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Experimental Neurology xxx (2010) xxx–xxx

YEXNR-10707; No. of pages: 8; 4C:

Contents lists available at ScienceDirect

Experimental Neurology

j ou rna l homepage: www.e lsev ie r.com/ locate /yexnr

Effect of hypothyroxinemia on thyroid hormone responsiveness and action duringrat postnatal neocortical development

Satish Babu a, Rohit Anthony Sinha a, Vishwa Mohan a, Geeta Rao a, Amit Pal a, Amrita Pathak a,Manish Singh b, Madan M. Godbole a,⁎a Department of Endocrinology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow 226014, Indiab Central Drug Research Institute, Lucknow 226014, India

Abbreviations: T3, triiodothyronine; T4, thyroxin; DTH, thyroid hormone; P16, postnatal day 16; MCT8, mMBP, myelin basic protein; Bax, Bcl-2-associated X prodria-derived activator of caspases.⁎ Corresponding author. Fax: +91 522 2668017.

E-mail address: madangodbole@yahoo.co.in (M.M. G

0014-4886/$ – see front matter © 2010 Published by Edoi:10.1016/j.expneurol.2010.12.012

Please cite this article as: Babu, S., et al., Eneocortical development, Exp. Neurol. (201

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Article history:Received 1 July 2010Revised 11 December 2010Accepted 14 December 2010Available online xxxx

Keywords:Iodine deficiencyHypothyroxinemiaBrain development

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Neurological deficits due tomaternal and neonatal hypothyroxinemia undermild–moderate iodine deficiencyare a major preventable health problem worldwide. The present study assesses the impact ofhypothyroxinemia on postnatal neocortical development and also compares it to the known effects of severehypothyroidism. Our results strongly suggest that even within elevated circulating triiodothyronine (T3)levels, hypothyroxinemia significantly impairs thyroid hormone responsiveness in developing rat neocortex.The significant compensatory alteration in deiodinase levels with unaltered monocarboxylate transporter8 (MCT8) and thyroid hormone receptors (TRs), although found to be similar in hypothyroxinemic andhypothyroid condition, is more pronounced under later condition. The resultant downregulation of nuclearmyelin binding protein (MBP) and mitochondrial transcripts Cytochrome oxidase III (Cox III) as well assignificantly enhanced mitochondrial localization of Bax and reduced Bcl-2 and Bcl-xL accompanied byenhanced release of Cytochrome c and Smac with activation of caspase-3 indicates pronounced apoptosisleading to compromised cellular survival. The similarities of this responsiveness albeit with difference indegree under hypothyroidism and hypothyroxinemic state with adequate availability of T3 are suggestive ofan independent role of thyroxine in neocortex development. Taken together, this study brings forth theneurophysiological aspects of hypothyroxinemia and underscores the importance of adequate iodinenutrition along with mandatory thyroxin monitoring during pregnancy and after birth.

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2 KO, deiodinase 2 knockout;onocarboxylate transporter 8;tein; Smac, second mitochon-

odbole).

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ffect of hypothyroxinemia on thyroid hormo0), doi:10.1016/j.expneurol.2010.12.012

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Introduction

Iodine deficiency disorders are still a challenge for the world'sendocrine community in ensuring normal brain development ofchildren worldwide (Aghini Lombardi et al., 1995; Laurberg et al.,2000; Delange, 2001; Vermiglio et al., 2004; Berbel et al., 2009). Thesedisorders which include severe hypothyroidism may also cause arelatively subtle form of thyroid hormone (TH) deficiency known ashypothyroxinemia (Royland et al., 2008). Hypothyroxinemia resultingfrom mild to moderate iodine deficiency is characterized by lowthyroxine (T4) yet normal or elevated triiodothyronine (T3) unlikehypothyroidism associated with both low T4 and T3. Mild iodinedeficiency leading to hypothyroxinemia is a much more prevalentscenario throughout the world compared to a classical hypothyroid

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condition (Escobar et al., 2000). Unfortunately, our understanding of THdeficiency in various experimental models like rats is primarily derivedfrom studies involving chemically induced hypothyroidism. Thesemodels, although providing ample mechanistic details of TH action onbrain, are not representative of hypothyroxinemic condition in humans.Recent studies (Lavado-Autric et al., 2003; Auso et al., 2004; Sinha et al.,2008) have convincingly addressed this point and have used differentmodels of hypothyroxinemia to study its effect during early embryoniccortical development. The present study investigates the effect of mildiodine deficiency induced hypothyroxinemia on postnatal cerebralcortex development and compares it with the known effects ofhypothyroidism. Our results show that, during postnatal development,both the conditions result in significantly altered expression of T3responsive genes in the developing neocortex. Some of these effectsalbeit relatively less compared to severe hypothyroid conditiondo showsignificant changes compared to euthyroid controls arguing that, underhypothyroxinemia circulating serum, T3 does not seem to substantiallycompensate for the reduced TH responsiveness in neonatal brain incontrast to what has been shown in D2 knockout (KO) mice (Galtonet al., 2007). The present study results shed light on some of the lessknown molecular effects of hypothyroxinemia on postnatal braindevelopment and discuss the possible reasons for these differences.

ne responsiveness and action during rat postnatal

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Material and methods

Iodine deficient model (hypothyroxinemic model)

Fifty-day-old female ratsweighing 120–150 gwere switched to a lowiodine diet (LID) and given 1% KClO4 in drinking water for 10 days.Animalswere then separated into two groups namely a) iodine sufficientgroups or euthyroid [LID+KI {10 μg iodine/20 g of diet+normaldrinking water}], b) low iodine diet or hypothyroxinemic group[LID+0.005% KClO4]. Both the groups were kept on above diet regimenfor three months.

The low iodine diet was prepared by mixing thoroughly 6 kg flour ofcorn grown inwell known iodine deficient areawith a recurrentfloodinghistory, 2.5 kg of wheat gluten, 1.0 kg brewer's yeast, and 0.15 kg NaCl,0.15 kg CaCO3 (constituents as per ICN protocol). Sufficient amount ofLID was prepared to cover a complete experiment with the same batch.

At the end of 3 months, ~1.0 ml of blood was obtained under slightether anesthesia from the jugular vein to determine T4 and T3. Theplasma was spun off and serum was kept at −20°C for hormoneestimation by specific radioimmunoassays. Total serum T4 (TT4) andtotal T3 (TT3) were measured in rat pups by RIA using Coat-A-CountSiemens Healthcare Diagnostics kits (cat no. TKT41 and TKT31).Urinary iodine of rats was estimated by an improved Sandell–Kolthoffammonium persulphate oxidation method. Based on the hormonalestimations and urinary iodine, female rats were divided into euthyroidand Hypothyroxinemic and were mated with normal males. In aseparate group of age-matched female rats, hypothyroidism wasinduced in rats by giving MMI (0.025% wt./vol.) in drinking water tothe pregnant rats from gestational day 8 and continued thereafter untilsacrifice of pups born to these dams. Blood of 0.8–1.0 ml was pooledfrom pups at P0 (n=6–8) and 1.5–2.0 ml blood was pooled at P8(n=5–7) and at P16 (n=3–4) for thyroid hormone analysis asmentioned above. Vaginal smears and microscopic visualization ofspermatozoa confirmed the day of mating (E0). The pregnant dams andpups were continued under the above conditions and neocortices weredissected at postnatal day 0 (P0) (n=18), P8 (n=15), and P16(n=15). All animal procedures performed abovewere approved by theInstitutional Animal Ethics Committee as per the international guide-lines for Animal Care and Research.

Protein extraction and Western blotting

Neocortices at P0 (n=6–8), P8 (n=5–7), and at P16 (n=3–4) from2–3 different litters (from 3 groups; euthyroid, hypothyroxinemic, andhypothyroid) were harvested snap-frozen in liquid nitrogen, and storedat−80 °Cuntil further investigation. The tissuewas collected inbuffer “A”(0.32 M sucrose, 1 mM K-EDTA, 10 mM Tris–HCl pH 7.4) and homoge-nized. The supernatant obtained from two successive centrifugations at1300g for 10 min was pooled and spun at 17,000g for 15 min to collectmitochondria. The absence of COX IV and actin was used to confirm thepurity of cytosolic (supernatant) and mitochondrial fraction (pellet),respectively. Mitochondrial pellet was resuspended in buffer “A” forfurther analysis. The protein concentration was determined by standardmethod. One hundredmicrograms of mitochondrial or cytosolic proteinsfromall three study groups at specified stageswere electrophoresed (12%SDS–PAGE) and transferred to nitrocellulose membrane and developedusing Amersham ECL Kit. Relative expression of each protein wasdetermined by densitometric analysis using LabWorks 4.0 software(UVP Ltd., Cambridge, UK). Rabbit polyclonal antibodies to cleavedcaspase-3, Bcl-xL, Bax, Bcl-2, b-actin, and MBP were obtained from CellSignaling Technology (Danvers, MA).

RNA extraction and real time PCR

Total mRNA was isolated from the cerebral cortex at P16 (n=4)from 2–3 different litters from 3 groups following single step mRNA

Please cite this article as: Babu, S., et al., Effect of hypothyroxinemia oneocortical development, Exp. Neurol. (2010), doi:10.1016/j.expneurol

isolation method using TRI reagent (MRC Inc., Cincinnati, OH). TotalmRNA (2 μg) was reverse transcribed to cDNA using ThermoScriptRT–PCR kit (Invitrogen) following manufacturer's instructions. Realtime analysis for D2, D3,MCT8, TRα, and β and Cox III and normalizinggene GAPDHwas performed using SYBR GreenMaster Mix or TaqManprobes as per the manufacturer's instruction (Applied Biosytems) andfold changes in gene expression were calculated using 2−ΔΔCT

method.

Immunofluorescence

In euthyroid, hypothyroxinemic, and hypothyroid groups, four pupsfrom two to three litters were taken at postnatal day 16 (P16). Pupswere anesthetized and transcardially perfusedwith normal saline (0.9%NaCl wt./vol., 15–20 ml for pups and 50 ml for adults) followed by 4%wt./vol. paraformaldehyde in 0.1 M phosphate buffer (20–25 ml) andpostfixed in 4% paraformaldehyde at 4°C for 4 h. Tissues were thendehydrated in an alcohol series for paraffin embedding by standardprotocol. Coronal sections (3 μm)were cut from the rat brain containingthe primary somatosensory cortex (S1) from the pups and adult rats atdifferent developmental stages and mounted on poly-L-lysine-coatedslides. For immunofluorescence, after deparaffinization and rehydrationsteps, antigen retrieval was performed in a microwave oven using10 mM citrate buffer (pH 6.0). Sections were blocked with 10% normalsheep serum for 20 min. To localize the apoptotic cells, sections wereincubated with a rabbit polyclonal cleaved caspase-3 antibody (1:250;Cell Signaling Technology) overnight at 4°C. Afterwashingwith PBS, thesections were incubated with Rhodamine Red-conjugated anti-rabbitIgG (red) secondary antibody for 1 h in the dark. To investigatewhetherthe apoptotic cells are neurons, the sections were double immuno-stained with cleaved caspase-3 antibody and a mouse monoclonalneurofilament antibody (1:50; Sigma). Then, the sections wereincubated with Rhodamine Red-conjugated anti-rabbit IgG (red) andfluorescein–isothiocyanate-conjugated anti-mouse IgG (green) antibo-dies for 1 h in the dark. The sections were then counterstained withHoechst 33258, mountedwith antifademedium, and visualized under afluorescencemicroscope. Sections not incubatedwith primary antibodywere used as negative controls. Image-Pro Plus 5.1 software (MediaCybernetics, Inc., Silver Spring, MD) was used for image capturing andcell counting.

Statistical analysis

Statistical analysis was performed using the SPSS package (SASInstitute Inc., Cary, NC). When indicated, the statistical significance ofthe differences was assessed using the ANOVA. The threshold forstatistical significance was set at pb0.05.

Results

Mild iodine deficiency induced hypothyroxinemia

Significant reduction in serum TT4 and TT3 levels of rat pupsadministered MMI confirmed induction of hypothyroidism at differentdevelopmental stages compared to euthyroid controls (Fig. 1A and B).However, pupson low iodine diet showeda reductiononly in serumTT4(hypothyroxinemia) and not in T3 which is significantly increasedcompared to euthyroid levels at P16 (Fig. 1A and B). The bodyweight ofpups born to hypothyroxinemic dams (22.24±2.77 g) was comparableto euthyroid pups (21.38±1.10 g; pN0.05); however, pups born tohypothyroid dams (16.0±0.67 g) had significantly lower weight incomparison to hypothyroxinemic and euthyroid pups (pb0.005). Inaddition to this, the urinary iodine content of hypothyroxinemic femalerats put on mating was b40±20 μg/l compared to control euthyroidlevels which was 350±50 μg/l, indicating a significant loss of iodinecontent in the diet regime.

n thyroid hormone responsiveness and action during rat postnatal.2010.12.012

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Fig. 1. (A, B) T3 and T4 levels in euthyroid, iodine deficient (hypothyroxinemic), andhypothyroid pups at P0, P8, and P16. Each bar represents the mean of the respectiveindividual ratios±SE at P0 (n=6–8), P8 (n=5–7), and at P16 (n=3–4). Significantdifferences compared with age-matched euthyroid counterpart are indicated (*pb0.05,**pb0.01, ANOVA).

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Effect of hypothyroxinemia on MCT8, deiodinases 2 and 3, and thyroidhormone receptors

Since developing brain possesses potent compensatorymechanismsto protect it frommoderate to drastic changes in circulating levels of THwhich also is the measure of stress due to low TH, we therefore studiedthe effect of hypothyroxinemia and chemically induced hypothyroidismon THmetabolizing enzymes deiodinases 2 and 3, TH transporterMCT8(Ceballos et al., 2009), andT3nuclear receptors by real timePCRanalysisat P16. Results showed that both hypothyroid and hypothyroxinemicpups demonstrated a significant increase in D2 levels compared tocontrols (Fig. 2A). This increase in D2 mRNA was higher in hypothy-roidism (~11 fold) than in hypothyroxinemia (~4 fold). The expressionof D3 mRNA also decreased significantly and suppressed (~3.3 fold)under hypothyroidism and (~3 fold) in hypothyroxinemic groupcompared to controls (Fig. 2B). However, the expression of MCT8 andTH nuclear receptorsα1 and β1 showed no significant alteration underboth hypothyroxinemic and hypothyroid condition (Fig. 2C–E).

Effect on nuclear and mitochondrial encoded T3 responsive genes

After looking at the alteration in the levels of genes controlling THavailability and action in brain under hypothyroxinemia, we nextassessed its effect on the key TH responsive genes such as nuclearencoded myelin basic protein (MBP) (Farsetti et al., 1992; Strait et al.,1997) and mitochondrial gene Cytochrome c oxidase III (Cox III)(Vega-Nunez et al., 1995; Sinha et al., 2010). Western blot and real

Please cite this article as: Babu, S., et al., Effect of hypothyroxinemia oneocortical development, Exp. Neurol. (2010), doi:10.1016/j.expneurol

time PCR results showed that, although less compared to hypothy-roidism, hypothyroxinemia caused a significant decrease in MBP(Fig. 3A and B) and Cox III (Fig. 3C) levels during neocorticaldevelopment.

Effect on genes regulating cell survival

Since the above result showed that hypothyroxinemia similar tohypothyroidism is associated with altered gene expression, we nextquestioned whether it also affects cellular processes altered underhypothyroidism. Western blot results reconfirmed that, in hypothyroidpups, the enhanced neocortical apoptosis and its nature is similar to thatwe had seen earlier under perinatal hypothyroidism in other areas ofneonatal brain (Singh et al., 2003; Kumar et al., 2006; Sinha et al., 2009).To investigate whether hypothyroxinemic condition can affect apoptoticsignaling, we performed immunoblotting using purified mitochondrialand cytosolic fractions of neocortical tissues at different developmentalstages. Analysis of pro-apoptotic protein Bax in mitochondrial fractionobtained from euthyroid pups showed that this protein showed a steeploss from themitochondrial fraction with increasing age. This loss of Baxis significantly blunted by hypothyroidism during all stages of develop-ment andBax levelswere also significantly increased at P0 andP16underhypothyroxinemia compared with euthyroid controls (Fig. 4A and B).The anti-apoptotic proteins Bcl-2 and Bcl-XL show an opposite trend toBax during development and progressively become more localized inmitochondria under euthyroid conditions whereas are significantlyreduced under hypothyroidism during all developmental stages(Fig. 4A–D). Under hypothyroxinemic condition, however, both Bcl-2and Bcl-xL are more evident in mitochondria at birth (P0) compared toeuthyroid controls but, later at P8 and P16, fall significantly below theirbasal levels (Fig. 4A–D).

Since the balance of pro- to anti-apoptotic Bcl-2 family proteins isindicative of mitochondrial dysfunction at P16 as evident by their levelsin hypothyroxinemic and hypothyroid conditions, therefore, we nextassessed the release of apoptogenic proteins frommitochondria and theactivation of caspase-3 at this stage in all three study groups. Resultsshowed that hypothyroxinemia similar to hypothyroidism caused anincreased release of apoptogenic proteins Cytochrome c and Smac incytosol (Fig. 5A–C) with enhanced activation of executioner caspase-3compared to euthyroid control indicating towards a pro-apoptoticsignaling under hypothyroxinemia (Fig. 5A and D). To investigate theradial distribution of the apoptotic cells in rat somatosensory corticalregion (S1) of the neocortex at P16, cleaved caspase-3 immunofluores-cence (red) was used as a marker of apoptosis (Srinivasan et al., 1998).To identify the cortical layers, sections were counterstained withHoechst 33258 (blue). In the euthyroid group, in S1, the majority ofthe cleaved caspase-3-immunoreactive (ir) cells were located in layersII–III and V (Fig. 6A). In contrast, increased number of apoptotic neuronswas evenly distributed in all the layers of the neocortex under bothhypothyroxinemic and hypothyroid conditions (Fig. 6B).

Discussion

The importanceof thyroidhormone forpostnatal brain developmentis well established and is indispensable during critical phases ofneurodevelopment. In the presence of high or normal circulating T3levels, the observeddecrease in serum thyroxine is generally consideredto be of little clinical relevance. However, with improved detection andresolution of biochemical assays in the clinical settings, identificationand characterization of more subtle degrees of thyroid dysfunctionunder varying levels of iodine intake can be observed and, as aconsequence, the concept of “hypothyroxinemia” (reduced T4, nochange in T3 or TSH) has emerged (Pedraza et al., 2006). In fact,statistically speaking, hypothyroxinemia is a more common andwidespread clinical situation than hypothyroidism per se in developingand developed countries alike (Escobar et al., 2000; Vermiglio et al.,

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Fig. 2. Real time PCR analysis for D2 (A), D3 (B), MCT8 (C), TRα1 (D), and TRβ1 (E) in each group at P16. Each bar represents the mean of the respective individual ratios±SE (n=3rats at each developmental stage). Significant differences compared with age-matched euthyroid pups are indicated (*pb0.05).

4 S. Babu et al. / Experimental Neurology xxx (2010) xxx–xxx

2004). Although there may be several causes of maternal and neonatalhypothyroxinemia, moderate iodine deficiency is the most commonfactor. Unfortunately, since elevated TSH levels still remain theprominentscreening parameter for assessing thyroid disorders in neonates, most ofthe hypothyroxinemic infants are missed out in routine checking.Although epidemiological studies (Aghini Lombardi et al., 1995; Laurberget al., 2000; Delange, 2001; Vermiglio et al., 2004; Berbel et al., 2009) doshow a strong correlation between maternal and neonatal hypothyrox-inemia with mental retardation in iodine deficient areas, experimentalevidenceshaveonly recentlybeen furnished(Ausoet al., 2004; Sinhaetal.,2008; Royland et al., 2008; Sharlin et al., 2008 and 2010; Axelstad et al.,2008). These studies provide a strong support for the effect of maternaland neonatal hypothyroxinemia on fetal and neonatal brain developmentin rats. We examined the effect of mild iodine deficiency induced

Please cite this article as: Babu, S., et al., Effect of hypothyroxinemia oneocortical development, Exp. Neurol. (2010), doi:10.1016/j.expneurol

combinedmaternal–neonatal hypothyroxinemia on postnatal neocorticaldevelopment. The model of hypothyroxinemia which we employ in ourstudy represents a “mild” iodine deficient state as evident by an increaseT3/T4 ratio and decreased urinary iodine content. Although we haveemployed the same treatment regime as described by Lavado-Autric et al.(2003), the iodine content in the source diet may differ to induce varyingdegree of iodine deficiency. Such a condition of iodine deficiency has beenpreviously described by Pedraza et al. (2006) where they report a similarincrease in serum T3 levels under their mild iodine deficient model. Thepossible reason for this increase has been attributed to changes inintrathyroid iodinemetabolism,which lead to a preferential synthesis andsecretion of T3 over T4, and an increased T3 to T4 ratio in the circulation.

Our results show that TH responsiveness in postnatal neocortex ismore sensitive to decrease in T4 levels rather than T3, which is

n thyroid hormone responsiveness and action during rat postnatal.2010.12.012

Fig. 3. (A) Immunoblot analysis of MBP levels in euthyroid, hypothyroxinemic, and hypothyroid cerebral cortical tissue fractions at P16. (B) Each bar represents the mean of the respectiveindividual ratios±SE (n=4 rats from each group). (C) Real time PCR analysis for Cox III in each group at P16. Each bar represents themean of the respective individual ratios±SE (n=3 rats ateach developmental stage). Significant differences compared with age-matched euthyroid pups are indicated (*pb0.05).

Fig. 4. (A) Immunoblot analysis of Bax, Bcl-2, and Bcl-xL levels in euthyroid (white bars), hypothyroxinemic (gray bars), and hypothyroid (black bars) cerebral cortical purifiedmitochondrial fractions from P0 to P16. Since most Cox protein levels are altered under hypothyroidism, therefore, equal loading was verified by Ponceau S staining. (B–D) Each barrepresents the mean of the respective individual ratios±SE at different developmental stages P0 (n=6–8), P8 (n=5–7), and at P16 (n=3–4) (*pb0.05 compared to euthyroid levels).

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Please cite this article as: Babu, S., et al., Effect of hypothyroxinemia on thyroid hormone responsiveness and action during rat postnatalneocortical development, Exp. Neurol. (2010), doi:10.1016/j.expneurol.2010.12.012

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Q5 Fig. 5. (A) Immunoblot analysis of Cytochrome c, Smac, and cleaved caspase-3 levels in euthyroid, hypothyroxinemic, and hypothyroid cerebral cortical purified cytosolic fractions.(B–D) Each bar represents the mean of the respective individual ratios±SE (n=4 rats from each group; *pb0.05 compared to euthyroid levels).

6 S. Babu et al. / Experimental Neurology xxx (2010) xxx–xxx

supported by a compensatory increase in D2 and decrease in D3 levelsunder hypothyroxinemia. However, contrary to earlier study inhippocampus, neocortex does not evoke a compensatory increase inMCT8 or nuclear TH receptors expression under iodine deficientcondition (Sharlin et al., 2010). Since TH responsiveness is assessed bythe levels of TH target genes, we, in this study, compared the changein some of the known TH regulated genes. Surprisingly, what appearsfrom our results is that, in spite of normal or even elevated serum T3levels and a compensatory D2 and D3 response, TH responsive nuclearand mitochondrial genes were still significantly decreased inhypothyroxinemia as under hypothyroidism. This was furthersupported by the similarity in the translocation of cellular proteinregulating neuronal survival under hypothyroxinemia which showeda similar pattern to what is well known under hypothyroidism andactivation of the major executioner caspase-3 in the somatosensorycortical region (Singh et al., 2003; Kumar et al., 2006; Sinha et al.,2009; Srinivasan et al., 1998). Although the initial increase in thelevels of the mitochondrial Bcl-2 and Bcl-xL at P0 stage underhypothyroxinemia is not understood, we can only speculate thatpossibly brain at birth under a mild TH deficiency retains its ability toshow an adaptable increase in the levels of anti-apoptotic genes likeBcl-2 which is gradually lost later on. However, the drastic lowering ofTH levels under hypothyroidism seems to completely impair thiscompensatory ability of the developing brain.

These results appeared very interesting to us because they are incomplete contrast to what one expects from the evidence of THcompensation in D2 KOmice (Galton et al., 2007). This study (Galton etal., 2007) very well shows that any loss of T4 to T3 conversion in D2 KOmice is sufficiently compensated by serum T3 which is evident fromnormal levels of T3 regulated genes and unimpaired behavioral indices.Therefore, it seems unlikely that T4 decrease under hypothyroxinemia

Please cite this article as: Babu, S., et al., Effect of hypothyroxinemia oneocortical development, Exp. Neurol. (2010), doi:10.1016/j.expneurol

would result in any gene alteration similar to hypothyroidism. Althoughone reason for this discrepancy could be species variation, but mostlikely, this suggests that possibly brain T4 other thanbeing a sourceof T3could in fact be independently corroborating T3 action in brain. Thereare now growing evidences that T4 could affect nuclear T3 action bycausing modification of TH nuclear receptors through acetylation(Hung-Yun et al., 2005) and phosphorylation (Davis et al., 2000), andperhaps, for this reason, the T4 converting enzyme D2 is preferentiallysilenced in neurons (Guadano-Ferraz et al., 1997) to preserve thisunique functionof T4.Although this hypothesis still needs tobe tested invivo, the results obtained in the present study suggest the significanceand potential downstream consequences of alteration in gene expres-sion and function under hypothyroxinemia caused by mild iodinedeficiency for ensuring adequate measures to protect the developingbrain of the fetus and neonates.

Uncited reference

Yadav et al., 2010

Acknowledgments

We are highly grateful to Dr. X. Wang (University of SouthwesternTexas Medical Center, Dallas, TX, USA) for the kind gift of antibody forSmac and to Dr. R. Jemmerson (University of Minnesota, USA) forCytochrome c antibodies. Financial grants from the Department ofScience and Technology, DST/FIST/LS-II/012/2003 and SR/SO/HS/95/2007, to MMG is also acknowledged. This work is the part of the PhDthesis of Satish Babu.

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Fig. 6. (A) Representative photomicrographs of coronal sections of primary somatosensory cortex showing the distribution of cleaved caspase-3-immunoreactivity (ir). It is shownwith white arrow (apoptotic, red) cells in the euthyroid, hypothyroxinemic, and hypothyroid rat pup at P16 stages. Sections were counterstained with Hoechst (blue). Note theincrease in apoptotic cells in the hypothyroxinemic and hypothyroid groups compared with the euthyroid group. Scale bar, 20 μm. (B) Proportion of cleaved caspase-3-ir cells in theprimary somatosensory cortex in the euthyroid, hypothyroxinemic, and hypothyroid rats at P16 stage expressed as fold increase above the basal euthyroid levels. Cleaved caspase-3-ir cells were counted in five different areas. At least 5000 cells in each section were scored. Results are expressed as means±SE of 4 rat pups. Significant differences compared withage-matched euthyroid pups are indicated; *pb0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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