the Founda-tion SICPA for fellowships to L.H.; Cure Autism Now FoundationFellowship to S.A.; NIH Grant 5-R350CA44339 to R.J.; and NIH-Interdepartmental Stroke Program Project Grant 5-P50NS10828.

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