Lack of extracellular superoxide dismutase (EC-SOD) in the microenvironment impacts...

Lack of EC-SOD in the Microenvironment Impacts Radiation-Induced Changes in Neurogenesis

Radoslaw Rola1,4, Yani Zou5, Ting-Ting Huang5,6, Kelly Fishman1, Jennifer Baure1,Susanna Rosi1,3, Heather Milliken3, Charles L. Limoli7, and John R. Fike1,2

1Department of Neurological Surgery, University of California, San Francisco, San Francisco, CA, USA2Department of Radiation Oncology, University of California, San Francisco, San Francisco, CA, USA3Physical Therapy and Rehabilitation Science, University of California, San Francisco, San Francisco, CA,USA 4Department of Neurological Surgery, F. Skubiszewski Medical University, Lublin, Poland 5Departmentof Neurology and Neurological Sciences, Stanford University, Stanford, CA, USA 6GRECC, VA Palo AltoHealth Care System, Palo Alto, CA, USA 7Department of Radiation Oncology, University of California, Irvine,CA, USA.

AbstractIonizing irradiation results in significant alterations in hippocampal neurogenesis that are associatedwith cognitive impairments. Such effects are influenced, in part, by alterations in themicroenvironment within which the neurogenic cells exist. One important factor that may affectneurogenesis is oxidative stress, and this study was done to determine if and how the extracellularisoform of superoxide dismutase (SOD3, EC-SOD) mediated radiation-induced alterations inneurogenic cells. Wild type (WT) and EC-SOD knock out (KO) mice were irradiated with 5 Gy andacute (8–48 hr) cellular changes and long-term changes in neurogenesis were quantified. Acuteradiation responses were not different between genotypes suggesting that the absence of EC-SODdid not influence mechanisms responsible for acute cell death after irradiation. On the other hand,the extent of neurogenesis was decreased by 39% in non-irradiated KO mice relative to WT controls.In contrast, while neurogenesis was decreased by nearly 85% in WT mice after irradiation, virtuallyno reduction in neurogenesis was observed in KO mice. These findings show that after irradiation,an environment lacking EC-SOD is much more permissive in the context of hippocampalneurogenesis. This finding may have a major impact in developing strategies to reduce cognitiveimpairment after cranial irradiation.

KeywordsRadiation; brain; neurogenesis; oxidative stress; inflammation; extracellular SOD

IntroductionRadiation-related brain injury has a variable character, involving multiple regions and cell andtissue types with a large number of physical and biological factors influencing its extent [1,2]. While overt tissue injury generally occurs after relatively high doses [3,4], less severe

Corresponding author: John R. Fike, Ph.D., Brain and Spinal Injury Center, Department of Neurological Surgery, University of California,San Francisco, San Francisco, CA 94110-0899, 415 206-3384, 415 206-3373 (fax), [email protected]'s Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customerswe are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resultingproof before it is published in its final citable form. Please note that during the production process errors may be discovered which couldaffect the content, and all legal disclaimers that apply to the journal pertain.

NIH Public AccessAuthor ManuscriptFree Radic Biol Med. Author manuscript; available in PMC 2007 July 30.

Published in final edited form as:Free Radic Biol Med. 2007 April 15; 42(8): 1133–1132.

NIH

-PA Author Manuscript

NIH


NIH


morphologic changes can occur after lower doses, resulting in differing degrees of cognitiveimpairment. This latter change often involves deficits in hippocampal-dependent functions oflearning and memory, including spatial information processing [5–9]. The underlyingmechanisms responsible for radiation-induced cognitive impairment have remained elusive,although recently it has been suggested that changes in neuronal precursor cells and alteredneurogenesis in the dentate subgranular zone (SGZ) of the hippocampus may be involved[10–14].

While radiation induced changes in neurogenesis involve a persistent decrease in precursorcells, there are data also showing that the ability of surviving cells to differentiate into maturecell phenotypes may involve an altered neurogenic environment [10,13,15–18]. One of themicroenvironmental factors that may have a significant effect on neurogenesis is oxidativestress, a biochemical mechanism that has been shown to regulate the fate of neural precursorcells [19]. The central nervous system (CNS) is inherently susceptible to oxidative injury, andbecause there are relatively low levels of endogenous antioxidants in the CNS [20,21] thatsensitivity has been implicated as playing a causative or contributory role in a number ofneurodegenerative conditions, aging, and in ischemic, traumatic and excitotoxic damage [22–26]. The cellular changes observed in the CNS after irradiation are very similar to changesobserved after other types of injury [27], and may involve increased production of reactiveoxygen species (ROS), which can contribute to the spread and ultimate expression of tissueinjury [2]. Recent studies show that the radiation response of hippocampal neural precursorcells in culture is characterized by early apoptosis, specific cell cycle blocks, and activation offunctional cell cycle checkpoints; these changes are coupled with elevated levels of ROS thatpersist for weeks after irradiation [28]. Indications of persistent oxidative stress after irradiationhave also been demonstrated in the brains of mice [29] and rats [30]. Taken together, thesedata suggest that ROS may constitute critical environmental cues to control precursor cellsurvival and differentiation, and may play an important role in altered neurogenesis and perhapscognitive impairment after irradiation [29].

There are several pathways that mitigate the physiological and pathological effects of ROS inmammalian cells [31]. One of the pathways involves the antioxidant enzyme, superoxidedismutase (SOD), which exists as three genetically and geographically distinct isoenzymes[32]. The SODs convert superoxide anions to hydrogen peroxide which is then enzymaticallyremoved by catalase and glutathione peroxidase. Although the physiological roles of variousisoforms of SOD in mammals are not completely understood, the extracellular isoform (EC-SOD, SOD3), was recently shown to be associated with certain cognitive functions [33,34],and its removal was shown to interfere with signaling cascades critical for learning [33]. Giventhe reported relationship between the effects of irradiation on SGZ precursor cells and changesin redox status within the hippocampal formation, along with recent evidence for a significantrole of EC-SOD in learning and memory, we were interested in determining if there was arelationship between EC-SOD expression and hippocampal neurogenesis. Understanding howEC-SOD expression affects radiation-induced changes in neurogenesis may provide animportant base for developing strategies to reduce cognitive impairment after cranialirradiation.

MethodsAnimals

Congenic EC-SOD KO mice [35] on the C57BL/6J (B6) background were initially obtainedfrom Dr. James Crapo at the National Jewish Medical and Research Center, Denver, Colorado.The colony is maintained by backcrossing to B6 mice purchased from the Jackson Laboratory(Bar Harbor, ME). Homozygous KO and their wild type littermate controls were generatedfrom the intercrosses of heterozygous KO, and two month old male mice (WT, n = 50; KO, n

Rola et al.

Free Radic Biol Med. Author manuscript; available in PMC 2007 July 30.

NIH


NIH


NIH


= 52) were used in all experiments. All animal handling procedures were done according toinstitutional IACUCs. Mice were kept in a temperature and light-controlled environment witha 12 hr light/dark cycle, and provided food and water ad libitum.

IrradiationsFor irradiation, animals were anesthetized with a mixture of ketamine hydrochloride (60 mg/kg, Abbott Laboratories, North Chicago, IL) and medetomidine hydrochloride (0.25 mg/kg,Orion Corp., Espoo, Finland), administered by intraperitoneal (i.p.) injection. Irradiations weredone using a Phillips orthovoltage X-ray system as previously described [11]. Briefly, animalswere exposed to a single dose of 5 Gy using a special positioning jig so 4 animals could beirradiated simultaneously; the heads were centered in a 5 × 6 cm treatment field. The beamwas directed down onto the head and the body was shielded with lead. Dosimetry was doneusing a Keithley electrometer ionization chamber calibrated using lithium fluoride thermalluminescent dosimeters. The corrected dose rate was approximately 175 cGy/min at a sourceto skin distance of 21 cm. Mice were irradiated at 8 weeks of age and were sacrificed, alongwith age-matched non-irradiated controls, at different times for various studies describedbelow.

Quantification of antioxidant enzymesTo determine if the major antioxidant enzymes were altered in EC-SOD KO mice, cortices andhippocampi were collected from irradiated and non-irradiated WT and KO mice one monthafter irradiation, which is the time when the neurogenesis study was carried out. For tissuecollection, mice were anesthetized with i.p. injection of a mixture of ketamine hydrochloride(120 mg/kg) and medetomidine hydrochloride (0.5 mg/kg) and decapitated. Brains wereremoved and dissected on ice, and the hippocampus and cortex were isolated, frozen in dry iceand stored at −80°C. Cortex and hippocampus were homogenized separately in four volumes(weight to volume ratio 1:4) of PBS, pH 7.4, containing Complete® protease inhibitor cocktail(Roche, Switzerland), followed by 3 short pulses of sonication (3×5 sec). To ensure completedisruption of mitochondrial membranes, 3 rounds of freeze-thaw cycles between liquidnitrogen and room-temperature water were carried out. The samples were centrifuged at 20,800g at 4°C for 5 minutes, and the supernatants were stored in 20 μl aliquots at −80°C. Proteinconcentration of each sample was measured in triplicates using the BCA Protein Assay Reagent(Pierce, Rockford, IL).

Non-denaturing isoelectric focusing gel analysis—CuZnSOD (SOD1) and MnSOD(SOD2) activities were determined by Ampholine PAGplate (Amersham Pharmacia Biotech,Inc. Piscataway, NJ), pH 3.5 – 9.5, as described previously [36]. For the CuZnSOD assay, 2.5,5.0 and 10 μg of each sample were analyzed on the gel; for the MnSOD assay, 15, 30 and 60μg of each sample were analyzed. The activity stain creates clear bands on a dark purplebackground and identifies the location of CuZnSOD and MnSOD in the gel. The band intensity,which is proportional to the enzyme activity, was quantified by Image J 1.36b (NIH, USA) andnormalized to total protein loading.

Western blot analysis—The protein levels of catalase, glutathione peroxidase 1 (GPx-1),CuZnSOD, and MnSOD were determined by western blot analysis. Equal amounts of protein(cortex, 30 μg; hippocampus, 50 μg) from each sample were separated by NuPAGE 4–12%Bis-Tris gel (Invitrogen) and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA).The following primary antibodies were used for the detection of specific proteins: catalase (C0979, 1:4,000, Sigma, St. Louis, MO), GPx-1 (LF-PA0019, 1:1,000, LabFrontier Co. Korea),CuZnSOD (LF-PA 0013, 1:2,000, LabFrontier), MnSOD (SOD-110, 1:4,000, Stressgen, AnnArbor, MI). Protein bands were visualized using DuoLuX Chemiluminescent/FluorescentSubstrate Kit (Lumigen, Southfield, MI) after incubation with HRP-conjugated secondary

Rola et al.


NIH


NIH


NIH


antibody. All blots were stripped with stripping buffer (Tris-HCl, 62.5 mM; β-mercaptoethanol, 90 mM; 1% SDS) and reprobed with an antibody against β-actin (A 3854,1:50,000, Sigma) as a loading control. Quantification of western blot results was done bynormalizing the signal intensity of each sample to that of β-actin.

In vivo assessment of oxidative stressAssuming that KO mice were under persistent oxidative stress, we made a qualitative appraisalof 4-hydroxynonenal (4HNE) and nitrotyrosine (NT) immunoreactivities using randomlyselected sections of the hippocampus. The sections were immunostained as describedpreviously [11] using anti-4HNE primary antibody (1:100, JaICA, Fukuroi, Shizuoka, Japan)or anti-NT primary antibody (1:100, Zymed, South San Francisco, CA) with subsequentvisualization with anti-mouse secondary antibody conjugated with FITC fluorophore (1:200,Jackson ImmunoResearch Labs, West Grove, PA). Nuclei were counterstained with To-Pro 3(Molecular Probes, Eugene, OR).

Acute post-irradiation response of neurogenic populationsTo determine if there was a general difference in the pattern of acute cell death (apoptosis)after irradiation of WT and KO mice, tissues were collected from 2–4 animals of each genotypeat 0, 8, 12, 24 and 48 hr after irradiation. For tissue collection, animals were anesthetized asdescribed above and perfused with 50 ml of 10% neutral buffered formalin into the ascendingaorta using a mechanical pump (Masterflex Model 7014; Cole Parmer, Chicago, IL) over a 5min period; brains were removed and immersed in 10% neutral buffered formalin for 3 days.Brains were then placed in a Mouse Brain Matrix (Harvard Apparatus, Natick, MA) and a 5mm-thick transverse section containing the hippocampus was taken for paraffin embedding.Five, 6 μm-thick sections were cut from the rostral face of each block using a rotary microtome,starting at a point 2.5 mm behind the bregma [37] and were placed onto polylysine coated glassmicroscopic slides. A second set of 5 sections were obtained 100 μm caudal to the first set ofsections, and a third set was taken after another 100 μm. Thus, sections were taken from 3distinct areas representing the rostral-mid dentate gyrus [11,13].

In the acute study we were only interested in determining relative changes in numbers of cellsdying within the dentate SGZ as a function of time after irradiation. To do this we used TUNEL(terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling) staining witha commercial kit (Apotag, Serological Corp., Norcross, GA.) as described before [11,38].Because not all apoptotic cells necessarily show TUNEL staining, we also used morphologiccriteria to estimate apoptosis [39]. The morphological changes used included fragmentation,or the compaction of chromatin into 2 or more dense, lobulated masses, and pyknosis, whichwas characterized by small, round, darkly staining nuclei. To minimize the impact of includingany normal cell profiles in our counts of apoptosis, if any cytoplasm was observed inconjunction with a small, dense nucleus, that cell was not considered as apoptotic.

To determine the overall early impact of irradiation on specific cellular components of the SGZwe quantified proliferating cells and immature neurons 48 hr after irradiation, when apoptosiswas complete [11]. We also quantified these cells from tissues obtained 1 month afterirradiation, at the time when our neurogenesis study was done (see below). Proliferating cellswere labeled with an antibody against Ki-67 (DakoCytomation, Caprinteria, CA, diluted 1:100in PBS with 2% normal rabbit serum), a nuclear antigen expressed during all stages of the cellcycle except Go [40,41]. Immature neurons were labeled using an antibody againstDoublecortin (DCx, Santa Cruz Biotechnology, Santa Cruz, CA; diluted 1:500 in PBS with5% normal horse serum), a tubulin-associated protein expressed in migrating neuroblasts[42–46] as described previously [11]. Briefly, sections were incubated overnight at 4°C withprimary antibodies and subsequently for 30 min at room temperature with biotinylated

Rola et al.


NIH


NIH


NIH


secondary antibodies (Vector Lab, Burlingame, CA; rabbit anti-rat IgG, diluted 1:200 in PBSwith 2% normal rabbit serum for Ki67 and anti-goat IgG, diluted 1:500 in 5% normal horseserum for DCx). Binding of secondary antibodies was detected using an avidin-biotinylatedperoxidase complex system (ABC; Vector Lab) and developed with 0.025% 3,3′-diaminobenzidine (DAB, Zymed) dissolved in double distilled water containing 0.005%H2O2. Sections were then counterstained with Gill’s hematoxylin, dehydrated and mounted.

Cell numbers positive for TUNEL/morphologic changes, Ki-67, and DCx were scored usinga histomorphometric approach with the genotype and treatment blinded to the researcher. Themethods used are well standardized and have been used effectively to determine responses ofcells in the dentate SGZ in both mice and rats after radiation and other lesions [11,47–49]. Forthe analysis of cell death, we counted all apoptotic cells in the suprapyramidal andinfrapyramidal blades of the dentate SGZ and granular cell layer (GCL). For the analyses ofKi-67 and DCx, we focused our counting on all positively labeled cells within the SGZ, definingit as the area immediately adjacent to the hilus and extending roughly 3 cells deep into thedentate granule cell layer. For each animal, 3 slides were counted, one from each of the 3regions of the dentate. For each marker and each mouse, the total number of positively labeledcells was determined by summing the values of all 3 sections; this approach of counting cellsin 3 evenly-spaced, non-contiguous tissue sections is referred to as our standardized countingarea.

NeurogenesisTo determine the effects of irradiation on the survival and fate of newly generated cells in theSGZ as a function of genotype, groups of WT (n = 4) and KO mice (n = 4) received a singlei.p. injection (50 mg/kg) of 5-bromo-2′ deoxyuridine (BrdU, Sigma, St. Louis, MO) daily for7 days starting at 30 days after irradiation. Three weeks after the last BrdU injection, mice wereanesthetized as described and perfused with ice-cold saline followed by freshly prepared, ice-cold 4% paraformaldehyde. The brain was removed, processed, and sectioned using a slidingmicrotome [11]. Fifty micrometer sections were stored at 4°C in cryoprotectant solution untilneeded. Free floating sections were immunostained as described [11,13] using the followingprimary antibodies and working concentrations: rat anti-BrdU (1:10; Oxford Biotechnology,Kidlington, Oxford, UK); mouse anti-NeuN (1:200; Chemicon, Temecula, CA); rabbit anti-NG2 (1:200; Chemicon); goat anti-GFAP (1:100, Santa Cruz Biotechnology); rat anti-CD68(1:20; Serotec, Inc. Raleigh, NC).

To calculate the numbers of BrdU-positive (BrdU+) cells in the dentate gyrus, at least 12sections of a one-in-six series were scored per animal. All counts were limited to the dentategranule cell layer and a 50 μm border along the hilar margin that included the dentate SGZ.Total numbers were obtained by multiplying the measured value by 6; overestimation wascorrected using the Abercrombie method [50]. Total numbers of BrdU+ cells displaying thevarious lineage-specific phenotypes were determined using confocal microscopy to score thecolocalization of BrdU and phenotypic markers in representative sections from each animal[11,13]. Confocal microscopy was performed using a Nikon C-1 confocal microscope(Melville, New York), using techniques previously described [10,11,13]. Appropriate gain andblack-level settings were obtained on control tissues stained with secondary antibodies alone.Upper and lower thresholds were always set using a range indicator function to minimize dataloss due to saturation. Each cell was manually examined in its full ‘z’ dimension with use ofsplit panel analysis, and only those cells for which the BrdU+ nucleus was unambiguouslyassociated with the lineage-specific marker were scored as positive. For each lineage-specificmarker, the percentage of BrdU+ cells expressing that marker was determined. Total numbersof lineage-specific BrdU+ cells were then calculated by multiplying this percentage by the totalnumber of BrdU+ cells in the dentate gyrus.

Rola et al.


NIH


NIH


NIH


Activated microgliaThe impact of irradiation on total numbers of activated microglia in the SGZ was determinedusing free floating brain sections from groups of WT (n = 4) and KO mice (n = 4). After 3washes with TBS and quenching in TBS with 3% H2O2 for 15 min, sections were incubatedin TSA blocking buffer (PerkinElmer Life Sciences, Emeryville, CA) for 30 min, followed byapplication of the polyclonal rabbit anti-CD68 antibody (1:20; Serotec, Inc. Raleigh, NC). After12hr at 4ºC, sections were incubated for 1 hr at room temperature with a secondary anti-rabbitbiotinylated antibody (Vector, Burlingame, CA), followed by incubation with an Avidin+Biotin amplification system (Vector) for 45 minutes. Cell staining was visualized using theTSA fluorescence system CY3 (PerkinElmer Life Sciences, Emeryville, CA); nuclearcounterstaining was with Sytox-Green (Molecular Probes, Eugene, OR). No staining wasdetected in the absence of the primary or secondary antibodies.

Total numbers of activated microglia were counted in each of 4 coronal sections of the dentategyrus and hilar region at the level of the dorsal hippocampus (~−3.6 mm posterior to theBregma). Images were reconstructed as described previously [51] and shown in Fig. 5. Themosaics were collected with a Zeiss AxioImager Apotome microscope using a 20X objective.The parameters were kept constant across sections. Regions of interest containing the dentategyrus and hilar region was selected using AxioImager imaging software (Zeiss) and thenumbers of positive cells were counted within the selected area. To avoid classification errors,we carefully verified that the staining belonged to the cell of interest and not to a dendrite orthe cell body of an adjacent cell. The final numbers of activated microglia were expressed asnumber of cells/per mm2.

StatisticsFor each endpoint, values for all animals of a given treatment group were averaged and standarderrors of mean (SEM) were calculated. Student’s t test was used to compare the results fromwestern blotting and IEF gel analyses. For the studies of cell numbers (when applicable), aone-way ANOVA with post-hoc Duncan “D” or Fisher tests with Bonferroni correction wereused to determine if there were significant differences between groups as a function of radiationtreatment and genotype. To directly compare the 2 genotypes (EC-SOD KO and WT), ifapplicable, a two-way ANOVA was used, with treatment, genotype and treatment-genotypeinteraction as factors.

ResultsNo differences in the level of major antioxidant enzymes between WT and EC-SOD KO mice

No differences in catalase, GPx-1, MnSOD, and CuZnSOD protein levels were detectedbetween WT and KO mice with or without irradiation (Fig. 1A and 1B). There was also nodifference in CuZnSOD and MnSOD activities (Fig. 1C and 1D). This suggested that at leastfor the enzymes analyzed here, there were no apparent compensatory changes in the majorantioxidant defense systems when EC-SOD was knocked out.

Higher baseline level of oxidative stress observed in EC-SOD KO miceImmunostaining of WT and KO mouse tissues for 4HNE showed a qualitative increase inimmunoreactivity in KO mice compared to their WT littermates (Fig. 2A and 2B); this wasconsistent with a higher baseline level of oxidative stress in the KO mice. Based strictly onmorphology, the localization of 4HNE appeared to be vascular and, perhaps, microglial (Fig.2B, inset). Given the relationship between excess superoxide and the formation ofperoxynitrites [52], we also used immunohistochemistry to qualitatively assess nitrotyrosine,and again, there was increased immunoreactivity in non-irradiated KO mice (Fig. 2C and 2D).

Rola et al.


NIH


NIH


NIH


No difference in the acute post-irradiation response between WT and EC-SOD KO miceIn non-irradiated animals, the total number of TUNEL-positive nuclei in our standardizedcounting area ranged from about 15 to 20 in both WT and KO mice; these values are slightlylower than reported by us previously [11] and there was no significant difference between WTand KO mice. Dying cells were seen in the SGZ of both blades of the dentate and onlyoccasionally in the hilus and GCL. After irradiation, however, the number of apoptotic nucleiincreased by 15–20 times in both WT and KO at the peak of apoptosis (8 hr after irradiation;data not shown). There was no apparent difference in the pattern or magnitude of apoptosisbetween WT and KO mice, and in both genotypes, the numbers of TUNEL-positive cellsreturned to control levels by 24 hr after exposure (data not shown).

In our standardized counting area, the number of Ki-67-positive cells averaged 49.8 ± 5.4 (n= 5) in non-irradiated WT mice and 49.5 ± 14.9 (n = 4) in non-irradiated KO mice (Fig. 3A).The numbers of DCx-positive immature neurons averaged 298.0 ± 19.2 (n = 5) and 321.7 ±51.8 (n = 4) in non-irradiated WT and KO animals, respectively (Fig. 3B). Forty-eight hoursafter irradiation there were highly significant reductions in the numbers of proliferating cells(p=0.003 and p=0.002 for WT and KO, respectively) and immature neurons (p=0.001 andp=0.002 for WT and KO, respectively) in both genotypes (Fig. 3A and 3B).

While our acute studies showed that there were substantial decreases in the numbers ofproliferating cells and their progeny shortly after irradiation (Fig. 3A and 3B), some precursorcells survived the x-ray treatment. To determine if the initial loss of cells translated into alonger-term impairment of neurogenic populations in the SGZ and if the condition of EC-SODdeficiency affected those populations, we assessed the numbers of proliferating cells andimmature neurons 1 month after irradiation. At that time there were still apparent reductionsin the numbers of proliferating cells and immature neurons after irradiation (Fig. 3C and 3D).However, technical problems resulted in the loss of some tissues and reduced the numbers ofanimals in some of the treatment groups; this compromised our ability to do complete statisticalanalyses. Given that caveat, the data in Fig. 3 suggest that while the radiation-induced loss ofproliferating cells and immature neurons appeared to be persistent, there might have been somerecovery in both genotypes 1 month after irradiation compared to that at 48 hr post irradiation.

Reduced baseline neurogenesis observed in non-irradiated EC-SOD KO miceBrdU+ cells observed 3 weeks after the last BrdU injection represent the long-term survivalof newly generated cells (Fig. 4A, B). In the non-irradiated mice, the total number of BrdU+cells in the dentate gyrus of WT mice averaged 4187 ± 614, while in KO mice the averagevalue was 3197 ± 325; the difference was not significant (p = 0.2). In WT mice, an average of2920 ± 301 newly generated cells differentiated into neurons (BrdU+/NeuN+; Fig. 4C, D). Innon-irradiated KO mice, the number of newly generated neurons averaged 1793 ± 226 (Fig.4C and D); compared to WT the difference was highly significant (p = 0.007). In non-irradiatedWT mice there was an average of 164.4 ± 43.1 BrdU+ cells that co-expressed GFAP (newlygenerated astrocytes) while in non-irradiated KO mice the average number of GFAP-positivecells was 292.2 ± 98.3; this difference was not significant (p = 0.3; Fig. 4E and F). The numbersof newly generated oligodendrocytes (BrdU+/NG2+) were not different between WT and KOmice before irradiation (Fig. 4G and H).

Total numbers and numbers of newly generated (BrdU+/CD68) activated microglia weredetected using the anti-CD68 antibody, and were present in the dentate SGZ of both non-irradiated WT and KO mice. In non-irradiated mice there were no differences in total numbersof activated microglia between WT and KO mice (Fig. 5) and while there appeared to be adifference in newly generated activated microglia (WT: 318.7±77.9, KO: 526.5±154.7), thevalues were not significantly different (p = 0.5). Finally, peripheral monocytes were detected

Rola et al.


NIH


NIH


NIH


by scoring BrdU+ cells that expressed both NG2 and CD68 [53,54]. The average numbers ofperipheral monocytes were relatively low, ranging from 14.1 ± 14.1 in WT to 34.1 ± 26.4 inKO mice; given inter-animal variability, the difference was not significant (p = 0.5).

EC-SOD KO mice are protected from the radiation-mediated reduction of neurogenesisIn WT mice after a single dose of 5 Gy, the average number of BrdU+ cells was reduced by76%, to 1003 ± 503 (p = 0.002; Fig. 4B), and this was translated into a 86% reduction in theBrdU+/NeuN+ population (p = 0.002; Fig. 4D). In contrast, the average number of BrdU+ cellsseen after irradiation of KO mice increased from 3197 ± 325 to 4140 ± 595 (p = 0.2; Fig. 4B).Consequently, irradiation resulted in no significant reduction (p = 0.4) in the number of BrdU+/NeuN+ cells in EC-SOD KO mice (Fig. 4D). The differences in the total number of BrdU+and BrdU+/NeuN+ cells between irradiated WT and KO mice were highly significant (p =0.002 and p = 0.007, respectively). A 2-way ANOVA showed that for new neuron productionthere was a highly significant (p = 0.001) interaction between irradiation and genotype In termsof newly generated astrocytes, 5 Gy resulted in about a 12% reduction in the number of BrdU+/GFAP+ cells in the WT group and in an apparent increase in KO mice (Fig. 4F) althoughthese changes were not significantly different from non-irradiated controls. However there wasa significant difference in the number of BrdU+/GFAP+ cells between irradiated WT andirradiated KO mice (p=0.03). While there appeared to be slightly fewer BrdU+/NG2+ cells inirradiated WT animals (p = 0.7) (Fig. 4H), there was a trend toward increasing numbers in KOmice (p = 0.1). Consequently, there was a significant difference in the number of BrdU+/NG2+ cells between irradiated WT and irradiated KO mice (p=0.03).

After irradiation there were significant increases in the total numbers of activated microglia inboth the WT (p = 0. 056) and KO mice (p = 0.01) when compared to non-irradiated controls(Fig. 5). Furthermore, there was a significant difference (P = 0.03) in the total numbers ofactivated microglia between irradiated WT and irradiated KO mice. In terms of newlygenerated activated microglia (BrdU+/CD68+), there were non-significant increases in bothgenotypes after irradiation; however there was a significant difference between irradiated WTand irradiated KO mice (p=0.03; not shown). Finally, there was a trend toward increasingnumbers of BrdU+ peripheral monocytes in the SGZ of both WT and KO mice, but thedifferences were not significant compared to non-irradiated controls, and the numbers ofperipheral monocytes were almost an order of magnitude lower than the numbers of BrdU+activated microglia (data not shown).

DiscussionThe main findings of the present study were: 1) EC-SOD KO mice had a lower baselinehippocampal neurogenesis compared to WT mice; 2) proliferating precursor cells andimmature neurons of the dentate SGZ in EC-SOD KO mice showed acute (6–48 hr) sensitivityto irradiation and the level was comparable to that of WT mice; 3) survival of newly generatedcells in the dentate SGZ after 5 Gy irradiation was substantially higher in EC-SOD KO micecompared to WT controls; and 4) contrary to what was seen in WT mice, irradiation did notreduce the survival of newly generated neurons and glia in EC-SOD KO mice. These findingsshowed that after irradiation, an environment lacking EC-SOD was much more permissive inthe context of hippocampal neurogenesis. The mechanism(s) behind this observation is not yetknown, but this effect might constitute a basis for future interventions aimed at rescue or atleast amelioration of the risks for cognitive dysfunction in individuals subjected to CNSirradiation.

The effects of irradiation on hippocampal structure and function have been extensively studied(reviewed in [5,55]) but only recently has it been suggested that radiation-induced injury ofthe neurogenic cell populations within dentate gyrus [10,11,39,48] may play a role in cognitive

Rola et al.


NIH


NIH


NIH


sequelae of cranial irradiation. Studies from our lab [10,11,13,14,48] and others [12,56] providea quantitative description of the impact that ionizing radiation exerts on hippocampalneurogenesis. Those studies show that the stem/precursor cell populations within theneurogenic areas are very vulnerable to injury and that changes are associated with radiation-related impairments of hippocampal-dependent cognitive tasks [12,14,57,58]. Furthermore,radiation-induced changes in neurogenesis have been shown to be associated withmicroenvironmental factors including neuroinflammation [56,59], vascular changes [10,60]and oxidative stress [28,61].

In this study we were interested in determining if and how the absence of EC-SOD wouldimpact neurogenesis. EC-SOD immunoreactivity is found throughout the brain, and isparticularly prominent in the hippocampus [62]. It is of particular interest that alterations inEC-SOD expression in mice is associated with impaired learning [34], and that over-expressionof EC-SOD protects synaptic plasticity and learning and memory against oxidative damage[63]. Here we found that the absence of EC-SOD was not associated with compensatorychanges in other SODs or other anti-oxidant enzymes (Fig. 1), although given the intracellularlocalization of these molecules it might be unlikely to expect them to have compensatoryfunctions in terms of affecting extracellular superoxide free radicals. It could be possible thatcirculating antioxidants, including ascorbic acid, tocopherol, uric acid, bilirubin, proteins andother compounds, could be increased as a compensatory mechanism, but we did not addressthis in the current study. While total antioxidant capacity in the serum can be assessedquantitatively [64], without knowing the amount of circulating EC-SOD and its contributionto total antioxidant capacity in the serum, how these compounds may or could affect regionaleffects in the SGZ may be difficult to interpret. However, a consideration of such ideas isworthy of further study. Regardless, our data show that at least qualitatively, 2-month-old EC-SOD KO mice showed indications of a persistent oxidative stress (Fig. 2). As a result, andbased on data relating EC-SOD with cognitive function [34,63], we expected neurogenesis inthe KO mice to be lower than that of WT mice. This was, in fact, what we observed (Fig. 4).Whether these changes are responsible for the cognitive impairment observed in EC-SOD KOmice is not clear. However, given the extensive data available associating changes inneurogenesis with cognitive performance [11,13,14,65–68], it certainly seems possible, and atthe very least may play a contributory role. The baseline reduction in new neuron productionin KO mice was different from what was seen in glia (Fig. 4) and inflammatory cells (Fig. 5),where there were apparent increases in cell number in KO mice relative to WT. While thesignificance of these latter findings may be affected by the variability in the data, increasednumbers of astrocytes and activated microglia would be consistent with an elevated level ofoxidative stress in the KO animals.

After irradiation with a relatively low dose of x-rays, the early responses, including apoptosis,altered cell proliferation, and changes in numbers of immature neurons, were similar betweenWT and KO mice. This suggested that the absence of EC-SOD and the increased oxidativestress status of KO animals did not influence mechanisms responsible for acute cell death afterirradiation. Furthermore, there were no major differences between WT and KO mice in cellproliferation and numbers of immature neurons at 1 month after irradiation (Fig. 3). It was ofinterest, therefore, to see substantial and significant differences in the survival of newlygenerated cells after irradiation as a function of genotype, with KO mice showing almost 4times the number of surviving newly generated cells than WT (Fig. 4). This surprising resultalso translated into a very significant and nearly 4-fold difference in the number of survivingnewly generated neurons in irradiated KO as compared to irradiated WT mice. In fact, whileradiation caused an 85% reduction in newly generated neurons in WT mice, the same doseresulted in virtually no difference in KO mice. To the best of our knowledge, this is the firstreport to show that an EC-SOD deficient environment provides a ‘protective’ effect inhippocampal neurogenesis after irradiation. In a general sense, this resembles the

Rola et al.


NIH


NIH


NIH


preconditioning seen in stroke [69], heart attack [70] or acute lung injury [71] where oxidativemechanisms plays a crucial role in the pathogenesis of the disease. While the precisemechanism(s) responsible for this effect has not yet been clarified, our study rules out simplecompensatory changes in expression and activities of other antioxidant enzymes (Fig. 1). Ourfindings clearly suggest that EC-SOD deficient mice have developed a resistance to radiation-induced inhibition of neurogenesis that may involve some type of adaptation within themicroenvironment, without compensatory changes in other major intracellular antioxidants.These ideas are being tested further using inducible in vitro and in vivo models of EC-SODexpression. Furthermore, it is possible that a specific set of trophic factors and signalingmolecules that favor differentiation and long-term survival of newly-generated neurons in theSGZ is up-regulated or activated in the irradiated EC-SOD KO brains to counter the chronicinflammatory environment created from irradiation. These trophic factors and signalingmolecules may include brain derived trophic factor (BDNF), vascular endothelial growth factor(VEGF), and nitric oxide (NO), all of which have been shown to favor differentiation andsurvival of neurons [72–75]. At the same time, the phenotype of the activated microglia [76,77] in the irradiated EC-SOD null environment may also play a role in enhanced neurogenesis.Whatever the mechanism(s) involved, our data clearly show that the production of new neuronsis more sensitive to irradiation in WT animals than in the EC-SOD KO mice.

Due to the relatively lower numbers of newly generated astrocytes and oligodendrocytes, thereis more variability in the data for these cells, but clearly there appears to be a trend towardincreased numbers in KO vs. WT mice after 5 Gy of x-rays (Fig. 4). This suggests that perhapsthe same mechanism responsible for the relative protective effect of neurons in EC-SOD KOmice is operative in glial cells as well. Given recent data showing that neural stem cells in theSGZ express GFAP [78], it is possible that the increased numbers of GFAP+ cells seen in KOmice represent, in part, increased stem cell or regenerative responses. On the other hand, theobserved changes in BrdU+/GFAP+ cells may very well be a sign of the well-describedastrogliosis seen in the oxidative stress conditions [79] as well as after irradiation [80].

Neurogenesis depends upon a complex microenvironment that involves signaling betweenmultiple cell types [81], and changes in the oxidative status along with irradiation could affectany or all of these cells or their interactions. While the precise nature of such effects has notyet been clarified, previous studies have suggested chronic inflammatory changes as one ofthe important factors [10,11,13,56]. We found significant increases in the total numbers ofactivated microglia in both WT and KO mice after irradiation as compared to non-irradiatedmice (Fig. 5). Furthermore, there appeared to be increases in numbers of newly generatedactivated microglia (BrdU+/CD68+) in both genotypes although the magnitude of thedifferences was not statistically significant. That increasing numbers of activated microgliaafter irradiation had no apparent association with neurogenesis in EC-SOD KO mice issomewhat surprising, given recent studies showing that increased neuroinflammation waslinked to an inhibition of hippocampal neurogenesis [13,56,59,82]. It is particularly interesting,therefore, that a recent study has suggested that microglial phenotype critically influences theirability of these cells to support or impair cell renewal processes in the adult brain [77]. It maybe that in a microenvironment characterized by persistent oxidative stress (i.e., EC-SOD KO),the subsequent activation of microglia may, in fact have a beneficial effect, at least in terms ofneurogenesis. This would suggest that microglia may respond differently to a given stimulus(irradiation) depending upon the presence of another and preceding stimulus (oxidative stress);such ideas have recently been reviewed [83]. While intriguing, such a hypothesis is speculativeat this time, but further studies seem warranted.

While the mechanistic relationship between EC-SOD and forebrain neurogenesis is not yetknown, the data shown here clearly suggest that the lack of EC-SOD provides some sort ofsurvival advantage to neurogenic cell populations after irradiation. This could have major

Rola et al.


NIH


NIH


NIH


implications with respect to protecting or ameliorating the adverse effects of radiation onspecific brain functions if the underlying changes in the molecular network can be identified.

Acknowledgements

The authors want to thank Dr. Kathleen Lamborn of the Brain Tumor Research Center, Dept. Neurological Surgery,UCSF, for assistance in the statistical analyses. This work was supported in part by NIH grant R01 NS46051 (JRF)NASCOR grant NNJ04HC90G (JRF), NIH grant AG24400 (TTH) and ACS grant RSG-00-036-04-CNE (CLL)

References1. Hopewell JW. Radiation injury to the central nervous system. Med Pediatr Oncol 1998;(Suppl 1):1–

9. [PubMed: 9659940]2. Tofilon PJ, Fike JR. The radioresponse of the central nervous system: a dynamic process. Radiat Res

2000;153:357–370. [PubMed: 10798963]3. Fike, JR.; Gobbel, GT. Central nervous system radiation injury in large animal models. In: Sheline,

GE., editor. Radiation Injury to the Nervous System. New York: Raven Press, Ltd.; 1991. p. 113-135.4. van der Kogel, AJ. Central nervous system radiation injury in small animal models. In: Sheline, GE.,

editor. Radiation Injury to the Nervous System. New York: Raven Press, Ltd.; 1991. p. 91-111.5. Abayomi OK. Pathogenesis of irradiation-induced cognitive dysfunction. Acta Oncol 1996;35:659–

663. [PubMed: 8938210]6. Crossen JR, Garwood D, Glatstein E, Neuwelt EA. Neurobehavioral sequelae of cranial irradiation in

adults: a review of radiation-induced encephalopathy. J Clin Oncol 1994;12:627–642. [PubMed:8120563]

7. Lee PW, Hung BK, Woo EK, Tai PT, Choi DT. Effects of radiation therapy on neuropsychologicalfunctioning in patients with nasopharyngeal carcinoma. J Neurol Neurosurg Psychiatry 1989;52:488–492. [PubMed: 2786925]

8. Roman DD, Sperduto PW. Neuropsychological effects of cranial radiation: current knowledge andfuture directions. Int J Radiat Oncol Biol Phys 1995;31:983–998. [PubMed: 7860415]

9. Surmaaho O, Niemela M, Vilkki J, Kouri M, Brander A, Salonen O, Paetau A, Kallio M, PyykkonenJ, Jaaskelainen J. Adverse long-term effects of brain radiotherapy in adult low-grade glioma patients.Neurology 2001;56:1285–1290. [PubMed: 11376174]

10. Monje ML, Mizumatsu S, Fike JR, Palmer TD. Irradiation induces neural precursor-cell dysfunction.Nat Med 2002;8:955–962. [PubMed: 12161748]

11. Mizumatsu S, Monje ML, Morhardt DR, Rola R, Palmer TD, Fike JR. Extreme Sensitivity of AdultNeurogenesis to Low Doses of X-Irradiation. Cancer Res 2003;63:4021–4027. [PubMed: 12874001]

12. Madsen TM, Kristjansen PE, Bolwig TG, Wortwein G. Arrested neuronal proliferation and impairedhippocampal function following fractionated brain irradiation in the adult rat. Neuroscience2003;119:635–642. [PubMed: 12809684]

13. Rola R, Raber J, Rizk A, Otsuka S, VandenBerg SR, Morhardt DR, Fike JR. Radiation-inducedimpairment of hippocampal neurogenesis is associated with cognitive deficits in young mice. ExpNeurol 2004;188:316–330. [PubMed: 15246832]

14. Raber J, Rola R, LeFevour A, Morhardt D, Curley J, Mizumatsu S, VandenBerg SR, Fike JR.Radiation-induced cognitive impairments are associated with changes in indicators of hippocampalneurogenesis. Radiat Res 2004;162:39–47. [PubMed: 15222778]

15. Suhonen JO, Peterson DA, Ray J, Gage FH. Differentiation of adult hippocampus-derived progenitorsinto olfactory neurons in vivo. Nature 1996;383:624–627. [PubMed: 8857538]

16. Luskin M. Neuroblasts of the postnatal mammalian forebrain: Their phenotype and fate. J Neurobiol1998;36:221–233. [PubMed: 9712306]

17. Palmer TD, Willhoite AR, Gage FH. Vascular niche for adult hippocampal neurogenesis. J CompNeurol 2000;425:479–494. [PubMed: 10975875]

18. Song H, Stevens CF, Gage FH. Astroglia induce neurogenesis from adult neural stem cells. Nature2002;417:39–44. [PubMed: 11986659]

19. Smith MA, Nunomura A, Zhu X, Takeda A, Perry G. Metabolic, metallic, and mitotic sources ofoxidative stress in Alzheimer disease. Antioxid Redox Signal 2000;2:413–420. [PubMed: 11229355]

Rola et al.


NIH


NIH


NIH


20. Smith KJ, Kapoor R, Felts PA. Demyelination: the role of reactive oxygen and nitrogen species. BrainPathol 1999;9:69–92. [PubMed: 9989453]

21. Peuchen S, Bolanos JP, Heales SJ, Almeida A, Duchen MR, Clark JB. Interrelationships betweenastrocyte function, oxidative stress and antioxidant status within the central nervous system. ProgNeurobiol 1997;52:261–281. [PubMed: 9247965]

22. Lin MT, Beal MF. Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases.Nature 2006;443:787–795. [PubMed: 17051205]

23. Forster MJ, Dubey A, Dawson KM, Stutts WA, Lal H, Sohal RS. Age-related losses of cognitivefunction and motor skills in mice are associated with oxidative protein damage in the brain. Proc NatlAcad Sci U S A 1996;93:4765–4769. [PubMed: 8643477]

24. Juurlink BH, Paterson PG. Review of oxidative stress in brain and spinal cord injury: suggestions forpharmacological and nutritional management strategies. J Spinal Cord Med 1998;21:309–334.[PubMed: 10096045]

25. Love S. Oxidative stress in brain ischemia. Brain Pathol 1999;9:119–131. [PubMed: 9989455]26. Schnell L, Fearn S, Klassen H, Schwab ME, Perry VH. Acute inflammatory responses to mechanical

lesions in the CNS: differences between brain and spinal cord. Eur J Neurosci 1999;11:3648–3658.[PubMed: 10564372]

27. Schultheiss TE, Stephens LC. Invited review: permanent radiation myelopathy. Br J Radiol1992;65:737–753. [PubMed: 1393407]

28. Limoli CL, Giedzinski E, Rola R, Otsuka S, Palmer TD, Fike JR. Radiation response of neuralprecursor cells: linking cellular sensitivity to cell cycle checkpoints, apoptosis and oxidative stress.Radiat Res 2004;161:17–27. [PubMed: 14680400]

29. Limoli CL, Rola R, Giedzinski E, Mantha S, Huang TT, Fike JR. Cell-density-dependent regulationof neural precursor cell function. Proc Natl Acad Sci U S A 2004;101:16052–16057. [PubMed:15522966]

30. Lonergan PE, Martin DS, Horrobin DF, Lynch MA. Neuroprotective effect of eicosapentaenoic acidin hippocampus of rats exposed to gamma-irradiation. J Biol Chem 2002;277:20804–20811.[PubMed: 11912218]

31. Riley PA. Free radicals in biology: oxidative stress and the effects of ionizing radiation. Int J RadiatBiol 1994;65:27–33. [PubMed: 7905906]

32. Huang TT, Carlson EJ, Raineri I, Gillespie AM, Kozy H, Epstein CJ. The use of transgenic and mutantmice to study oxygen free radical metabolism. Ann N Y Acad Sci 1999;893:95–112. [PubMed:10672232]

33. Thiels E, Urban NN, Gonzalez-Burgos GR, Kanterewicz BI, Barrionuevo G, Chu CT, Oury TD,Klann E. Impairment of long-term potentiation and associative memory in mice that overexpressextracellular superoxide dismutase. J Neurosci 2000;20:7631–7639. [PubMed: 11027223]

34. Levin ED, Brady TC, Hochrein EC, Oury TD, Jonsson LM, Marklund SL, Crapo JD. Molecularmanipulations of extracellular superoxide dismutase: functional importance for learning. BehavGenet 1998;28:381–390. [PubMed: 9926619]

35. Carlsson LM, Jonsson J, Edlund T, Marklund SL. Mice lacking extracellular superoxide dismutaseare more sensitive to hyperoxia. Proc Natl Acad Sci U S A 1995;92:6264–6268. [PubMed: 7603981]

36. Huang TT, Raineri I, Eggerding F, Epstein CJ. Transgenic and mutant mice for oxygen free radicalstudies. Methods Enzymol 2002;349:191–213. [PubMed: 11912909]

37. Slotnick, BM.; Leonard, CM. A stereotaxic atlas of the albino mouse forebrain. Rockville, Md.: U.S.Dept. of Health Education and Welfare Public Health Service Alcohol Drug Abuse and Mental HealthAdministration.; 1975.

38. Shinohara C, Gobbel GT, Lamborn KR, Tada E, Fike JR. Apoptosis in the subependyma of youngadult rats after single and fractionated doses of X-rays. Cancer Res 1997;57:2694–2702. [PubMed:9205079]

39. Tada E, Parent JM, Lowenstein DH, Fike JR. X-irradiation causes a prolonged reduction in cellproliferation in the dentate gyrus of adult rats. Neuroscience 2000;99:33–41. [PubMed: 10924950]

40. Fisher BJ, Naumova E, Leighton CC, Naumov GN, Kerklviet N, Fortin D, Macdonald DR, CairncrossJG, Bauman GS, Stitt L. Ki-67: a prognostic factor for low-grade glioma? Int J Radiat Oncol BiolPhys 2002;52:996–1001. [PubMed: 11958894]

Rola et al.


NIH


NIH


NIH


41. Kee N, Sivalingam S, Boonstra R, Wojtowicz JM. The utility of Ki-67 and BrdU as proliferativemarkers of adult neurogenesis. J Neurosci Methods 2002;115:97–105. [PubMed: 11897369]

42. Mizuguchi M, Qin J, Yamada M, Ikeda K, Takashima S. High expression of doublecortin andKIAA0369 protein in fetal brain suggests their specific role in neuronal migration. Am J Pathol1999;155:1713–1721. [PubMed: 10550327]

43. Nacher J, Crespo C, McEwen BS. Doublecortin expression in the adult rat telencephalon. Eur JNeurosci 2001;14:629–644. [PubMed: 11556888]

44. Englund U, Bjorklund A, Wictorin K. Migration patterns and phenotypic differentiation of long-termexpanded human neural progenitor cells after transplantation into the adult rat brain. Brain Res DevBrain Res 2002;134:123–141.

45. Kempermann G, Gast D, Kronenberg G, Yamaguchi M, Gage FH. Early determination and long-termpersistence of adult-generated new neurons in the hippocampus of mice. Development2003;130:391–399. [PubMed: 12466205]

46. Jessberger S, Kempermann G. Adult-born hippocampal neurons mature into activity-dependentresponsiveness. Eur J Neurosci 2003;18:2707–2712. [PubMed: 14656319]

47. Tada E, Yang C, Gobbel GT, Lamborn KR, Fike JR. Long-term impairment of subependymalrepopulation following damage by ionizing irradiation. Exp Neurol 1999;160:66–77. [PubMed:10630191]

48. Parent JM, Tada E, Fike JR, Lowenstein DH. Inhibition of dentate granule cell neurogenesis withbrain irradiation does not prevent seizure-induced mossy fiber synaptic reorganization in the rat. JNeurosci 1999;19:4508–4519. [PubMed: 10341251]

49. Rola R, Mizumatsu S, Otsuka S, Morhardt DR, Noble-Haeusslein LJ, Fishman K, Potts MB, FikeJR. Alterations in hippocampal neurogenesis following traumatic brain injury in mice. Exp Neurol.2006

50. Abercrombie M. Estimation of nuclear population from microtomic sections. Anat Rec 1946;94:239–247.

51. Rosi S, Ramirez-Amaya V, Vazdarjanova A, Worley PF, Barnes CA, Wenk GL. Neuroinflammationalters the hippocampal pattern of behaviorally induced Arc expression. J Neurosci 2005;25:723–731.[PubMed: 15659610]

52. Halliwell, BGJ. Free Radicals in Biology and Medicine. Oxford University Press; 1999.53. Bu J, Akhtar N, Nishiyama A. Transient expression of the NG2 proteoglycan by a subpopulation of

activated macrophages in an excitotoxic hippocampal lesion. Glia 2001;34:296–310. [PubMed:11360302]

54. Jones LL, Yamaguchi Y, Stallcup WB, Tuszynski MH. NG2 is a major chondroitin sulfateproteoglycan produced after spinal cord injury and is expressed by macrophages and oligodendrocyteprogenitors. J Neurosci 2002;22:2792–2803. [PubMed: 11923444]

55. Abayomi OK. Pathogenesis of cognitive decline following therapeutic irradiation for head and necktumors. Acta Oncol 2002;41:346–351. [PubMed: 12234025]

56. Monje ML, Toda H, Palmer TD. Inflammatory Blockade Restores Adult Hippocampal Neurogenesis.Science 2003;302:1760–1765. [PubMed: 14615545]

57. Raber J, Fan Y, Matsumori Y, Liu Z, Weinstein PR, Fike JR, Liu J. Irradiation attenuates neurogenesisand exacerbates ischemia-induced deficits. Ann Neurol 2004;55:381–389. [PubMed: 14991816]

58. Winocur G, Wojtowicz JM, Sekeres M, Snyder JS, Wang S. Inhibition of neurogenesis interfereswith hippocampus-dependent memory function. Hippocampus 2006;16:296–304. [PubMed:16411241]

59. Rola R, Sarkissian V, Obenaus A, Nelson GA, Otsuka S, Limoli CL, Fike JR. High-LET radiationinduces inflammation and persistent changes in markers of hippocampal neurogenesis. Radiat Res2005;164:556–560. [PubMed: 16187787]

60. Otsuka S, Coderre JA, Micca PL, Morris GM, Hopewell JW, Rola R, Fike JR. Depletion of neuralprecursor cells after local brain irradiation is due to radiation dose to the parenchyma, not thevasculature. Radiat Res 2006;165:582–591. [PubMed: 16669713]

61. Giedzinski E, Rola R, Fike JR, Limoli CL. Efficient production of reactive oxygen species in neuralprecursor cells after exposure to 250 MeV protons. Radiat Res 2005;164:540–544. [PubMed:16187784]

Rola et al.


NIH


NIH


NIH


62. Oury TD, Card JP, Klann E. Localization of extracellular superoxide dismutase in adult mouse brain.Brain Res 1999;850:96–103. [PubMed: 10629753]

63. Hu D, Serrano F, Oury TD, Klann E. Aging-dependent alterations in synaptic plasticity and memoryin mice that overexpress extracellular superoxide dismutase. J Neurosci 2006;26:3933–3941.[PubMed: 16611809]

64. Benzie IF, Strain JJ. Ferric reducing/antioxidant power assay: direct measure of total antioxidantactivity of biological fluids and modified version for simultaneous measurement of total antioxidantpower and ascorbic acid concentration. Methods Enzymol 1999;299:15–27. [PubMed: 9916193]

65. Kempermann G. Why new neurons? Possible functions for adult hippocampal neurogenesis. JNeurosci 2002;22:635–638. [PubMed: 11826092]

66. Kempermann, G. Ernst Schering Res Found Workshop. 2002. Neuronal stem cells and adultneurogenesis; p. 17-28.

67. Kempermann G, Kuhn HG, Gage FH. More hippocampal neurons in adult mice living in an enrichedenvironment. Nature 1997;386:493–495. [PubMed: 9087407]

68. van Praag H, Schinder AF, Christie BR, Toni N, Palmer TD, Gage FH. Functional neurogenesis inthe adult hippocampus. Nature 2002;415:1030–1034. [PubMed: 11875571]

69. Schaller B, Graf R. Cerebral ischemic preconditioning. An experimental phenomenon or a clinicalimportant entity of stroke prevention? . J Neurol 2002;249:1503–1511. [PubMed: 12420088]

70. Gori T, Forconi S. The role of reactive free radicals in ischemic preconditioning--clinical andevolutionary implications. Clin Hemorheol Microcirc 2005;33:19–28. [PubMed: 16037629]

71. Pespeni M, Hodnett M, Pittet JF. In vivo stress preconditioning. Methods 2005;35:158–164.[PubMed: 15649842]

72. Scharfman H, Goodman J, Macleod A, Phani S, Antonelli C, Croll S. Increased neurogenesis and theectopic granule cells after intrahippocampal BDNF infusion in adult rats. Exp Neurol 2005;192:348–356. [PubMed: 15755552]

73. Cao L, Jiao X, Zuzga DS, Liu Y, Fong DM, Young D, During MJ. VEGF links hippocampal activitywith neurogenesis, learning and memory. Nat Genet 2004;36:827–835. [PubMed: 15258583]

74. Cardenas A, Moro MA, Hurtado O, Leza JC, Lizasoain I. Dual role of nitric oxide in adultneurogenesis. Brain Res Brain Res Rev 2005;50:1–6. [PubMed: 16291071]

75. Gibbs SM. Regulation of neuronal proliferation and differentiation by nitric oxide. Mol Neurobiol2003;27:107–120. [PubMed: 12777682]

76. Battista D, Ferrari CC, Gage FH, Pitossi FJ. Neurogenic niche modulation by activated microglia:transforming growth factor beta increases neurogenesis in the adult dentate gyrus. Eur J Neurosci2006;23:83–93. [PubMed: 16420418]

77. Butovsky O, Ziv Y, Schwartz A, Landa G, Talpalar AE, Pluchino S, Martino G, Schwartz M.Microglia activated by IL-4 or IFN-gamma differentially induce neurogenesis andoligodendrogenesis from adult stem/progenitor cells. Mol Cell Neurosci 2006;31:149–160.[PubMed: 16297637]

78. Seri B, Garcia-Verdugo JM, McEwen BS, Alvarez-Buylla A. Astrocytes give rise to new neurons inthe adult mammalian hippocampus. J Neurosci 2001;21:7153–7160. [PubMed: 11549726]

79. Baydas G, Tuzcu M. Protective effects of melatonin against ethanol-induced reactive gliosis inhippocampus and cortex of young and aged rats. Exp Neurol 2005;194:175–181. [PubMed:15899254]

80. Calvo W, Hopewell JW, Reinhold HS, Yeung TK. Time- and dose-related changes in the white matterof the rat brain after single doses of X rays. The British Journal of Radiology 1988;61:1043–1052.[PubMed: 3208008]

81. Seki T. Microenvironmental elements supporting adult hippocampal neurogenesis. Anat Sci Int2003;78:69–78. [PubMed: 12828419]

82. Ekdahl CT, Claasen JH, Bonde S, Kokaia Z, Lindvall O. Inflammation is detrimental for neurogenesisin adult brain. Proc Natl Acad Sci U S A 2003;100:13632–13637. [PubMed: 14581618]

83. Schwartz M, Butovsky O, Bruck W, Hanisch UK. Microglial phenotype: is the commitmentreversible? . Trends Neurosci 2006;29:68–74. [PubMed: 16406093]

Rola et al.


NIH


NIH


NIH


Figure 1.Deficiency in EC-SOD does not induce compensatory changes in other antioxidant enzymes.Western blot analyses of antioxidant enzymes showed no obvious differences in tissue extractsfrom WT (Sod3+/+) and EC-SOD KO (Sod3−/−) mice in non-irradiated (A) and irradiated (B)mice. Extracts were obtained from the hippocampi and cortices, and enzymes assessed includecatalase, glutathione peroxidase (GPx-1), manganese SOD (MnSOD) and copper zinc SOD(CuZnSOD). Each column of bands represents the analyses of 1 mouse, and the bands labeled‘C’ represent a positive liver control. Isoelectric focusing (IEF) gel analysis of CuZnSOD andMnSOD activities in non-irradiated (C) and irradiated (D) tissue extracts showed no apparentdifferences between WT and KO mice. For each genotype and treatment, samples were loadedin exactly the same order; that is, for a specific mouse, the protein samples and the enzymeactivity analyses were at the same location in the gels.

Rola et al.


NIH


NIH


NIH


Figure 2.EC-SOD deficiency leads to indications of oxidative stress in the dentate gyrus. Confocalmicroscopic images of the dentate gyrus in non-irradiated WT (A & C) and EC-SOD KO mice(B & D) immunostained for 4-hydroxynonenal (4HNE, A & B) and nitrotyrosine (NT, C &D). These images show clear differences in staining between WT and KO, and the qualitativecharacter suggests, in part, vascular staining. The inset in B suggests that some 4HNE stainingmay also be in microglia (arrow). The extensive blue staining represents granule cell neurons,while the green represents either 4HNE (B) or NT (D). Magnification is 200 X without digitalzoom (Inset 600 X with 1.5 × digital zoom).

Rola et al.


NIH


NIH


NIH


Figure 3.Irradiation decreases neurogenic populations in the dentate subgranular zone (SGZ) 48 hr (A& B), and 1 month (C & D) after irradiation with 5 Gy. Numbers of proliferating cells (Ki-67positive cells) and immature neurons (DCx-positive cells) in the SGZ are the same for non-irradiated WT and EC-SOD KO mice at both time points (solid bars). 48 hr after a single doseof 5 Gy of X-rays (striped bars), proliferating cells (A) and immature neurons (B) aresignificantly decreased in both strains of mice; the magnitudes of cell loss are over 90% forproliferating cells and over 80% for immature neurons. One month following irradiation thereappeared to be some recovery in numbers of proliferating cells (C) and immature neurons (D)when compared to the earlier time point. All columns in A and B and the columns showingerror bars in C and D represent an average of 3–5 mice each. The columns without error barsrepresent an average of only 2 mice each; the individual values making up those averages areshown as black diamonds. a – irradiated vs. non irradiated WT (p=0.003); b – irradiated vs.non irradiated KO (p=0.002); c –irradiated vs. non irradiated WT (p=0.001); d –irradiated vs.non irradiated KO (p=0.002).

Rola et al.


NIH


NIH


NIH


Figure 4.Radiation effects on survival and fate of newly generated cells in WT and EC-SOD KO mice.Confocal images (left) and quantification of cell phenotype of newly generated cells in thedentate subgranular zone (right) before (solid bars) and after a single 5 Gy dose of x-rays(striped bars). The fluorescent images show newly generated (BrdU+) only (A) or cells thatco-express mature cell markers (C, E, G). Cells quantified include: neurons (C) immunostainedwith BrdU and NeuN; astrocytes (E) immunostained with BrdU and GFAP; and immatureoligodendrocytes (G) immunostained with BrdU and NG2. In A, BrdU+ cells are red; in E andG, BrdU+ cells are yellow/orange against the green background of the cell specific stain. In E,astrocytes stain blue while BrdU stains magenta. The total numbers of BrdU+ cells wereslightly lower in non-irradiated EC-SOD KO mice as compared to non-irradiated WT mice(B). After irradiation there was a highly significant decrease in BrdU+ cells in WT and a modestincrease in EC-SOD KO mice. The difference between numbers of BrdU+ cells in irradiatedWT and irradiated KO mice was highly significant. In terms of cell fate, the most significant

Rola et al.


NIH


NIH


NIH


differences between WT and KO mice were observed for newly generated neurons (D), where5 Gy caused an 85% reduction in WT and virtually no change in KO mice. Magnification was400X without (A) or with (C, E, G) 2X digital zoom. a – non irradiated KO vs. non irradiatedWT (p=0.007); b – irradiated vs. non irradiated WT (p=0.008); c –irradiated WT vs. irradiatedKO (p=0.002); d – irradiated WT vs. irradiated KO (p=0.03). a – irradiated vs. non irradiatedWT (p=0.002); b – irradiated WT vs. irradiated KO (p=0.002).

Rola et al.


NIH


NIH


NIH


Figure 5.Representative flat images of reconstructed dentate gyri and the hilar region of thehippocampus of WT (A, B) and EC-SOD KO mice (C, D) before (A, C) or after irradiation(B, D). Activated microglia stain red and nuclei stain green. Activated microglia showed thecharacteristic bushy morphology with contracted and ramified processes (Inset). (E) In bothgenotypes, irradiated animals had more microglia when compared to non-irradiated controls.a – irradiated vs. non irradiated WT (p=0.056); b – irradiated vs. non irradiated KO (p=0.03).

Rola et al.


NIH


NIH


NIH


Lack of extracellular superoxide dismutase (EC-SOD) in the microenvironment impacts...

Documents

Transcript of Lack of extracellular superoxide dismutase (EC-SOD) in the microenvironment impacts...