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Neurotrophin potentiation of iron-induced spinal cord injury
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Neuroscience Vol. 115, No. 3, pp. 931^939, 2002 D 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 02 $22.00+0.00
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NEUROTROPHIN POTENTIATION OF IRON-INDUCED SPINAL CORD INJURY J. W. MCDONALD, V. G. STEFOVSKA, X. Z. LIU, H. SHIN, S. LIU and D. W. CHOI Center for the Study of Nervous System Injury and Department of Neurology, Washington University School of Medicine, P.O. Box 8111, 660 S. Euclid Avenue, St. Louis, MO 63110-1093, USA
Abstract8Previous studies have shown that pretreatment with neurotrophins can potentiate the vulnerability of cultured neurons to excitotoxic and free radical-induced necrosis, in contrast to their well known neuroprotective e¡ects against apoptosis. Here we tested the hypothesis that this unexpected injury-potentiating e¡ect of neurotrophins would also take place in the adult rat spinal cord. Fe3þ -citrate was injected stereotaxically into spinal cord gray matter in adult rats in amounts su⁄cient to produce minimal tissue injury 24 h later. Twenty-four-hour pretreatment with brain-derived neurotrophic factor, neurotrophin-3, or neurotrophin-4/5, but not nerve growth factor, markedly enhanced tissue injury in the gray matter as evidenced by an increase in the damaged area, as well as the loss of neurons and oligodendrocytes. Consistent with maintained free radical mediation, the neurotrophin-potentiated iron-induced spinal cord damage was blocked by co-application of the antioxidant N-tert-butyl-(2-sulfophenyl)-nitrone. These data support the hypothesis that the overall neuroprotective properties of neurotrophins in models of acute injury to the spinal cord may be limited by an underlying potentiation of free radical-mediated necrosis. D 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4/5, excitotoxicity, free radicals.
and glial cells (Schwann cells, microglia, astrocytes and oligodendrocytes) (Dreyfus et al., 1999; Krenz and Weaver, 2000; Huang and Reichardt, 2001). In development and models of nervous system injury, neurotrophins have a powerful ability to reduce neuronal cell death, in particular apoptosis mediated by growth factor deprivation, such as induced by axotomy in vivo (Hefti, 1986; Tuszynski et al., 1990; Yan et al., 1992; Kobayashi et al., 1997; Houle and Ye, 1999; Novikova et al., 2000), or neuronal injury induced by exposure to excitotoxins, glucose or serum deprivation in vitro (Alderson et al., 1990; Shigeno et al., 1991; Davies and Beardsall, 1992; Frim et al., 1993; Cheng et al., 1994; Cheng and Mattson, 1994). However, several in vitro studies have raised the possibility that the neuroprotective e⁄cacy of neurotrophins may be limited by an underlying propensity to potentiate neuronal death under some conditions. Pretreatment with BDNF or NT-3 enhanced glutamate-induced death of cultured cerebellar neurons, a death possibly apoptotic since basic ¢broblast growth factor-induced survival was linked to increased calcium in£ux (Ferna¤ndez-Sa¤nchez and Novelli, 1993; see also Ankarcrona et al., 1995). Conversely, we found that pretreatment with NT-3, NT-4/5, or BDNF reduced the vulnerability of cultured cortical neurons to apoptosis, but enhanced necrosis induced by N-methyl-D-aspartate (NMDA), oxygen^glucose deprivation, or iron-induced oxidative stress (Gwag et al., 1995; Koh et al., 1995). This necrosis-speci¢c injury potentiation was likely dependent on new protein synthesis since it required pretreatment for several hours and was abol-
Neurotrophins regulate development, maintenance and plasticity in the vertebrate nervous system. There are four neurotrophin families identi¢ed in mammals: nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5). These neurotrophins activate two separate classes of receptors, the tyrosine kinase (Trk) receptors and p75NTR, a member of the tumor necrosis factor receptor superfamily (Giehl et al., 2001; Huang and Reichardt, 2001). NGF is the preferred ligand for TrkA, BDNF and NT-4/5 are the preferred ligands for TrkB, and NT-3 binds primarily to TrkC, although it can also interact with TrkA or TrkB (Yan et al., 1997). Both neurotrophins and their receptors are widely expressed on neurons, including cortical (Pitts and Miller, 2000; Schu«tte et al., 2000), spinal cord (Scarisbrick et al., 1999), and dorsal root ganglia neurons (Heppenstall and Lewin, 2001; Lever et al., 2001). In addition, neurotrophins are produced by target organs
*Corresponding author. Present address: Merck Research Labs, West Point, PA 19486, USA. Tel.: +1-314-362-9460; fax: +1-314747-0422. E-mail address: [email protected] (D. W. Choi). Abbreviations : APC CC1, adenomatous polyposis coli gene; BDNF, brain-derived neurotrophic factor; HpE, hematoxylin and eosin; LFB-PAS, Luxol-Fast Blue-PAS ; NeuN, neuron-speci¢c nuclear protein; NGF, nerve growth factor; NMDA, N-methyl-D-aspartate; NT-3, neurotrophin-3; NT-4/5, neurotrophin-4/5; PBS, phosphate-bu¡ered saline; S-PBN, N-tert-butyl(2-sulfophenyl)-nitrone; Trk, tyrosine kinase. 931
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ished by concurrent exposure to protein synthesis inhibitors (Lobner and Choi, 1996). Other groups have also reported observations consistent with neurotrophin injury potentiation (Prehn, 1996; Samdani et al., 1997; Park et al., 1998; Glazner and Mattson, 2000). In addition, Won et al. (2000) found that injection of NT-4/5 potentiated the striatal damage induced by co-injection of Fe2þ in the adult rat brain. The idea that neurotrophin mediated injury enhancement may be a common component of neurotrophin action does not necessarily con£ict with the many in vivo studies demonstrating net bene¢ts by neurotrophins against oxidative (Kirschner et al., 1996), ischemic (Chan et al., 1996; Cheng et al., 1997; Scha«bitz et al., 1997; Andsberg et al., 1998; Kiprianova et al., 1999) or traumatic (Dixon et al., 1997; Grill et al., 1997; Sinson et al., 1997; Houweling et al., 1998a,b; Jakeman et al., 1998) insults. Since programmed cell death likely contributes to neuronal loss after acute insults (Johnson et al., 1996; Lee et al., 1999; Johnston et al., 2000), the anti-apoptotic e¡ects of neurotrophins may be large enough to override any concurrent enhancement of necrosis. The goal of the present study was to test the hypothesis that the observed ability of neurotrophins to potentiate neuronal necrosis in vitro and in striatum in vivo would also apply to the adult spinal cord, noting that neurotrophin administration is speci¢cally of interest as a possible therapy for spinal cord injury (Grill et al., 1997; Houweling et al., 1998a,b; Jakeman et al., 1998; McTigue et al., 1998).
EXPERIMENTAL PROCEDURES
Preparation of Fe 3+-citrate solution A pH balanced, iso-osmotic Fe3þ -citrate solution was prepared using methods adapted from Sengstock et al. (1992). Stock solutions were made for the four components of the Fe3þ -citrate solution: #1 ferric chloride hexahydrate ^
FeCl3 W6H2 0 (molecular weight (MW) 270.3, 100 mM; Sigma, St. Louis, MO, USA); #2 sodium citrate-dihydrate ^ C6 H5 Na3 O7 W2H2 O (MW 294.1, 180 mM; Sigma); #3 sodium bicarbonate ^ NaHCO3 (MW 84.01, 11.4 mM; Sigma); #4 Trizma Base ^ C4 H11 NO3 (MW 121.1, 0.455 M; Sigma). The Fe3þ -citrate solution was always prepared immediately before each experiment by combining the above stock solutions: 15 Wl #1, 15 Wl #2, 100 Wl #3, 6 Wl #4, then phosphate-bu¡ered saline (PBS), pH 7.4 balance to a total volume of 1 ml. Animal care and spinal cord injury All surgical interventions and animal care were provided in accordance with the Laboratory Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996) and the Guidelines and Policies for Rodent Survival Surgery provided by the Animal Studies Committee of Washington University School of Medicine. Adult Long^Evans female rats (275 T 25 g, Simonsen Laboratories ^ Gilroy, CA, USA) were anesthetized with ketamine/medetomidine (75:0.5 mg/kg i.p.). Ketamine was from Fort Dodge Animal Health (Fort Dodge, IW, USA), medetomidine was from Burns Veterinary supply (Rockville, NY, USA). A laminectomy was performed at T9 and microstereotaxic injections of vehicle, NGF, BDNF, NT-3, or NT-4/5 were completed at two sites, 1 mm apart just to the right of midline (marked by the dorsal vein) 1.0 mm below the dura into the central gray matter. NGF, NT-3 and NT-4/5 were gifts from Regeneron (Tarrytown, NY, USA), Amgen (Thousand Oaks, CA, USA), and Genentech (South San Francisco, CA, USA), respectively. BDNF was from Amgen and RpD Systems (Minneapolis, MN, USA). The treatment paradigm used in these studies is shown in Fig. 1. The amounts of neurotrophin injected at each site were 0.5 Wg for NT-4/5 and 1 Wg for NGF, BDNF, NT-3 in 0.5 Wl (volumes were always kept constant). The wound was closed and sedation was reversed with antisedane (Atipamezole, 1 mg/kg s.c., Animal Health, Exton, PA, USA). Rectal temperature was maintained at 37.0 T 0.5‡C using a thermostatically regulated heating pad (Versa-Therm 2156; Cole-Parmer, Chicago, IL, USA) and heating lamp system during surgery and for 2 h during recovery. Twenty-four hours after the initial injections, the wounds were reopened and all animals received a single microstereotaxic injection of a threshold injury dose of Fe3þ -citrate (0.75 nmol in 0.5 Wl PBS, pH 7.4) midway between the two previous injections. The antioxidant N-tert-butyl-(2-sulfophenyl)-nitrone (S-PBN) (Sigma), 200 mg/kg i.p. or saline of a
Fig. 1. Schematic representation of the intra-cord injection paradigm. Adult Long^Evans female rats, deeply anesthetized, received a microstereotaxic injection of vehicle media (0.5 Wl), NGF, BDNF, NT-3 (1 Wg/0.5 Wl) or NT-4/5 (0.5 Wg/0.5 Wl) at two sites each 1 mm apart just to the right of midline in the central gray matter on day 1. Twenty-four hours later, on day 2, all animals received a single intra-cord injection of a minimally toxic amount of iron (0.75 nmol Fe3þ -citrate in 0.5 Wl vehicle media, pH 7.4) or only vehicle media (0.5 Wl) midway between the two previous injections, n = 5^12 for all groups. Twenty-four hours later, on day 3, animals were killed. Tissues were processed for para⁄n embedding, routine staining with HpE or LFB-PAS and speci¢c immunohistochemistry. Injury was quanti¢ed by two methods : area measurements of the injured tissue and cell counts of neurons and oligodendrocytes.
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Fig. 2. NT-3 potentiation of Fe3þ -induced spinal cord injury. Experiments were performed as described in Fig. 1. Panels A and B illustrate representative amounts of tissue injury present in longitudinal para⁄n sections, stained with anti-NeuN, from animals that were pretreated with vehicle media (vehicle/Fe ; panel A) and NT-3 (NT-3/Fe; panel B) on day 1 followed by intra-cord injection of iron on day 2. Panel C illustrates the quantitative analysis of lesion area measurements from comparison of four groups: group 1 ^ vehicle/vehicle, intra-cord injections of vehicle media on days 1 and 2; group 2 ^ NT-3/ vehicle, intra-cord injections of NT-3 (1 Wg) on day 1 and vehicle media on day 2; group 3 ^ vehicle/Fe, injections of vehicle media on day 1 and Fe3þ on day 2; group 4 ^ NT-3/Fe, injections of NT-3 on day 1 and Fe3þ on day 2. *P 6 0.05, ANOVA followed by the Student^Newman^Keuls test, n = 6 for each group, mean T S.D. Scale bar = 1 mm. Vehicle = vehicle media, Fe = Fe3þ -citrate.
similar volume i.p., was applied on day 1 and day 2, each twice daily. A set of preliminary studies was completed to determine a threshold injury amount of iron when injected into the adult rat spinal cord. Microstereotaxic injection of 0.75 nmol Fe3þ / 0.5 Wl PBS into the central gray matter at the T9 level of the spinal cord produced a discrete gray matter lesion present in all animals. Instead of iron injection, control groups of animals treated with neurotrophins the ¢rst day received only vehicle injections of a similar volume on the second day. The number of animals used per group ranged between ¢ve and 12. Histology Twenty-four hours after the ¢nal injection, animals were killed with a lethal dose of phenobarbital (100 mg/kg i.p.), and perfused intracardially with physiological saline followed by 4% paraformaldehyde in 0.1 M PBS, pH 7.4. Tissue specimens using 2 cm of the spinal cord surrounding the injection
sites were then processed for para⁄n embedding and 7-Wm para⁄n sections were cut longitudinally. Serial sections were collected from the anterior horns through the entire dorsal columns. Sections were routinely stained for hematoxylin and eosin (HpE) and Luxol-Fast Blue-PAS (LFB-PAS). Immunohistochemistry Primary antibodies were used to visualize neurons (NeuN, against neuron-speci¢c nuclear protein, mIgG1 , 1:500, Chemicon, Temecula, CA, USA), oligodendrocytes (APC CC1, against adenomatous polyposis coli gene, mIgG1 , 1:400, Calbiochem Oncogene Sciences, La Jolla, CA, USA) and astrocytes (GFAP, against glial ¢brillary acidic protein, rabbit polyclonal, 1:4, Incstar, Stillwater, MI, USA). Sections for APC CC1 staining were treated with antigen unmasking solution (Vector Laboratories, Burlingame, CA, USA) in a microwave. Blocking of endogenous peroxidase with 3% H2 O2 in methanol and nonspeci¢c antigenic sites with 10% horse or goat serum was per-
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formed before the ¢rst antibody. Biotinylated species-speci¢c secondary antibodies (1:200 dilution, Vector) were applied, followed by ABC complex (Vector). Detection of peroxidase activity was carried out by 3,3P-diaminobenzidine tetrahydrochloride (DAB kit, Vector). Quantitative analysis of injury Area measurements of the injured tissue were completed in three longitudinal sections, one centered on the central canal and the others separated by 200 Wm, from six to 12 animals in each group in a blinded fashion using a video-based image analysis system (McDonald et al., 1989). Values were summed within animals and averaged group values were compared amongst groups using Student’s independent t-tests for two group comparisons, and one-way analysis of variants (ANOVA) with post-hoc test for comparisons of three or more groups. Data are mean T S.E.M. values. Area data were further con¢rmed counting the number of surviving neurons and oligodendrocytes in the BDNF group, as our original observations of neurotrophin potentiation were made with BDNF (Koh et al., 1995). Cell counts for NeuN and APC CC1 positive cells were performed on three di¡erent longitudinal sections, one centered on the central canal and the others separated by 150 Wm, from four animals in each group in a blinded fashion. A 0.16-mm2 area in the gray matter at U200 magni¢cation was examined in the epicenter (the middle point between both sites of the injection), 400 Wm, 800 Wm and 1600 Wm rostrally and caudally. The average number of cells from each area at the respective distance in every animal was calculated. Only intensively labeled cells with clearly de¢ned cell body and nucleus were used. Statistical evaluation was performed with one-way ANOVA and Bonferroni t-test for multiple comparisons.
Fig. 3. Selective neurotrophin potentiation of Fe3þ -induced spinal cord injury. NT-4/5 but not NGF or vehicle media potentiated Fe3þ injury. Experiments were performed as outlined in Figs. 1 and 2. Area measurements of injured tissue were completed in a blind fashion among four groups: group 1 ^ vehicle/vehicle ; group 2 ^ vehicle/Fe; group 3 ^ NGF or NT-4/5/vehicle; group 4 ^ NGF or NT-4/5/Fe. *P 6 0.05, ANOVA followed by the Student^ Newman^Keuls test, n = 8^12, mean T S.D. Vehicle = vehicle media, Fe = Fe3þ -citrate.
potential role of oxidative stress to the neurotrophin injury potentiation in vivo, we tested the ability of the antioxidant S-PBN to abolish the potentiation. We found that co-treatment with S-PBN 200 mg/kg, i.p. twice daily, begun with onset of neurotrophin delivery reduced both BDNF and NT-4/5 potentiation of iron injury (Fig. 4D^F). Cell count documentation of injury
RESULTS
E¡ect of neurotrophins and co-treatment with S-PBN on Fe3+-induced spinal cord injury Intra-cord injection of iron (0.75 nmol Fe3þ -citrate in 0.5 Wl PBS, pH 7.4) alone in animals that had received vehicle pretreatment injected intra-cord 24 h earlier produced a small lesion con¢ned to the central gray matter as assessed 24 h later (Fig. 2A). The pretreatment injection of vehicle per se produced local injury near the injection tract. In contrast, in animals pretreated 24 h with intra-cord injections of NT-3 (1 Wg, each of two sites), intra-cord injection of iron induced markedly increased tissue damage with substantial cell loss, assessed 24 h later (Fig. 2B, C). Injury potentiation was also observed with NT-4/5 (0.5 Wg, each of two sites) pretreatment, but not NGF (1 Wg, each of two sites) pretreatment (Fig. 3). Pretreatment with NT-3 or NT-4/5 followed by intra-cord injection of vehicle produced more tissue injury than injections of vehicle alone. Iron injury potentiation similar to NT-3 and NT-4/5 was induced also by BDNF pretreatment (Fig. 4A^C, E) (P 6 0.05, ANOVA followed by the Student^Newman^Keuls test, n = 6^12, mean T S.D.). Previous in vitro studies indicate that oxidative stress plays an important role in the neurotrophin injury potentiation (Koh et al., 1995; Prehn, 1996; Samdani et al., 1997; Glazner and Mattson, 2000). To examine the
The observations of tissue damage were then extended to examination of immunohistochemically identi¢ed neurons, oligodendrocytes, and astrocytes in ¢ve groups: group 1 ^ vehicle/vehicle/saline; group 2 ^ BDNF/ vehicle/saline; group 3 ^ vehicle/Fe/saline; group 4 ^ BDNF/Fe/saline; group 5 ^ BDNF/Fe/S-PBN. The injection of iron alone (group 3) induced modestly increased neuronal loss compared to vehicle near the epicenter. However, BDNF pretreatment markedly potentiated the iron injury, resulting in widespread neuronal loss extending as far as studied, 1600 Wm from the injection epicenter (P 6 0.05) (Fig. 5). Co-application of the S-PBN eliminated the BDNF enhancement of neuronal loss (P 6 0.05) (Fig. 5). Iron induced a similar pattern of oligodendrocyte loss in gray matter. BDNF-potentiated iron-induced oligodendrocyte loss also extended up to 1600 Wm away from the injection epicenter (P 6 0.05) (Fig. 6). BDNFpotentiated oligodendrocyte loss was also eliminated by co-treatment with S-PBN (P 6 0.05) (Fig. 6). No substantial di¡erences in astrocyte number were observed among the treatment groups.
DISCUSSION
Although the ability of neurotrophins to promote neuronal survival and attenuate neuronal apoptosis is well recognized, several recent studies have demonstrated that neurotrophin pretreatment can potentiate excitotoxic and oxidative necrosis of cultured neurons. Previous
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NSC 5766 4-11-02 Fig. 4. Reduction of BDNF and NT-4/5 mediated potentiation of iron toxicity by the S-PBN. All experiments were performed as described in Fig. 1. Panels A^D : representative longitudinal para⁄n sections stained with anti-NeuN. (A) Vehicle/Fe/saline i.p.; (B) BDNF/vehicle/saline i.p.; (C) BDNF/Fe/saline i.p.; (D) BDNF/Fe/S-PBN i.p. Injection of vehicle media alone produced mild injury con¢ned to the area adjacent to the injection tract (data not shown). (E) illustration of the quantitative data of ¢ve groups and their comparison : group 1 ^ vehicle/vehicle/saline i.p.; group 2 ^ vehicle/Fe/saline i.p.; group 3 ^ BDNF/vehicle/saline i.p.; group 4 ^ BDNF/Fe/saline i.p.; group 5 ^ BDNF/Fe/S-PBN i.p. (200 mg/kg, twice a day); positions 1 and 2 refer to what was injected into the cord on days 1 and 2, respectively ; position 3 refers to what was delivered i.p. on both days 1 and 2, each twice daily. Twenty-four hours after the ¢nal intra-cord injection on day 2, tissues were processed and area measurements were completed. The S-PBN reduced BDNF-mediated potentiation of iron toxicity. *P 6 0.05, ANOVA followed by the Student^Newman^Keuls test, n = 5, mean T S.D.; (F) the S-PBN also reduced NT-4/5-mediated potentiation of iron toxicity. Three groups were compared : group 1 ^ vehicle/Fe/saline i.p.; group 2 ^ NT-4/5/Fe/saline i.p.; group 3 ^ NT-4/5/Fe/S-PBN i.p. (200 mg/kg, twice a day). *P 6 0.05, ANOVA followed by the Student^Newman^Keuls test, n = 8, mean T S.D. Scale bar = 1 mm. Vehicle = vehicle media, Fe = Fe3þ -citrate.
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work, by our group and others, suggests that oxidative stress may be an important mechanism contributing to the neurotrophin injury potentiation (Gwag et al., 1995; Lobner and Choi, 1996; Samdani et al., 1997; Park et al., 1998; Glazner and Mattson, 2000; Won et al., 2000). The present study provides direct demonstration that neurotrophins can also have a paradoxical injury-potentiating e¡ect in vivo in the adult rat spinal cord. We found that pretreatment with NT-3, NT-4/5 and BDNF potentiated the free radical injury induced by intra-cord injection of iron. Two di¡erent quantitative methods for indexing injury severity, measurement of gross lesion area and cell counts of immunohistochemically identi¢ed neurons or oligodendrocytes, revealed evidence that neurotrophins potentiated iron-induced gray matter injury. The injury potentiation was not induced by pretreatment with either vehicle or NGF. This selectivity is similar to that previously found in cultured murine neocortical cultures, where pretreatment with NT-3, NT-4/5 or BDNF, but not NGF, potentiated neuronal necrosis after oxygen^glucose deprivation or NMDA exposure (Koh et al., 1995). Necrosis induced by a 24-h exposure to iron in cortical cell cultures was also potentiated by BDNF pretreatment (Gwag et al., 1995).
Current observations in vivo thus support the hypothesis formulated based on in vitro observations (Gwag et al., 1995; Koh et al., 1995) that neurotrophins may have two opposing actions in the injured CNS: reduction of neuronal apoptosis, but potentiation of neuronal necrosis. The observation that co-treatment with the putative antioxidant S-PBN (although see Atamna et al., 2000) negated the neurotrophin injury potentiation in the spinal cord is consistent with a critical role for free radicals in this injury potentiation. These data are also compatible with observations that S-PBN or other antioxidant treatments can produce synergistic protection when combined with BDNF in an in vivo model of axotomy-induced retinal ganglion death (Isenmann et al., 1998; Klo«cker et al., 1998). Further studies will be needed to £esh out understanding of the mechanism(s) underlying neurotrophin injury potentiation. It may be mediated in part by up-regulation of NMDA receptor expression or function (Bai and Kusiak, 1997; Jarvis et al., 1997; Suen et al., 1997; Levine et al., 1998; Lin et al., 1998; Small et al., 1998) or by enhancement of glutamate release (Takei et al., 1998). Regardless of the underlying mechanism, the broad therapeutic promise of neurotrophins in neu-
Fig. 5. Cell counts of neurons immunostained with anti-NeuN were performed in the gray matter. A 0.16-mm2 area at U200 magni¢cation was examined at 400 Wm, 800 Wm and 1600 Wm rostrally (R) and caudally (C) from the epicenter (0), the middle point between both sites of the injection. Values were averaged from three sections through the gray matter, one centered on the central canal and the others separated by 150 Wm. Five groups were investigated : group 1 ^ vehicle/vehicle/ saline i.p.; group 2 ^ BDNF/vehicle/saline i.p. ; group 3 ^ vehicle/Fe/saline i.p.; group 4 ^ BDNF/Fe/saline i.p.; group 5 ^ BDNF/Fe/S-PBN i.p. Statistical evaluation was performed with one-way ANOVA and Bonferroni t-test for multiple comparisons. At all distances investigated BDNF potentiated iron injury and the co-application of S-PBN reduced this potentiation, i.e. group 4 di¡ers from all other groups, *P 6 0.05 vs. every other group at each speci¢c distance. #P 6 0.05 for group 1 vs. other groups as indicated. n = 4, mean T S.D. Vehicle = vehicle media, Fe = Fe3þ -citrate.
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Fig. 6. Cell counts of APC CC1 immunolabeled oligodendrocytes were performed in the gray matter as discussed in Fig. 5 legend. The same ¢ve groups were examined : group 1 ^ vehicle/vehicle/saline i.p.; group 2 ^ BDNF/vehicle/saline i.p.; group 3 ^ vehicle/Fe/saline i.p.; group 4 ^ BDNF/Fe/saline i.p.; group 5 ^ BDNF/Fe/S-PBN i.p. Statistical evaluation was performed with one-way ANOVA and Bonferroni t-test for multiple comparisons. At all distances investigated BDNF potentiated iron injury and the co-application of S-PBN reduced the potentiation, except for the epicenter, *P 6 0.05 for group 4 vs. each other group at all measurement points. #P 6 0.05 for group 1 vs. other groups as indicated. n = 4, mean T S.D. Vehicle = vehicle media, Fe = Fe3þ -citrate.
rological diseases makes it compelling to explore the potential of neurotrophins to enhance CNS damage under some conditions. It is conceivable that unsuspected injury-potentiating e¡ects may have contributed to some negative results seen to date with the testing of neurotrophins in human clinical trials of motor neuron disease.
Acknowledgements)This work was funded by grants from the NIH (NS32636 to D.W.C.; NS01931, NS36265 to J.W.M.), and the Christopher Reeve Paralysis Foundation (D.W.C. and J.W.M.). NGF, NT-3 and NT-4/5 were kind gifts from Regeneron (Tarrytown, NY, USA), Amgen (Thousand Oaks, CA, USA), and Genentech (South San Francisco, CA, USA), respectively. We gratefully acknowledge helpful comments from Dr. B. Joy Snider.
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Novel treatments after experimental brain injury. Semin. Neonatol. 5, 75^86. Kiprianova, I., Freiman, T.M., Desiderato, S., Schwab, S., Galmbacher, R., Gillardon, F., Spranger, M., 1999. Brain-derived neurotrophic factor prevents neuronal death and glial activation after global ischemia in the rat. J. Neurosci. Res. 56, 21^27. Kirschner, P.B., Jenkins, B.G., Schulz, J.B., Finkelstein, S.P., Matthews, R.T., Rosen, B.R., Beal, M.F., 1996. NGF, BDNF and NT-5, but not NT-3 protect against MPPþ toxicity and oxidative stress in neonatal animals. Brain Res. 713, 178^185. Klo«cker, N., Cellerino, A., Ba«hr, M., 1998. Free radical scavenging and inhibition of nitric oxide synthase potentiates the neurotrophic e¡ects of brain-derived neurotrophic factor on axotomized retinal ganglion cells in vivo. J. Neurosci. 18, 1038^1046. Kobayashi, N.R., Fan, D.-P., Giehl, K.M., Bedard, A.M., Wiegand, S.J., Tetzla¡, W., 1997. BDNF and NT-4/5 prevent atrophy of rat rubrospinal neurons after cervical axotomy, stimulate GAP-43 and TK1-tubulin mRNA expression, and promote axonal regeneration. J. Neurosci. 17, 9583^9595. Koh, J.-Y., Gwag, B.J., Lobner, D., Choi, D.W., 1995. Potentiated necrosis of cultured cortical neurons by neurotrophins. Science 268, 573^575. Krenz, N.R., Weaver, L.C., 2000. Nerve growth factor in glia and in£ammatory cells of the injured rat spinal cord. J. Neurochem. 74, 730^739. Lee, J.-M., Zipfel, G.J., Choi, D.W., 1999. The changing landscape of ischaemic brain injury mechanisms. Nature 399, A7^A14. Lever, I.J., Bradbury, E.J., Cunningham, J.R., Adelson, D.W., Jones, M.G., McMahon, S.B., Marvizo¤n, J.C.G., Malcangio, M., 2001. Brainderived neurotrophic factor is released in the dorsal horn by distinctive patterns of a¡erent ¢ber stimulation. J. Neurosci. 21, 4469^4477. Levine, E.S., Crozier, R.A., Black, I.B., Plummer, M.R., 1998. Brain-derived neurotrophic factor modulates hippocampal synaptic transmission by increasing N-methyl-D-aspartic acid receptor activity. Proc. Natl. Acad. Sci. USA 95, 10235^10239. Lin, S.-Y., Wu, K., Levine, E.S., Mount, H.T., Suen, P.C., Black, I.B., 1998. BDNF acutely increases tyrosine phosphorylation of the NMDA receptor subunit 2B in cortical and hippocampal postsynaptic densities. Mol. Brain Res. 55, 20^27. Lobner, D., Choi, D.W., 1996. Preincubation with protein synthesis inhibitors protects cortical neurons against oxygen^glucose deprivationinduced death. Neuroscience 72, 335^341. McDonald, J.W., Roeser, N.F., Silverstein, F.S., Johnston, M.V., 1989. Quantitative assessment of neuroprotection against NMDA-induced brain injury. Exp. Neurol. 106, 289^296. McTigue, D.M., Horner, P.J., Stokes, B.T., Gage, F.H., 1998. Neurotrophin-3 and brain-derived neurotrophic factor induce oligodendrocyte proliferation and myelination of regenerating axons in the contused adult rat spinal cord. J. Neurosci. 18, 5354^5365. Novikova, L.N., Novikov, L.N., Kellerth, J.-O., 2000. Survival e¡ects of BDNF and NT-3 on axotomized rubrospinal neurons depend on the temporal pattern of neurotrophin administration. Eur. J. Neurosci. 12, 776^780. Park, E.C., Jou, I., Gwag, B.J., 1998. Nerve growth factor potentiates the oxidative necrosis of striatal cholinergic neurons. NeuroReport 9, 687^ 690. Pitts, A.F., Miller, M.W., 2000. Expression of nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3 in the somatosensory cortex of the mature rat: coexpression with high-a⁄nity neurotrophin receptors. J. Comp. Neurol. 418, 241^254. Prehn, J.H.M., 1996. Marked diversity in the action of growth factors on N-methyl-D-aspartate-induced neuronal degeneration. Eur. J. Pharmacol. 306, 81^88. Samdani, A.F., Newcamp, C., Resink, A., Facchinetti, F., Ho¡man, B.E., Dawson, V.L., Dawson, T.M., 1997. Di¡erential susceptibility to neurotoxicity mediated by neurotrophins and neuronal nitric oxide synthase. J. Neurosci. 17, 4633^4641. Scarisbrick, I.A., Isackson, P.J., Windebank, A.J., 1999. Di¡erential expression of brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4/5 in the adult rat spinal cord: regulation by the glutamate receptor agonist kainic acid. J. Neurosci. 19, 7757^7769. Scha«bitz, W.-R., Schwab, S., Spranger, M., Hacke, W., 1997. Intraventricular brain-derived neurotrophic factor reduces infarct size after focal cerebral ischemia in rats. J. Cereb. Blood Flow Metab. 17, 500^506. Schu«tte, A., Yan, Q., Mestres, P., Giehl, K.M., 2000. The endogenous survival promotion of axotomized rat corticospinal neurons by brainderived neurotrophic factor is mediated via paracrine, rather than autocrine mechanisms. Neurosci. Lett. 290, 185^188. Sengstock, G.J., Olanow, C.W., Dunn, A.J., Arendash, G.W., 1992. Iron induces degeneration of nigrostriatal neurons. Brain Res. Bull. 28, 645^ 649. Shigeno, T., Mima, T., Takakura, K., Graham, D.I., Kato, G., Hashimoto, Y., Furukawa, S., 1991. Amelioration of delayed neuronal death in the hippocampus by nerve growth factor. J. Neurosci. 11, 2914^2919.
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Sinson, G., Perri, B.R., Trojanowski, J.Q., Flamm, E.S., McIntosh, T.K., 1997. Improvement of cognitive de¢cits and decreased cholinergic neuronal cell loss and apoptotic cell death following neurotrophin infusion after experimental traumatic brain injury. J. Neurosurg. 86, 511^ 518. Small, D.L., Murray, C.L., Mealing, G.A.R., Poulter, M.O., Buchan, A.M., Morley, P., 1998. Brain derived neurotrophic factor induction of N-methyl-D-aspartate receptor subunit NR2A expression in cultured rat cortical neurons. Neurosci. Lett. 252, 211^214. Suen, P.-C., Wu, K., Levine, E.S., Mount, H.T.J., Xu, J.-L., Lin, S.-Y., Black, I.B., 1997. Brain-derived neurotrophic factor rapidly enhances phosphorylation of the postsynaptic N-methyl-D-aspartate receptor subunit 1. Proc. Natl. Acad. Sci. USA 94, 8191^8195. Takei, N., Numakawa, T., Kozaki, S., Sakai, N., Endo, Y., Takahashi, M., Hatanaka, H., 1998. Brain-derived neurotrophic factor induces rapid and transient release of glutamate through the non-exocytotic pathway from cortical neurons. J. Biol. Chem. 273, 27620^27624. Tuszynski, M.H., U, H.S., Amaral, D.G., Gage, F.H., 1990. Nerve growth factor infusion in the primate brain reduces lesion-induced cholinergic neuronal degeneration. J. Neurosci. 10, 3604^3614. Won, S.J., Park, E.C., Ryu, B.R., Ko, H.W., Sohn, S., Kwon, H.J., Gwag, B.J., 2000. NT-4/5 exacerbates free radical-induced neuronal necrosis in vitro and in vivo. Neurobiol. Dis. 7, 251^259. Yan, Q., Elliott, J., Snider, W.D., 1992. Brain-derived neurotrophic factor rescues spinal motor neurons from axotomy-induced cell death. Nature 360, 753^755. Yan, Q., Radeke, M.J., Matheson, C.R., Talvenheimo, J., Welcher, A.A., Feinstein, S.C., 1997. Immunocytochemical localization of TrkB in the central nervous system of the adult rat. J. Comp. Neurol. 378, 135^157. (Accepted 12 June 2002)
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Neuroscience Vol. 115, No. 3, pp. 931^939, 2002 D 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 02 $22.00+0.00
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NEUROTROPHIN POTENTIATION OF IRON-INDUCED SPINAL CORD INJURY J. W. MCDONALD, V. G. STEFOVSKA, X. Z. LIU, H. SHIN, S. LIU and D. W. CHOI Center for the Study of Nervous System Injury and Department of Neurology, Washington University School of Medicine, P.O. Box 8111, 660 S. Euclid Avenue, St. Louis, MO 63110-1093, USA
Abstract8Previous studies have shown that pretreatment with neurotrophins can potentiate the vulnerability of cultured neurons to excitotoxic and free radical-induced necrosis, in contrast to their well known neuroprotective e¡ects against apoptosis. Here we tested the hypothesis that this unexpected injury-potentiating e¡ect of neurotrophins would also take place in the adult rat spinal cord. Fe3þ -citrate was injected stereotaxically into spinal cord gray matter in adult rats in amounts su⁄cient to produce minimal tissue injury 24 h later. Twenty-four-hour pretreatment with brain-derived neurotrophic factor, neurotrophin-3, or neurotrophin-4/5, but not nerve growth factor, markedly enhanced tissue injury in the gray matter as evidenced by an increase in the damaged area, as well as the loss of neurons and oligodendrocytes. Consistent with maintained free radical mediation, the neurotrophin-potentiated iron-induced spinal cord damage was blocked by co-application of the antioxidant N-tert-butyl-(2-sulfophenyl)-nitrone. These data support the hypothesis that the overall neuroprotective properties of neurotrophins in models of acute injury to the spinal cord may be limited by an underlying potentiation of free radical-mediated necrosis. D 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4/5, excitotoxicity, free radicals.
and glial cells (Schwann cells, microglia, astrocytes and oligodendrocytes) (Dreyfus et al., 1999; Krenz and Weaver, 2000; Huang and Reichardt, 2001). In development and models of nervous system injury, neurotrophins have a powerful ability to reduce neuronal cell death, in particular apoptosis mediated by growth factor deprivation, such as induced by axotomy in vivo (Hefti, 1986; Tuszynski et al., 1990; Yan et al., 1992; Kobayashi et al., 1997; Houle and Ye, 1999; Novikova et al., 2000), or neuronal injury induced by exposure to excitotoxins, glucose or serum deprivation in vitro (Alderson et al., 1990; Shigeno et al., 1991; Davies and Beardsall, 1992; Frim et al., 1993; Cheng et al., 1994; Cheng and Mattson, 1994). However, several in vitro studies have raised the possibility that the neuroprotective e⁄cacy of neurotrophins may be limited by an underlying propensity to potentiate neuronal death under some conditions. Pretreatment with BDNF or NT-3 enhanced glutamate-induced death of cultured cerebellar neurons, a death possibly apoptotic since basic ¢broblast growth factor-induced survival was linked to increased calcium in£ux (Ferna¤ndez-Sa¤nchez and Novelli, 1993; see also Ankarcrona et al., 1995). Conversely, we found that pretreatment with NT-3, NT-4/5, or BDNF reduced the vulnerability of cultured cortical neurons to apoptosis, but enhanced necrosis induced by N-methyl-D-aspartate (NMDA), oxygen^glucose deprivation, or iron-induced oxidative stress (Gwag et al., 1995; Koh et al., 1995). This necrosis-speci¢c injury potentiation was likely dependent on new protein synthesis since it required pretreatment for several hours and was abol-
Neurotrophins regulate development, maintenance and plasticity in the vertebrate nervous system. There are four neurotrophin families identi¢ed in mammals: nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5). These neurotrophins activate two separate classes of receptors, the tyrosine kinase (Trk) receptors and p75NTR, a member of the tumor necrosis factor receptor superfamily (Giehl et al., 2001; Huang and Reichardt, 2001). NGF is the preferred ligand for TrkA, BDNF and NT-4/5 are the preferred ligands for TrkB, and NT-3 binds primarily to TrkC, although it can also interact with TrkA or TrkB (Yan et al., 1997). Both neurotrophins and their receptors are widely expressed on neurons, including cortical (Pitts and Miller, 2000; Schu«tte et al., 2000), spinal cord (Scarisbrick et al., 1999), and dorsal root ganglia neurons (Heppenstall and Lewin, 2001; Lever et al., 2001). In addition, neurotrophins are produced by target organs
*Corresponding author. Present address: Merck Research Labs, West Point, PA 19486, USA. Tel.: +1-314-362-9460; fax: +1-314747-0422. E-mail address: [email protected] (D. W. Choi). Abbreviations : APC CC1, adenomatous polyposis coli gene; BDNF, brain-derived neurotrophic factor; HpE, hematoxylin and eosin; LFB-PAS, Luxol-Fast Blue-PAS ; NeuN, neuron-speci¢c nuclear protein; NGF, nerve growth factor; NMDA, N-methyl-D-aspartate; NT-3, neurotrophin-3; NT-4/5, neurotrophin-4/5; PBS, phosphate-bu¡ered saline; S-PBN, N-tert-butyl(2-sulfophenyl)-nitrone; Trk, tyrosine kinase. 931
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ished by concurrent exposure to protein synthesis inhibitors (Lobner and Choi, 1996). Other groups have also reported observations consistent with neurotrophin injury potentiation (Prehn, 1996; Samdani et al., 1997; Park et al., 1998; Glazner and Mattson, 2000). In addition, Won et al. (2000) found that injection of NT-4/5 potentiated the striatal damage induced by co-injection of Fe2þ in the adult rat brain. The idea that neurotrophin mediated injury enhancement may be a common component of neurotrophin action does not necessarily con£ict with the many in vivo studies demonstrating net bene¢ts by neurotrophins against oxidative (Kirschner et al., 1996), ischemic (Chan et al., 1996; Cheng et al., 1997; Scha«bitz et al., 1997; Andsberg et al., 1998; Kiprianova et al., 1999) or traumatic (Dixon et al., 1997; Grill et al., 1997; Sinson et al., 1997; Houweling et al., 1998a,b; Jakeman et al., 1998) insults. Since programmed cell death likely contributes to neuronal loss after acute insults (Johnson et al., 1996; Lee et al., 1999; Johnston et al., 2000), the anti-apoptotic e¡ects of neurotrophins may be large enough to override any concurrent enhancement of necrosis. The goal of the present study was to test the hypothesis that the observed ability of neurotrophins to potentiate neuronal necrosis in vitro and in striatum in vivo would also apply to the adult spinal cord, noting that neurotrophin administration is speci¢cally of interest as a possible therapy for spinal cord injury (Grill et al., 1997; Houweling et al., 1998a,b; Jakeman et al., 1998; McTigue et al., 1998).
EXPERIMENTAL PROCEDURES
Preparation of Fe 3+-citrate solution A pH balanced, iso-osmotic Fe3þ -citrate solution was prepared using methods adapted from Sengstock et al. (1992). Stock solutions were made for the four components of the Fe3þ -citrate solution: #1 ferric chloride hexahydrate ^
FeCl3 W6H2 0 (molecular weight (MW) 270.3, 100 mM; Sigma, St. Louis, MO, USA); #2 sodium citrate-dihydrate ^ C6 H5 Na3 O7 W2H2 O (MW 294.1, 180 mM; Sigma); #3 sodium bicarbonate ^ NaHCO3 (MW 84.01, 11.4 mM; Sigma); #4 Trizma Base ^ C4 H11 NO3 (MW 121.1, 0.455 M; Sigma). The Fe3þ -citrate solution was always prepared immediately before each experiment by combining the above stock solutions: 15 Wl #1, 15 Wl #2, 100 Wl #3, 6 Wl #4, then phosphate-bu¡ered saline (PBS), pH 7.4 balance to a total volume of 1 ml. Animal care and spinal cord injury All surgical interventions and animal care were provided in accordance with the Laboratory Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996) and the Guidelines and Policies for Rodent Survival Surgery provided by the Animal Studies Committee of Washington University School of Medicine. Adult Long^Evans female rats (275 T 25 g, Simonsen Laboratories ^ Gilroy, CA, USA) were anesthetized with ketamine/medetomidine (75:0.5 mg/kg i.p.). Ketamine was from Fort Dodge Animal Health (Fort Dodge, IW, USA), medetomidine was from Burns Veterinary supply (Rockville, NY, USA). A laminectomy was performed at T9 and microstereotaxic injections of vehicle, NGF, BDNF, NT-3, or NT-4/5 were completed at two sites, 1 mm apart just to the right of midline (marked by the dorsal vein) 1.0 mm below the dura into the central gray matter. NGF, NT-3 and NT-4/5 were gifts from Regeneron (Tarrytown, NY, USA), Amgen (Thousand Oaks, CA, USA), and Genentech (South San Francisco, CA, USA), respectively. BDNF was from Amgen and RpD Systems (Minneapolis, MN, USA). The treatment paradigm used in these studies is shown in Fig. 1. The amounts of neurotrophin injected at each site were 0.5 Wg for NT-4/5 and 1 Wg for NGF, BDNF, NT-3 in 0.5 Wl (volumes were always kept constant). The wound was closed and sedation was reversed with antisedane (Atipamezole, 1 mg/kg s.c., Animal Health, Exton, PA, USA). Rectal temperature was maintained at 37.0 T 0.5‡C using a thermostatically regulated heating pad (Versa-Therm 2156; Cole-Parmer, Chicago, IL, USA) and heating lamp system during surgery and for 2 h during recovery. Twenty-four hours after the initial injections, the wounds were reopened and all animals received a single microstereotaxic injection of a threshold injury dose of Fe3þ -citrate (0.75 nmol in 0.5 Wl PBS, pH 7.4) midway between the two previous injections. The antioxidant N-tert-butyl-(2-sulfophenyl)-nitrone (S-PBN) (Sigma), 200 mg/kg i.p. or saline of a
Fig. 1. Schematic representation of the intra-cord injection paradigm. Adult Long^Evans female rats, deeply anesthetized, received a microstereotaxic injection of vehicle media (0.5 Wl), NGF, BDNF, NT-3 (1 Wg/0.5 Wl) or NT-4/5 (0.5 Wg/0.5 Wl) at two sites each 1 mm apart just to the right of midline in the central gray matter on day 1. Twenty-four hours later, on day 2, all animals received a single intra-cord injection of a minimally toxic amount of iron (0.75 nmol Fe3þ -citrate in 0.5 Wl vehicle media, pH 7.4) or only vehicle media (0.5 Wl) midway between the two previous injections, n = 5^12 for all groups. Twenty-four hours later, on day 3, animals were killed. Tissues were processed for para⁄n embedding, routine staining with HpE or LFB-PAS and speci¢c immunohistochemistry. Injury was quanti¢ed by two methods : area measurements of the injured tissue and cell counts of neurons and oligodendrocytes.
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∗
Fig. 2. NT-3 potentiation of Fe3þ -induced spinal cord injury. Experiments were performed as described in Fig. 1. Panels A and B illustrate representative amounts of tissue injury present in longitudinal para⁄n sections, stained with anti-NeuN, from animals that were pretreated with vehicle media (vehicle/Fe ; panel A) and NT-3 (NT-3/Fe; panel B) on day 1 followed by intra-cord injection of iron on day 2. Panel C illustrates the quantitative analysis of lesion area measurements from comparison of four groups: group 1 ^ vehicle/vehicle, intra-cord injections of vehicle media on days 1 and 2; group 2 ^ NT-3/ vehicle, intra-cord injections of NT-3 (1 Wg) on day 1 and vehicle media on day 2; group 3 ^ vehicle/Fe, injections of vehicle media on day 1 and Fe3þ on day 2; group 4 ^ NT-3/Fe, injections of NT-3 on day 1 and Fe3þ on day 2. *P 6 0.05, ANOVA followed by the Student^Newman^Keuls test, n = 6 for each group, mean T S.D. Scale bar = 1 mm. Vehicle = vehicle media, Fe = Fe3þ -citrate.
similar volume i.p., was applied on day 1 and day 2, each twice daily. A set of preliminary studies was completed to determine a threshold injury amount of iron when injected into the adult rat spinal cord. Microstereotaxic injection of 0.75 nmol Fe3þ / 0.5 Wl PBS into the central gray matter at the T9 level of the spinal cord produced a discrete gray matter lesion present in all animals. Instead of iron injection, control groups of animals treated with neurotrophins the ¢rst day received only vehicle injections of a similar volume on the second day. The number of animals used per group ranged between ¢ve and 12. Histology Twenty-four hours after the ¢nal injection, animals were killed with a lethal dose of phenobarbital (100 mg/kg i.p.), and perfused intracardially with physiological saline followed by 4% paraformaldehyde in 0.1 M PBS, pH 7.4. Tissue specimens using 2 cm of the spinal cord surrounding the injection
sites were then processed for para⁄n embedding and 7-Wm para⁄n sections were cut longitudinally. Serial sections were collected from the anterior horns through the entire dorsal columns. Sections were routinely stained for hematoxylin and eosin (HpE) and Luxol-Fast Blue-PAS (LFB-PAS). Immunohistochemistry Primary antibodies were used to visualize neurons (NeuN, against neuron-speci¢c nuclear protein, mIgG1 , 1:500, Chemicon, Temecula, CA, USA), oligodendrocytes (APC CC1, against adenomatous polyposis coli gene, mIgG1 , 1:400, Calbiochem Oncogene Sciences, La Jolla, CA, USA) and astrocytes (GFAP, against glial ¢brillary acidic protein, rabbit polyclonal, 1:4, Incstar, Stillwater, MI, USA). Sections for APC CC1 staining were treated with antigen unmasking solution (Vector Laboratories, Burlingame, CA, USA) in a microwave. Blocking of endogenous peroxidase with 3% H2 O2 in methanol and nonspeci¢c antigenic sites with 10% horse or goat serum was per-
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formed before the ¢rst antibody. Biotinylated species-speci¢c secondary antibodies (1:200 dilution, Vector) were applied, followed by ABC complex (Vector). Detection of peroxidase activity was carried out by 3,3P-diaminobenzidine tetrahydrochloride (DAB kit, Vector). Quantitative analysis of injury Area measurements of the injured tissue were completed in three longitudinal sections, one centered on the central canal and the others separated by 200 Wm, from six to 12 animals in each group in a blinded fashion using a video-based image analysis system (McDonald et al., 1989). Values were summed within animals and averaged group values were compared amongst groups using Student’s independent t-tests for two group comparisons, and one-way analysis of variants (ANOVA) with post-hoc test for comparisons of three or more groups. Data are mean T S.E.M. values. Area data were further con¢rmed counting the number of surviving neurons and oligodendrocytes in the BDNF group, as our original observations of neurotrophin potentiation were made with BDNF (Koh et al., 1995). Cell counts for NeuN and APC CC1 positive cells were performed on three di¡erent longitudinal sections, one centered on the central canal and the others separated by 150 Wm, from four animals in each group in a blinded fashion. A 0.16-mm2 area in the gray matter at U200 magni¢cation was examined in the epicenter (the middle point between both sites of the injection), 400 Wm, 800 Wm and 1600 Wm rostrally and caudally. The average number of cells from each area at the respective distance in every animal was calculated. Only intensively labeled cells with clearly de¢ned cell body and nucleus were used. Statistical evaluation was performed with one-way ANOVA and Bonferroni t-test for multiple comparisons.
Fig. 3. Selective neurotrophin potentiation of Fe3þ -induced spinal cord injury. NT-4/5 but not NGF or vehicle media potentiated Fe3þ injury. Experiments were performed as outlined in Figs. 1 and 2. Area measurements of injured tissue were completed in a blind fashion among four groups: group 1 ^ vehicle/vehicle ; group 2 ^ vehicle/Fe; group 3 ^ NGF or NT-4/5/vehicle; group 4 ^ NGF or NT-4/5/Fe. *P 6 0.05, ANOVA followed by the Student^ Newman^Keuls test, n = 8^12, mean T S.D. Vehicle = vehicle media, Fe = Fe3þ -citrate.
potential role of oxidative stress to the neurotrophin injury potentiation in vivo, we tested the ability of the antioxidant S-PBN to abolish the potentiation. We found that co-treatment with S-PBN 200 mg/kg, i.p. twice daily, begun with onset of neurotrophin delivery reduced both BDNF and NT-4/5 potentiation of iron injury (Fig. 4D^F). Cell count documentation of injury
RESULTS
E¡ect of neurotrophins and co-treatment with S-PBN on Fe3+-induced spinal cord injury Intra-cord injection of iron (0.75 nmol Fe3þ -citrate in 0.5 Wl PBS, pH 7.4) alone in animals that had received vehicle pretreatment injected intra-cord 24 h earlier produced a small lesion con¢ned to the central gray matter as assessed 24 h later (Fig. 2A). The pretreatment injection of vehicle per se produced local injury near the injection tract. In contrast, in animals pretreated 24 h with intra-cord injections of NT-3 (1 Wg, each of two sites), intra-cord injection of iron induced markedly increased tissue damage with substantial cell loss, assessed 24 h later (Fig. 2B, C). Injury potentiation was also observed with NT-4/5 (0.5 Wg, each of two sites) pretreatment, but not NGF (1 Wg, each of two sites) pretreatment (Fig. 3). Pretreatment with NT-3 or NT-4/5 followed by intra-cord injection of vehicle produced more tissue injury than injections of vehicle alone. Iron injury potentiation similar to NT-3 and NT-4/5 was induced also by BDNF pretreatment (Fig. 4A^C, E) (P 6 0.05, ANOVA followed by the Student^Newman^Keuls test, n = 6^12, mean T S.D.). Previous in vitro studies indicate that oxidative stress plays an important role in the neurotrophin injury potentiation (Koh et al., 1995; Prehn, 1996; Samdani et al., 1997; Glazner and Mattson, 2000). To examine the
The observations of tissue damage were then extended to examination of immunohistochemically identi¢ed neurons, oligodendrocytes, and astrocytes in ¢ve groups: group 1 ^ vehicle/vehicle/saline; group 2 ^ BDNF/ vehicle/saline; group 3 ^ vehicle/Fe/saline; group 4 ^ BDNF/Fe/saline; group 5 ^ BDNF/Fe/S-PBN. The injection of iron alone (group 3) induced modestly increased neuronal loss compared to vehicle near the epicenter. However, BDNF pretreatment markedly potentiated the iron injury, resulting in widespread neuronal loss extending as far as studied, 1600 Wm from the injection epicenter (P 6 0.05) (Fig. 5). Co-application of the S-PBN eliminated the BDNF enhancement of neuronal loss (P 6 0.05) (Fig. 5). Iron induced a similar pattern of oligodendrocyte loss in gray matter. BDNF-potentiated iron-induced oligodendrocyte loss also extended up to 1600 Wm away from the injection epicenter (P 6 0.05) (Fig. 6). BDNFpotentiated oligodendrocyte loss was also eliminated by co-treatment with S-PBN (P 6 0.05) (Fig. 6). No substantial di¡erences in astrocyte number were observed among the treatment groups.
DISCUSSION
Although the ability of neurotrophins to promote neuronal survival and attenuate neuronal apoptosis is well recognized, several recent studies have demonstrated that neurotrophin pretreatment can potentiate excitotoxic and oxidative necrosis of cultured neurons. Previous
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NSC 5766 4-11-02 Fig. 4. Reduction of BDNF and NT-4/5 mediated potentiation of iron toxicity by the S-PBN. All experiments were performed as described in Fig. 1. Panels A^D : representative longitudinal para⁄n sections stained with anti-NeuN. (A) Vehicle/Fe/saline i.p.; (B) BDNF/vehicle/saline i.p.; (C) BDNF/Fe/saline i.p.; (D) BDNF/Fe/S-PBN i.p. Injection of vehicle media alone produced mild injury con¢ned to the area adjacent to the injection tract (data not shown). (E) illustration of the quantitative data of ¢ve groups and their comparison : group 1 ^ vehicle/vehicle/saline i.p.; group 2 ^ vehicle/Fe/saline i.p.; group 3 ^ BDNF/vehicle/saline i.p.; group 4 ^ BDNF/Fe/saline i.p.; group 5 ^ BDNF/Fe/S-PBN i.p. (200 mg/kg, twice a day); positions 1 and 2 refer to what was injected into the cord on days 1 and 2, respectively ; position 3 refers to what was delivered i.p. on both days 1 and 2, each twice daily. Twenty-four hours after the ¢nal intra-cord injection on day 2, tissues were processed and area measurements were completed. The S-PBN reduced BDNF-mediated potentiation of iron toxicity. *P 6 0.05, ANOVA followed by the Student^Newman^Keuls test, n = 5, mean T S.D.; (F) the S-PBN also reduced NT-4/5-mediated potentiation of iron toxicity. Three groups were compared : group 1 ^ vehicle/Fe/saline i.p.; group 2 ^ NT-4/5/Fe/saline i.p.; group 3 ^ NT-4/5/Fe/S-PBN i.p. (200 mg/kg, twice a day). *P 6 0.05, ANOVA followed by the Student^Newman^Keuls test, n = 8, mean T S.D. Scale bar = 1 mm. Vehicle = vehicle media, Fe = Fe3þ -citrate.
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work, by our group and others, suggests that oxidative stress may be an important mechanism contributing to the neurotrophin injury potentiation (Gwag et al., 1995; Lobner and Choi, 1996; Samdani et al., 1997; Park et al., 1998; Glazner and Mattson, 2000; Won et al., 2000). The present study provides direct demonstration that neurotrophins can also have a paradoxical injury-potentiating e¡ect in vivo in the adult rat spinal cord. We found that pretreatment with NT-3, NT-4/5 and BDNF potentiated the free radical injury induced by intra-cord injection of iron. Two di¡erent quantitative methods for indexing injury severity, measurement of gross lesion area and cell counts of immunohistochemically identi¢ed neurons or oligodendrocytes, revealed evidence that neurotrophins potentiated iron-induced gray matter injury. The injury potentiation was not induced by pretreatment with either vehicle or NGF. This selectivity is similar to that previously found in cultured murine neocortical cultures, where pretreatment with NT-3, NT-4/5 or BDNF, but not NGF, potentiated neuronal necrosis after oxygen^glucose deprivation or NMDA exposure (Koh et al., 1995). Necrosis induced by a 24-h exposure to iron in cortical cell cultures was also potentiated by BDNF pretreatment (Gwag et al., 1995).
Current observations in vivo thus support the hypothesis formulated based on in vitro observations (Gwag et al., 1995; Koh et al., 1995) that neurotrophins may have two opposing actions in the injured CNS: reduction of neuronal apoptosis, but potentiation of neuronal necrosis. The observation that co-treatment with the putative antioxidant S-PBN (although see Atamna et al., 2000) negated the neurotrophin injury potentiation in the spinal cord is consistent with a critical role for free radicals in this injury potentiation. These data are also compatible with observations that S-PBN or other antioxidant treatments can produce synergistic protection when combined with BDNF in an in vivo model of axotomy-induced retinal ganglion death (Isenmann et al., 1998; Klo«cker et al., 1998). Further studies will be needed to £esh out understanding of the mechanism(s) underlying neurotrophin injury potentiation. It may be mediated in part by up-regulation of NMDA receptor expression or function (Bai and Kusiak, 1997; Jarvis et al., 1997; Suen et al., 1997; Levine et al., 1998; Lin et al., 1998; Small et al., 1998) or by enhancement of glutamate release (Takei et al., 1998). Regardless of the underlying mechanism, the broad therapeutic promise of neurotrophins in neu-
Fig. 5. Cell counts of neurons immunostained with anti-NeuN were performed in the gray matter. A 0.16-mm2 area at U200 magni¢cation was examined at 400 Wm, 800 Wm and 1600 Wm rostrally (R) and caudally (C) from the epicenter (0), the middle point between both sites of the injection. Values were averaged from three sections through the gray matter, one centered on the central canal and the others separated by 150 Wm. Five groups were investigated : group 1 ^ vehicle/vehicle/ saline i.p.; group 2 ^ BDNF/vehicle/saline i.p. ; group 3 ^ vehicle/Fe/saline i.p.; group 4 ^ BDNF/Fe/saline i.p.; group 5 ^ BDNF/Fe/S-PBN i.p. Statistical evaluation was performed with one-way ANOVA and Bonferroni t-test for multiple comparisons. At all distances investigated BDNF potentiated iron injury and the co-application of S-PBN reduced this potentiation, i.e. group 4 di¡ers from all other groups, *P 6 0.05 vs. every other group at each speci¢c distance. #P 6 0.05 for group 1 vs. other groups as indicated. n = 4, mean T S.D. Vehicle = vehicle media, Fe = Fe3þ -citrate.
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Fig. 6. Cell counts of APC CC1 immunolabeled oligodendrocytes were performed in the gray matter as discussed in Fig. 5 legend. The same ¢ve groups were examined : group 1 ^ vehicle/vehicle/saline i.p.; group 2 ^ BDNF/vehicle/saline i.p.; group 3 ^ vehicle/Fe/saline i.p.; group 4 ^ BDNF/Fe/saline i.p.; group 5 ^ BDNF/Fe/S-PBN i.p. Statistical evaluation was performed with one-way ANOVA and Bonferroni t-test for multiple comparisons. At all distances investigated BDNF potentiated iron injury and the co-application of S-PBN reduced the potentiation, except for the epicenter, *P 6 0.05 for group 4 vs. each other group at all measurement points. #P 6 0.05 for group 1 vs. other groups as indicated. n = 4, mean T S.D. Vehicle = vehicle media, Fe = Fe3þ -citrate.
rological diseases makes it compelling to explore the potential of neurotrophins to enhance CNS damage under some conditions. It is conceivable that unsuspected injury-potentiating e¡ects may have contributed to some negative results seen to date with the testing of neurotrophins in human clinical trials of motor neuron disease.
Acknowledgements)This work was funded by grants from the NIH (NS32636 to D.W.C.; NS01931, NS36265 to J.W.M.), and the Christopher Reeve Paralysis Foundation (D.W.C. and J.W.M.). NGF, NT-3 and NT-4/5 were kind gifts from Regeneron (Tarrytown, NY, USA), Amgen (Thousand Oaks, CA, USA), and Genentech (South San Francisco, CA, USA), respectively. We gratefully acknowledge helpful comments from Dr. B. Joy Snider.
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