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Neuroscience Letters 568 (2014) 67–71

Contents lists available at ScienceDirect

Neuroscience Letters

jo ur nal ho me page: www.elsev ier .com/ locate /neule t

hanges in ascorbate, glutathione and �-tocopherol concentrations inhe brain regions during normal development and moderateypoglycemia in rats

nirudh R. Raoa, Hung Quachb, Ed Smithb, Govind T. Vatasseryb,1, Raghavendra Raoa,c,∗

Division of Neonatology, Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USAResearch Service and the Geriatric Research Education and Clinical Center (GRECC), Minneapolis VA Health Care System, Minneapolis, MN 55417, USACenter for Neurobehavioral Development, University of Minnesota, Minneapolis, MN 55455, USA

i g h l i g h t s

Postnatal changes in antioxidant concentrations in the brain regions are unknown.Ascorbate, glutathione and �-tocopherol were studied in 7, 14 and 60 day old rats.The effect of acute hypoglycemia on the three antioxidants also was determined.All three antioxidants were 100–600% higher during development than at adulthood.Hypoglycemia led to less antioxidant decrease in developing brain than adult brain.

r t i c l e i n f o

rticle history:eceived 19 December 2013eceived in revised form 26 February 2014ccepted 20 March 2014

eywords:-Tocopherolscorbaterainlutathione

a b s t r a c t

Ascorbate, glutathione and �-tocopherol are the major low molecular weight antioxidants in the brain.The simultaneous changes in these compounds during normal development, and under a pro-oxidantcondition are poorly understood. Ascorbate, glutathione and �-tocopherol concentrations in the olfactorybulb, cerebral cortex, hippocampus, striatum, hypothalamus, midbrain, cerebellum, pons and medullaoblongata were determined in postnatal day (P) 7, P14 and P60 male rats. A separate group of P14 andP60 rats were subjected to acute hypoglycemia, a pro-oxidant condition, prior to tissue collection. Theconcentrations of all three antioxidants were 100–600% higher in the brain regions at P7 and P14, relativeto P60. The neuron-rich anterior brain regions (cerebral cortex and hippocampus) had higher concentra-tions of all three antioxidants than the myelin-rich posterior regions (pons and medulla oblongata) at P14

ypoglycemiaat

and P60. Hypoglycemia had a differential effect on the antioxidants. Glutathione was decreased at bothP14 and P60. However, the decrease was localized at P14 and global at P60. Hypoglycemia had no effecton ascorbate and �-tocopherol at either age. Higher antioxidant concentrations in the developing brainmay reflect the risk of oxidant stress during the early postnatal period and explain the relative resistanceto oxidant-mediated injury at this age.

© 2014 Elsevier Ireland Ltd. All rights reserved.

. Introduction

Oxidants play a major role in acute brain injury and neurodegen-rative disorders [1–3]. A system of antioxidant enzymes and lowolecular compounds protects the brain against oxidant-mediated

Abbreviations: GSH, glutathione; P, postnatal day.∗ Corresponding author at: Mayo Mail Code 391, 420 Delaware Street, SE, Min-eapolis, MN 55455, USA. Tel.: +1 612 625 3260; fax: +1 612 624 8176.

E-mail addresses: raghurao@umn.edu, raghubkrao@gmail.com (R. Rao).1 Deceased.

ttp://dx.doi.org/10.1016/j.neulet.2014.03.035304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.

injury. Ascorbate, glutathione (GSH) and �-tocopherol are themajor low molecular weight antioxidants in the brain. Ascorbateand GSH are water-soluble, while �-tocopherol is lipid-soluble [1].Ascorbate and �-tocopherol are transported from plasma across theblood brain barrier via specialized transport systems, while GSH islocally synthesized from its constituents, glutamate, glycine andcysteine [4,5]. The three compounds differ in their cellular local-ization. Ascorbate is primarily localized in neurons and GSH in

glia [6]. A primary localization site for �-tocopherol has yet to bedetermined, although its concentration is higher in the gray matterthan the white matter [7]. Supplementation studies confirm that allthree compounds independently protect against oxidant-mediated

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njury [8–10]. Additionally, they interact synergistically and helpesynthesize those depleted during oxidant exposure [1,6,11]. Thehree compounds also have non-antioxidant properties and influ-nce neuronal and glial maturation and function [6,12–14].

Ascorbate, GSH and �-tocopherol concentrations vary amonghe brain regions in adult rodents, with the concentrations beingigher in the neuron-rich anterior regions (cerebral cortex andippocampus) than the myelin-rich posterior regions (pons andedulla oblongata) [7,15–18]. Whether similar interregional vari-

tions exist during development is not well understood. In humansnd rats, peak brain development occurs postnatally with the pos-erior brain regions maturing earlier than the anterior regions [19].nergy-demanding processes, such as synaptogenesis and myeli-ation are primarily postnatal events in both species. Free radicalsroduced during this metabolically active period could impair brainevelopment by altering gene expression, DNA replication and cellivision, suggesting the need for an effective antioxidant systemuring development.

Previous studies demonstrate that ascorbate concentrationsre indeed higher in the developing brain than the mature brain15,18,20]. A higher brain GSH concentration during developmentas also been demonstrated in some, but not all studies [15,21].hese studies have evaluated the changes in the whole brain or in

limited number of brain regions. To our knowledge, the devel-pmental changes in �-tocopherol have never been evaluated.imilarly, no studies have compared the effects of a pro-oxidantondition on the three antioxidants in the developing and maturerains. The first aim of this study was to compare the ascorbate, GSHnd �-tocopherol concentrations in nine brain regions in postnatalay (P) 7, P14 and P60 rats. The brain is still developing at P7 and14, while at P60 it is mature [19]. The second aim was to comparehe effect of acute hypoglycemia of equivalent severity and dura-ion on the three antioxidants at P14 and P60. Free radicals initiateeuronal injury during hypoglycemia [2,3]. Previous studies haveemonstrated that the developing brain is more resistant to injuryhan the mature brain during hypoglycemia [22,23], likely due toess severe oxidative stress [24]. Our purpose was to gain a betternderstanding of the role of antioxidants in the age-specific vulner-bility, so that neuroprotective strategies can be developed usinghese compounds.

. Materials and methods

.1. Animal preparation

The Institutional Animal Care and Use Committee approved allrocedures. Male P7 (body weight, 16 ± 1 g), P14 (39 ± 4 g) and P60311 ± 20 g) Sprague-Dawley rats were used. Pregnant rats wereurchased (Harlan Laboratories, Indianapolis, IN, USA) and allowedo deliver spontaneously. The litter size was culled to eight on P3.nly males were studied, in order to avoid the confounding effectsf sex on the antioxidants [16,25]. Animals in Aim 1 were killedfter overnight fasting (n = 6 at P14 and P60, and n = 14 at P7). Aeparate set of P14 and P60 rats (n = 6) was subjected to overnightasting and hypoglycemia before tissue collection in Aim 2. P7 ratsere not subjected to hypoglycemia, as they do not sustain brain

njury in this model [22].

.2. Induction of hypoglycemia

Acute hypoglycemia was induced using previously published

rotocol [22,24]. In brief, after overnight fasting, rats were injectedubcutaneously with regular insulin in a dose of 10 IU/kg. Fastingas continued and the rats were maintained at an ambient tem-erature of 34 ± 1 ◦C. Blood glucose was monitored every 30 min

etters 568 (2014) 67–71

using a glucometer (Accu-Check®, Roche Laboratories, Indianapo-lis, IN, USA) and maintained between 20 mg/dL and 40 mg/dL as inour previous studies [22,24]. In this model, hypoglycemia (bloodglucose <40 mg/dL) is achieved 30 min after the insulin adminis-tration and is maintained until 240 min [22,24,26]. The animalswere killed 240 min after the insulin injection without correctionof hypoglycemia and the brain was collected.

2.3. Tissue preparation

Animals were deeply anesthetized using pentobarbital(120 mg/kg ip) and perfused with ice-cold saline. The brainwas removed and the following nine regions were dissectedas previously described [7,16]: olfactory bulb, cerebral cortex,hippocampus, striatum, hypothalamus (anterior brain regions),cerebellum, pons, medulla oblongata (posterior brain regions)and midbrain. The tissue samples were manually homogenizedand extractions for ascorbate and GSH analyses were performedimmediately. The remainder of the homogenate was stored at−70 ◦C for protein, cholesterol and �-tocopherol analyses. Samplesfrom individual rats were used at P14 and P60, resulting in n = 6. AtP7, samples from two rats were combined to augment the tissuequantity, resulting in n = 7.

2.4. Determination of antioxidants

Tissue antioxidant concentrations were determined by HPLCusing published methods described in Supplementary Material.Briefly, ascorbate was determined using the procedure of Margolis[27]. The modified method of Ubbink et al. [28,29] with minor revi-sions was used to determine free and total GSH. Cholesterol and�-tocopherol were determined in the hexane extracts of the tis-sue homogenate as previously described [30,31]. Total protein wasdetermined using modified Lowry technique [32].

2.5. Statistical analysis

The effects of postnatal age and hypoglycemia on the regionalantioxidant concentrations were determined using ANOVA.The intergroup differences were determined using Bonferroni-corrected unpaired t tests. Data are presented as mean ± SD.The ascorbate and GSH data are reported as nmol/mg protein.The �-tocopherol data are reported as nmol �-tocopherol/mmolcholesterol as is conventional [33]. A p value <0.05 was consideredsignificant.

3. Results

3.1. Developmental changes in the antioxidant concentrations inthe brain regions

There was a main effect of postnatal age on all three antiox-idants in all the nine brain regions (p < 0.001; Fig. 1). Ascorbateconcentrations were higher at P7, compared with those at P14 andP60 (p < 0.01; Fig. 1a). Between P7 and P14, ascorbate concentrationdecreased markedly in all the brain regions except the cerebellum(p < 0.001). The concentration decreased further, but less markedly,between P14 and P60 in all the brain regions, except the olfac-tory bulb (p < 0.001). There were inter-regional variations in theascorbate concentrations at all the three ages. Overall, the con-centrations were higher in the anterior regions than the posterior

regions, particularly at P14 and P60 (p < 0.001; Fig. 1a).

Free GSH concentrations were also higher during developmentin all the brain regions (p < 0.001; Fig. 1b). Unlike ascorbate, GSHconcentrations did not differ between P7 and P14; a P7 > P14

A.R. Rao et al. / Neuroscience Le

Fig. 1. Developmental changes in ascorbate (a), free GSH (b), and �-tocopherol (c)concentrations in the brain regions of postnatal day (P) 7, P14 and P60 rats. Valuesare mean ± SD, n = 6–7. There is a main effect of postnatal age on all three antiox-idants in all the brain regions (p < 0.001 for each, ANOVA). The concentration ofeach compound differs between P7, P14 and P60 in all the brain regions (p < 0.01,Bonferroni-adjusted unpaired t tests) except, there are no difference in GSH con-centrations between P7 and P14 in ctx, hip, str, crb, hyp and mid, and between P7and P60 in olf, and no difference in �-tocopherol concentrations between P7 andP14 in olf and str, and between P7 and P60 in hyp. Abbreviations: olf, olfactory bulb;cbG

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tocopherol concentrations in any of the brain regions at either P14or P60 (Supplementary Figure).

Fig. 2. Effect of hypoglycemia on free glutathione concentration in the brain regionsof 14-day-old rats (a) and adult rats (b). Values are mean ± SD, n = 6. There is a

tx, cerebral cortex; hip, hippocampus; str, striatum; hyp, hypothalamus; mid, mid-rain; crb, cerebellum; pns, pons; mob, medulla oblongata; chol, cholesterol; andSH, glutathione.

ifference was present only in the pons and medulla oblongatap < 0.005). Between P14 and P60, free GSH decreased in all therain regions (p < 0.001). An overall decrease along the anterior-o-posterior axis was also present at P14 and P60 (Fig. 1b). The agend region-specific variations in total GSH mirrored those of freeSH (not shown).

Similar to ascorbate and GSH, the �-tocopherol concentrationsere higher in all the brain regions at P7, compared with those at

14 and P60 (p < 0.005; Fig. 1c). As with ascorbate, a P7 > P14 > P60radient was present, except in the olfactory bulb and striatum

tters 568 (2014) 67–71 69

(P7 = P14 > P60), and the hypothalamus (P14 > P7 = P60). However,unlike ascorbate, the decrease in �-tocopherol concentrationbetween P7 and P14 was less pronounced, except in the cerebralcortex and the posterior brain regions (Fig. 1c). There were min-imal inter-regional variations in �-tocopherol concentrations atP7. Similar to ascorbate and GSH, an overall decrease along theanterior-to-posterior axis was present at P14 and P60 (Fig. 1c).

3.2. Effect of hypoglycemia on the antioxidant concentrations inthe brain regions

The severity of hypoglycemia at P14 and P60 was compara-ble (mean blood glucose concentration: P14, 28.5 ± 3.9 mg/dL; P60,28.2 ± 1.5 mg/dL). Hypoglycemia had a differential effect on thethree antioxidants. A main effect of hypoglycemia was present onfree and total GSH concentrations at both P14 and P60 (p ≤ 0.002for each). Free GSH decreased in the olfactory bulb, striatum, mid-brain and medulla oblongata at P14 (p < 0.05; Fig. 2a), and in all thebrain regions, except the hypothalamus, at P60 (p < 0.05; Fig. 2b).This was accompanied by decreased total GSH concentration in theolfactory bulb (−8%) and medulla oblongata (−8%) at P14, and inthe olfactory bulb (−13%), hippocampus (−19%), striatum (−14%),cerebellum (−11%) and pons (−18%) at P60 (p < 0.05 for all; notshown). There was no effect of hypoglycemia on ascorbate and �-

main effect of hypoglycemia on the glutathione concentrations at both postnatalages (p < 0.001 for each, ANOVA). *p < 0.05, Control group vs. Hypoglycemia group(Bonferroni-adjusted unpaired t tests). Abbreviations: olf, olfactory bulb; ctx, cere-bral cortex; hip, hippocampus; str, striatum; hyp, hypothalamus; mid, midbrain; crb,cerebellum; pns, pons; mob, medulla oblongata; and GSH, glutathione.

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. Discussion

In this comprehensive study, we found that the concentra-ions of all three major low molecular weight antioxidants variedetween the developing and mature brain regions under basalonditions in rats. With few exceptions, the ascorbate, GSH and �-ocopherol concentrations were higher during development withome brain regions displaying six fold higher concentrations thanhe adult brain regions. The concentrations of all three antioxidantsecreased postnatally. However, the rate of decline was not uni-orm, and varied among the antioxidants and the brain regions.ypoglycemia also had a differential effect on the antioxidants

n the developing and mature brains. Collectively, these data sug-est that postnatal age influences antioxidant concentrations in therain regions during normal development and under pro-oxidantonditions.

There were interregional variations in ascorbate, GSH and �-ocopherol at all the three ages. The variations at P7 were not inny particular order. In contrast, there was a progressive decreaselong the anterior-to-posterior axis in all three antioxidants at P60.nterestingly, following just one week of development, the adultattern was already established for ascorbate and �-tocopherol inhe P14 brain with few exceptions. Overall, these results can bexplained by the developmental stage of a brain region, such as thearying neuronal and glial densities and the stage of myelinationt a given postnatal age [15,16,19]. However, this is unlikely to behe sole explanation, as discussed below.

Ascorbate contributed more than GSH to the water-solublentioxidant pool, resulting in an ascorbate:GSH ratio as high as 4:1t P7 and 2:1 at P60 in most regions. Higher ascorbate concen-rations at P7 likely reflect the higher density of neurons, wherescorbate is localized, at this age [15,20]. However, even in theelatively myelin-dense regions, such as the P60 medulla oblon-ata, the ascorbate:GSH ratio was >1.0. Previous studies attributedhe postnatal decrease in the ascorbate:GSH ratio to the progres-ive increase in glia, where GSH is localized [6]. However, our dataemonstrate that both ascorbate and GSH decrease postnatally,lbeit at different rates, in both neuron-rich and glia-rich regions.n this respect, our data are similar to previous whole brain studies18,21].

The �-tocopherol concentrations also were higher during devel-pment. Since data are reported in relation to cholesterol, thisould be a reflection of the relatively unmyelinated state of theeveloping brain. Between P7 and P60, �-tocopherol concentra-ion decreased in all regions, except the hypothalamus. The percentecrease from P7 to P60 was similar to that of ascorbate, althoughhe rates of decline of the two compounds were not identical.

hereas a marked decrease was already evident by P14 for ascor-ate in all regions, except the cerebellum, �-tocopherol decreasedinimally between P7 and P14 in most regions. The inter-regional

ariations at P60 are similar to previous data from our lab [7] with arogressive decrease along the neuron-rich anterior brain regionso myelin-rich posterior brain regions. Collectively, these data sug-est that similar to ascorbate, �-tocopherol is primarily localizedn neurons [15]. Additional studies are necessary to confirm thisossibility.

Thus, the brain has a substantial pool of low molecular weightntioxidants during its peak development. This contrasts with theelatively low concentrations of the antioxidant enzymes, super-xide dismutase and glutathione peroxidase, at the correspondingostnatal period [34,35]. Higher antioxidant concentrations areotentially necessary to prevent oxidant-mediated injury during

he metabolically active phase of brain development. The robustxpression of the nuclear enzyme poly(ADP-ribose)polymerase-1hat repairs oxidant-mediated DNA damage at the correspondingostnatal period [24] supports this interpretation. An additional

etters 568 (2014) 67–71

explanation for the higher ascorbate, GSH and �-tocopherol con-centrations during development is their role in neuronal and glialmaturation and function [6,12]. While all three compounds likelyhave major roles during brain development, the data suggest thatascorbate may be particularly important during the early postnatalperiod.

Hypoglycemia had a differential effect on the three antioxidantsin the developing and mature brains. GSH was the only compoundaffected at both ages. The effect was localized at P14 involving onlya few brain regions. Conversely, a more global involvement wasseen at P60 and free GSH decreased in all brain regions, except thehypothalamus, a region known to be resistant to hypoglycemia-induced injury [22]. Further, in those regions affected at both ages(olfactory bulb, striatum, midbrain and medulla oblongata), thepercent GSH decrease relative to the control was greater at P60 thanat P14 (12–22% vs. 7–10%). These age-specific differences betweenP14 and P60 parallel the known vulnerability to injury during hypo-glycemia and other pro-oxidant conditions at the correspondingages [22–24,36]. Decreased GSH during hypoglycemia has beenpreviously demonstrated in adult rats [37]. GSH is an electrondonor during the reduction of hydrogen peroxide by glutathioneperoxidase, an enzyme that is upregulated in hypoglycemia [37].However, total GSH was also decreased in several brain regions,suggesting inadequate GSH synthesis [11] also may be responsiblefor our results. Such a scenario is likely, since glutamate, a precur-sor of GSH is consumed for energy production during hypoglycemia[24].

In vitro studies demonstrate that ascorbate and �-tocopherol areconsumed prior to GSH during oxidant exposure [38,39]. This orderof decrease was not present in our study. Our results are similar toprevious in vivo studies in adult rats demonstrating decreased GSHwithout altered ascorbate during hypoxia-ischemia [11,40], a con-dition characterized by cerebral energy failure, excitotoxicity andoxidative stress, similar to hypoglycemia. Thus, GSH likely plays agreater role than the other two antioxidants during a pathologicalstress. However, this possibility remains to be explained by study-ing other models of hypoglycemia, such as more severe, recurrentand prolonged hypoglycemia, or varying combinations of these, andby incorporating measurements of oxidant stress in the model.

5. Conclusions

The postnatal age influenced ascorbate, GSH and �-tocopherolconcentrations in the brain regions during normal developmentand acute hypoglycemia in rats. The higher antioxidant concen-trations in the early postnatal period may be reflective of thepotential for oxidative stress during this metabolically active periodor the necessity of these compounds for normal neurodevelop-ment. The antioxidant profile in the hypoglycemic mature brainimplies the potential for oxidative stress and neuronal injury. Ther-apies directed at augmenting antioxidants, particularly GSH, maybe beneficial in this setting.

Conflicts of interest

The authors declare that they have no conflicts of interest.

Author contributions

ARR conceived the study, designed and participated in all

and ES performed tissue analyses and assisted with manuscriptpreparation. GTV assisted with study design, data analysis andinterpretation. RR was in overall charge of the project, data analysisand manuscript preparation.

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cknowledgements

The authors gratefully acknowledge Maurice W. Dysken, MD,irector, GRECC Program, Minneapolis VA Health Care System foris support, and Kathleen Czerniak for assistance with the exper-

ments. This project was supported with resources and the use ofacilities at the Minneapolis VA Health Care System, and by grantsrom National Institute of Child Health and Human DevelopmentHD47276) and Viking Children’s Fund. The manuscript is dedicatedo the memory of Govind T. Vatassery, PhD.

ppendix A. Supplementary data

Supplementary data associated with this article can beound, in the online version, at http://dx.doi.org/10.1016/j.neulet.014.03.035.

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