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Research Report

Comparison of the effects of maternal protein malnutrition andintrauterine growth restriction on redox state of centralnervous system in offspring rats

Mehmet Tatlia,⁎, Aslan Guzela, Goksel Kizilb, Vatan Kavakc, Murat Yavuzb, Murat Kizilb

aDepartment of Neurosurgery, Faculty of Medicine, University of Dicle, 21280 Diyarbakir, TurkeybDepartment of Chemistry, Faculty of Science, University of Dicle, 21280 Diyarbakir, TurkeycDepartment of Anatomy, Faculty of Medicine, University of Dicle, 21280 Diyarbakir, Turkey

A R T I C L E I N F O

⁎ Corresponding author. Fax: +90 412 248 85 2E-mail address: mtatli@dicle.edu.tr (M. TaAbbreviations: CAT, catalase; CNS, central n

protein restriction; NTD, neural tube defectreactive substance

0006-8993/$ – see front matter © 2007 Elsevidoi:10.1016/j.brainres.2007.04.036

A B S T R A C T

Article history:Accepted 13 April 2007Available online 20 April 2007

Both maternal protein malnutrition and intrauterine growth restriction (IUGR) havedeleterious effects on brain development, but a comparison of these effects has not beenpreviously reported. The objectives of this study were to investigate and compare the effectsof both factors on the oxidative status of the central nervous system (CNS), including thespinal cord, in offspring rats. We evaluated various parameters of oxidative status andantioxidant enzyme activities of superoxide dismutase and catalase (CAT) in differentregions of the CNS from 60-day-old rats subjected to prenatal and postnatal proteinrestrictions [middle protein restriction 12%, severe protein restriction (SPR) 4%] or IUGRproduced by uterine artery ligation. Furthermore, we compared these study groups to eachother and to control rats fed an isocaloric 24% protein diet. Results were analyzed using one-way ANOVA followed by Tukey's post hoc test. Both protein restrictions and IUGR alteredvarious parameters of oxidative status. In all evaluated structures, protein restrictionsresulted in increases in thiobarbituric acid-reactive substances level and index of lipidperoxidation (Pb0.001), and in decreases in antioxidant enzyme activities (Pb0.005). IUGRalso increased lipid peroxidation levels in the blood samples (Pb0.04) and protein oxidativedamage in the cerebellum and cerebral cortex (Pb0.005); however, no effects were detectedon the spinal cord. The greatest decrease in CAT activity was in the cerebellum of rats fedwith SPR diet (Pb0.001). This study suggests that not only severe but also middle proteinmalnutrition have deleterious effects on CNS structures, including the spinal cord. Proteinrestriction has a greater effect on the redox state of the CNS than IUGR.

© 2007 Elsevier B.V. All rights reserved.

Keywords:Central nervous systemIntrauterine growth restrictionProtein restrictionRedox state

3.tli).ervous system; CPD, control protein diet; IUGR, intrauterine growth restriction; MPR, middle; SOD, superoxide dismutase; SPR, severe protein restriction; TBARS, thiobarbituric acid-

er B.V. All rights reserved.

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1. Introduction

Table 1 – The mean body and brain weights of all groups

Group Body weight (g) N P Brainweight

(g)2nd day 60th day

1 5.2±0.2 210±10 10 3±0.2 ⁎, ⁎⁎2 3±0.1 160±12 10 2±0.1 ⁎⁎, ⁎⁎⁎3 2.5±0.1 100±15 10 b0.001a 1±0.3 ⁎4 3.7±0.2 180±10 10 2±0.2 ⁎⁎, ⁎⁎⁎

a Statistical significance between the control group and the othergroups (one-way ANOVA test).⁎ According to one-way ANOVA test, Pb0.001.⁎⁎ According to one-way ANOVA test, Pb0.001.⁎⁎⁎ According to one-way ANOVA test, PN0.05.

Nutrition plays a crucial role in the maturation and functionaldevelopment of the central nervous system (CNS). However,malnutrition during pregnancy is still a serious healthproblem in underdeveloped and developing countries. Mater-nal protein malnutrition is able to induce deleterious effectson brain structures (King et al., 2002, King et al., 2004, Rubio etal., 2004; Soto-Moyano et al., 1999), resulting in brain dysfunc-tion, and decreases brain weight in pups (Bennis-Taleb et al.,1999; Desai et al., 1996; Feoli et al., 2006; Joshi et al., 2003;Morgane et al., 1993; Rotta et al., 2003; Rotta et al., 2003;Winickand Rosso, 1969) particularly in the neonatal and earlychildhood periods (Joshi et al., 2003). Intrauterine growthrestriction (IUGR) also has a negative effect on brain develop-ment (Alkalay et al., 1998; Geva et al., 2006; Huizinga et al.,2004; Lane et al., 2001; Tan and Yeo, 2005; Tolsa et al., 2004).

Dietary protein is an important source of essential aminoacids that can be used as intracellular antioxidants. Therefore,its restriction may lead to an increase in oxidative damage bydiminishing antioxidant defenses of the tissue (Bonatto et al.,2005; Feoli et al., 2006; Li et al., 2002). Free radicals arephysiologically generated by biological systems during meta-bolism and can induce oxidative damage in organic molecules(Bonatto et al., 2005). The main enzymatic defenses againstfree radicals are superoxide dismutase (SOD) and catalase(CAT). This coupled enzymatic activity is able to reduce the ionsuperoxide to H2O (Halliwell and Gutteridge, 2006). Whenthere is an imbalance between free radical generation and thescavenging system, cellular damage occurs. Because of itshigh rate of oxidative metabolic activity, high content ofphospholipids andmoderate levels of antioxidant, the brain isespecially vulnerable to free radicals (Garcia et al., 2005).

Fetus receives its nutrients from the maternal uterinecirculation via the placenta. Any disturbance in the placental–fetal circulation will therefore have severe consequences onthe supply of important nutrients such as oxygen, glucose,and amino acids (McMillen et al., 2001).

Follow-up studies of infants with IUGR have shown thatfetal IUGR is associated with significant neurodevelopmentaldisabilities in fine and gross motor skills, cognitive function,activity, self-regulation, language, abstract reasoning, recog-nition memory, concentration, attention, mood, and schoolperformance in both preterm and term infants (Alkalay et al.,1998; Fattal-Valevski et al., 1999; Van Wassenaer, 2005, Zuk etal., 2004). Animal studies have clearly demonstrated thenegative impact of chronic intrauterine hypoxia on cellnumber and cell size, with overall lighter brain weight andlower DNA content as well as reduced synapse numbers(Cragg, 1972; Huizinga et al., 2004; Lane et al., 2001; Mallard etal., 1998; Xu et al., 2005).

Many investigators found biochemical alterations in thenervous system in experimentalmalnutritionmodels (Bonattoet al., 2005; 2006; Feoli et al., 2006; Garcia et al., 2005), especiallythose related to neurotransmitter systems (Feoli et al., 2006;Rotta et al., 2003; Steiger et al., 2003), phosphorylation ofsynaptic membrane proteins (Singh and Shankar, 1999), andneuronal connections (Morgane et al., 2002; Perry and Souza,2003). In some studies, the effects of protein malnutrition on

oxidative status in the rat brain were investigated (Bonattoet al., 2005, 2006; Feoli et al., 2006), however, to our bestknowledge, the oxidative status of the spinal cord has notbeen evaluated in the event of protein malnutrition andIUGR.

Various maternal animal models such as pharmacological(streptozotocin), dietary (global food restriction, low-proteindiet), or surgical (uterine artery ligation) have been used toinduce IUGR and to evaluate its consequences in differentsystem (Holemans et al., 2003; Huizinga et al., 2004; Kollee etal., 1979; Vuguin, 2002). However, none of them focused oncomparison of the effects of maternal protein malnutritionand IUGR on oxidative status of the CNS.

The aims of the present study were to evaluate the effectsof both maternal protein restriction, a preventable cause ofuteroplacental insufficiency, and IUGR produced by uterineartery ligation on oxidative status of the CNS, includingcerebral cortex, cerebellum and spinal cord in offspringWistarrats, and to compare their effects using various oxidativestatus parameters such as tissue lipid peroxidation level andCAT and SOD activities.

2. Results

2.1. Body and brain weights

Both protein restriction and IUGR decreased in body and brainweights of offspring rats, but the highest decreased was ingroup 3. On the 2nd and 60th experimental days, thedifferences in body weights between the control group andthe other groups were statistically significant (Pb0.001). Thedifferences between their brain weights were also statisticallysignificant (Pb0.001). The differences in both body and brainweights between protein restricted groups and control groupwere significant as well (Pb0.001). However, in terms of brainweight there was no significance between group 2 and group 4(PN0.05) (Table 1).

2.2. Lipid peroxidation levels

Both protein malnutrition and IUGR increased lipid peroxida-tion levels in the plasma samples (Fig. 1). However, the highestlipid peroxidation level (1.85 nmol/ml) was in group 3, whereas

Fig. 1 – Comparison of serum TBARS levels among groups.Data are expressed as mean±SD of the mean of 10 animalsper group. Means with different letters at a time differsignificantly, p<0.05, while the values sharing commonletters are not significantly different, at p>0.05 (ANOVA test,bP<0.001, cP<0.04).

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the lowest lipid peroxidation level was found in the controlgroup. When the results were compared to each other and tothe control group, the mean differences were statisticallysignificant in the protein-malnourished group (Pb0.001) andin the IUGR group (Pb0.04).

Tissue lipid peroxidation levels were also analyzed.Increased lipid peroxidation levels were observed only in therats subjected to severe protein restriction, in all evaluated

Fig. 2 – Lipid peroxidation levels of studied structures in offsprinanimals per group. In the studied CNS areas, means with differensharing common letters are not significantly different, at p<0.05

structures (Fig. 2). Surprisingly, and in contrast to resultsobtained from the plasma samples, the differences among theother groups were not statistically significant.

2.3. Protein oxidative damage

Fig. 3 shows protein oxidative damage in offspring rats. In thecerebral cortex, no statistical differencewas observed betweengroup 2 and group 4 (P=1), but when analyses were repeatedfor the cerebellum, a statistical significance was found(Pb0.001). SPR-fed animals showed an increase in oxidative-damaged proteins (Pb0.001).

Protein malnutrition altered parameters of protein oxida-tive damage, and specifically damagedmacromolecules, in allregions of the spinal cord. However, IUGR produced nooxidative damage in the spinal cord.

2.4. Antioxidant protective enzyme activities

2.4.1. Catalase activityThe effects of protein malnutrition and IUGR on CAT activityare presented in Fig. 4. CAT activity decreased in allevaluated structures and experiment groups; however, thehighest decrease was in the cerebellum. Moreover, thelowest CAT activity level (Pb0.001) was detected in thegroup 3, in all studied structures. In cerebral cortex andspinal cord, CAT activities of group 2 and group 4 weresimilar, and statistical significance was not detected. How-ever, their cerebellar CAT enzyme activities were statisticallydifferent (Pb0.02).

2.4.2. Superoxide dismutase activityFig. 5 reveals the effects of different protein content diets andof IUGR on SOD activity. Similar to that observed in CAT

g rats. Data are expressed as mean±SD of the mean of 10t letters at a time differ significantly, p<0.05, while the values(ANOVA test, bP<0.001).

Fig. 3 – Protein oxidative damage in offspring rats. Data are expressed asmean±SD of themean of 10 animals per group. In thestudied CNS areas, means with different letters at a time differ significantly, p<0.05, while the values sharing commonletters are not significantly different, at p>0.05 (ANOVA test, bP<0.001, cP<0.0001, dP<0.05).

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activity, the most affected group was group 3 (Pb0.001). In allstudied structures, SOD activities of group 2 and group 4 weresimilar, and statistical significance was not detected. How-ever, their SOD enzyme activities were significantly differentwhen compared to the control group.

Fig. 4 – Catalase activity of all groups in the studied CNS areas in10 animals per group. Data are expressed as mean±SD of the mewith different letters at a time differ significantly, p<0.05, whiledifferent, at p>0.05 (ANOVA test, bP<0.001, cP<0.0001, dP<0.02).

3. Discussion

The present study is the first to evaluate the effects of bothprotein malnutrition and IUGR produced by uterine artery

offspring rats. Data are expressed asmean±SD of themean ofan of 10 animals per group. In the studied CNS areas, meansthe values sharing common letters are not significantly

Fig. 5 – Superoxide dismutase activity of groups 1, 2, 3 and 4 in the studied CNS areas in offspring rats. Data are expressedas mean±SD of the mean of 10 animals per group. In studied CNS areas, means with different letters at a time differsignificantly, p<0.05, while the values sharing common letters are not significantly different, at p>0.05 (ANOVA test, bP<0.001,cP<0.0001, dP<0.05).

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ligation on redox state of the CNS, including the spinal cord,and to compare these effects. Furthermore, the study is alsothe first work to include a middle-protein diet (12% protein)and to evaluate its effect on oxidative status of the CNS in sucha research. In other words, the effect of MPR on the oxidativestatus of the CNS has not been reported previously.

Though the studies of nutritional effects on CNS develop-ment with animal models have usually used diets that areformulated to be adequate (24–25%) or low (6–8% protein) inprotein content, in reality, malnutrition demonstrates a greatvariability among people and societies. Moreover, its severitychanges among pregnants and newborns, ranging from mildto severe. Therefore, in our opinion, the food restrictionmodels evaluating the effect of maternal food on braindevelopment might be different. Just as, various diets havebeenused in different studies (Molina-Perez et al., 2000; Reeveset al., 1993; Yahya et al., 1994). The middle-protein diet of 12%protein and low-protein diet of 4% protein used in this studywas used in studies evaluating the effects of malnutrition onmuscle and bone growth (Tirapegui, 1999) growth trajectoriesof the craniofacial skeleton (Miller and German, 1999), andbrain fatty acid levels (Rao et al., 2006). In the present study, weevaluated the effects of middle and severe protein restrictionson oxidative status of the CNS. Further studies are required toverify the reliability of these protein concentrations.

We hypothesized that if a study uses a MPR diet to producefetal growth restriction and evaluate its consequences, itmight improve our ability to understand some outcomes ofIUGR. The study demonstrated that MPR also has detrimentaleffects on oxidative status of the CNS. Though maternal milkprotein concentration could be measured by some methods

(Lowry et al., 1951), we did notmeasure it in the study, as in theother diets and studies (Bonatto et al., 2005, 2006; Feoli et al.,2006). Therefore, this may be considered as a limitation of thestudy. However, because of the extended experimental period,we accepted that maternal milk had a middle protein content.Further study may be conducted to determine the exactmaternal milk protein content in the event of middle proteinrestriction.

The primary environmental factor that regulates fetalgrowth in the third trimester in animals and humans issubstrate delivery to the fetus. This delivery depends onmaternal substrate levels, and on adequatematernal and fetalblood flow, which is essential for placental function andtherefore also for efficient nutrient supply to the fetus(Holemans et al., 2003). Maternal protein restriction anduterine artery ligation are characterized by both deficientsubstrate delivery to the fetoplacental unit and decreaseduteroplacental blood flow (Holemans et al., 2003; Rosso andKava, 1980). Furthermore, total milk volume is decreasedduring lactation (Rasmussen and Warman, 1983), whichhampers normal neonatal growth (Holemans et al., 2003).

Maternal protein restriction is a frequently used model toinduce fetal growth restriction and to study its consequencesin various systems and organs such as the brain andcerebellum (Bonatto et al., 2005, 2006; De Souza et al., 2004;Feoli et al., 2006). Maternal uterine artery ligation in the rat,originally described by Wigglesworth (Wigglesworth, 1964), isone of the most frequently used methods to study theconsequences of uteroplacental insufficiency. Inmost studies,the uterine artery is ligated between gestational days 14 and19 (Huizinga et al., 2004; Ogata et al., 1990). The period around

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day 17 coincides with the onset of rapid fetal growth inpregnancy (Wigglesworth, 1964). This time window maycorrespond to the beginning of the third trimester in pregnantwomen, although the developmental regulatory profile of aspecific organ system of the rat may differ from that inhumans. The rat fetus is neurologically immature comparedto the human fetus (Huizinga et al., 2004), and the cerebellardevelopment occurs mainly postnatally (Bonatto et al., 2006).It was demonstrated that uterine artery ligation at gestationalday 17 leads to a persistent growth restriction until day 80without any sign of catch-up in body weight in both male andfemale rats (Huizinga et al., 2004). Due to the previouslymentioned reasons, the effects of maternal protein malnutri-tion can be comparedwith those of IUGR produced by bilateraluterine artery ligation.

Oxidative damage may occur following a traumatic orischemic injury or malnutrition (Hill et al., 2003; Xu et al.,2005). The oxidative status of the spinal cord has beenevaluated in the event of traumatic spinal cord injury,ischemic injury and in some vitamin deficiency studies(Bul'on et al., 2005; Hill et al., 2003; Tirapegui, 1999). Moreover,in experimental spinal cord studies, some oxidative stressparameters have been used as an indicator demonstrating theactivity of some pharmacological agents (Bul'on et al., 2005; Xuet al., 2005). Nonetheless, the oxidative status of the spinalcord has not been evaluated in the event of maternal proteinrestriction.

It is known that spina bifida, anencephaly, and encepha-locele are commonly grouped together and termedneural tubedefects (NTD). Failure of closure of the neural tube duringdevelopment results in anencephaly or spina bifida aperta.The exact etiology of NTD remains complex and poorlyunderstood (Padmanabhan, 2006); however, maternal nutri-tional factors have been implicated in the complex etiology ofNTD. Moreover, nutrition has been demonstrated to influenceNTD risk. Best known is the role of folic acid. Other nutritionparameters have also been implicated, including various othernutrients, such as protein restriction, low maternal weightgain, long-term restrictive diets, andweight loss due to dietingduring early pregnancy (Carmichael et al., 2003).

The normal fetal growth is a result of complex interactionamong the three components of maternal–placental–fetalunit. Nutritional status of the mother is the most importantmaternal factor leading to intrauterine growth retardation.Malnutrition involves deficiency of the macronutrients i.e.fats, proteins, carbohydrates. Proteins provide amino acids forsynthesis of antioxidant defense enzymes, reduced glu-tathione and albumin (as sacrificial antioxidant protein)(Gupta et al., 2004). Therefore, one would expect a putativecorrelation between these congenital lesions and the antiox-idant defense mechanisms in protein restriction or IUGR.

No study is available on the oxidative stress in spinal cordas a consequence of maternal protein malnutrition andintrauterine growth restriction produced by uterin arteryligation. We hypothesized that if protein restriction has anegative effect on spinal cord development and changesamong spinal cord regions, this might be helpful in explainingthe main cause underlying some congenital spinal cordlesions such as spina bifida. In addition, it might explainwhy spina bifida occurs mainly in the lumbar region.

In summary, we tried in this study to answer the followingquestions.

(1) Do proteinmalnutrition and IUGR have any effect on theredox state of the spinal cord?

(2) Do these effects change among spinal cord areas?

Toward these aims, we evaluated the oxidative status ofthe spinal cord in the event ofmaternal protein restriction andIUGR produced by uterine artery ligation, and the oxidativeresponses of the spinal cord regions were studied. The studydemonstrated that IUGR has no effect on spinal cord oxidativeparameters. Although protein restriction demonstrated adeleterious effect on the redox state of the spinal cord, thiseffect was not statistically different among spinal cordregions. The study also suggested that there is a strongcorrelation between protein restriction and the oxidativestatus of spinal cord, whereas no correlation between IUGRand the oxidative status of spinal cord. This variousness maypartly be explained by different effect periods of these twofactors. Our investigation might be considered as a prelimin-ary study. Further data are required and could be obtained byperforming ultrastructural histopathological investigationsand more specific designed biochemical studies.

The present data confirmed the working hypothesis andsupport the idea that oxidative damage may be related tobrain changes induced by protein malnutrition, as reportedearlier (Feoli et al., 2006). In addition, the study revealed thatnot only SPR but also MPR has a detrimental effect onoxidative status of the CNS, including the spinal cord. More-over, negative effects of IUGR on redox states of the cerebralcortex and cerebellum were detected, and both proteinmalnutrition (in different contents) and IUGR increased lipidperoxidation levels in the plasma samples.

CAT activity decreased in all studied structures and experi-ment groups; however, the lowest CAT enzyme activity was inthe cerebellum, as in a study presented by Feoli et al. (2006).This condition may be explained in part by the differentmaturation period and different physiological and biochem-ical characteristics of different brain structures (Bonatto et al.,2006). Cerebellar development occurs mainly postnatally,while cortical development occurs during the fetal period(Huizinga et al., 2004). Differences in CAT activities in thegroup 2 and group 4 in the cerebral cortex and spinal cordwerenot statistically significant, but statistical significance wasobserved in cerebellar CAT activities. The group 2 showedbetter CAT enzyme activity compared to the group 4. This alsomight be explained by the different development periods ofthe brain structures. In our opinion, this result might demons-trate that uterine artery ligation-produced IUGR affects moreimmature than mature organs. Further studies are required tosupport this idea.

Although some similarities were found between our studyand other work presented by Feoli et al. (2006), in our study theSOD activity was found to be decreased in cerebellum. Thisdiscrepancy might be explained by methodological differ-ences. On the other hand, our result related to SOD activity iscompatible with published literature (Bonatto et al., 2005;Partadiredja et al., 2005). We detected a decrease in SODactivity in the cerebellum as result of protein restriction. This

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result corroborates with a recent work that found a greatdecrease in SOD activity comparing with normal diet (Bonattoet al., 2005), and confirm the results of the other study thatfound a deficit in the expression of Cu/Zn-SOD in cerebellumof malnourished mice (Partadiredja et al., 2005).

There are only a few studies related to oxidative stress inthe brain tissue of rats subjected to protein malnutrition(Bonatto et al., 2005, 2006; Ehrenbrink et al., 2006; Feoli et al.,2006). Feoli et al. (2006) reported that prenatal and postnatallow protein malnutrition increased oxidative damage to lipidsand proteins from the studied brain areas (cortex, hippocam-pus, and cerebellum). Bonatto et al. suggested that both lowprotein content in the diet and the essential amino acidmethionine might alter the antioxidant system and the redoxstate of the hippocampus (Bonatto et al., 2005), brain andcerebellum (Bonatto et al., 2006).

In addition to the results of the studies mentioned above,our study showed that not only SPR, but also MPR, mayincrease oxidative damage to lipids and proteins from thestudied CNS areas, including the spinal cord, and may destroytheir antioxidant defense systems. Moreover, the resultsobtained from the IUGR group demonstrated that it alsocaused harmful effects, similar to MPR but less than thoseobserved in SPR, on the oxidative status of the cerebral cortexand cerebellum, and may destroy their antioxidant defensesystem. However, the effect of IUGR produced by bilateraluterine artery ligation on protein oxidative damage of thespinal cord could not be demonstrated. This might beexplained by the different rates of oxidative metabolicactivities and content of phospholipids of the spinal cord andcerebral cortex aswell as by their different antioxidant defensemechanisms. Based on these results, we hypothesize thatprenatal proteinmalnutrition is more important than IUGR onthe development of the brain and cerebellum. Further studiesand data are required to support and verify this conclusion.

Table 2 – Contents of protein diets

Ingredient (g/kg) Low (4%protein)diet

Middle (12%protein) diet

Control(24%

protein)diet

4. Conclusion

Our data indicated that both protein malnutrition and IUGRincreased oxidative damage to lipids and proteins from thestudied CNS areas. Moreover, the study suggested that notonly severe but also middle protein malnutrition havedeleterious effects on CNS structures including the spinalcord. Protein restriction has a greater effect on the redox stateof the CNS than IUGR.

Casein 46.00 141.00 276.00Cornstarch 500.90 432.86 329.90Dyetrose 167.00 144.20 110.00Sucrose 100.00 100.00 100.00Cellulose 50.00 50.00 50.00Soybean oil 70.00 70.00 70.00t-Butylhydroquinone 0.014 0.014 0.014Salt mix #213266 35.00 35.00 35.00Calcium phosphatedibasic

11.66 8.63 4.08

Calcium carbonate 3.91 6.14 9.49Vitamin mix #310025 10.00 10.00 10.00L-Cystine 0.70 0.75 4.10Choline bitartrate 2.50 2.50 2.50Blue dye – 0.03 0.05

5. Experimental procedure

5.1. Reagents

Bovine serum albumin, Folin–Ciocalteu phenol reagent,1,1,3,3-tetramethoxypropane (TMP), 2,4-dinitrophenylhydra-zine (DNPH), guanidine hydrochloride, xanthine, xanthineoxidase (EC: 1.1.3.22), nitro blue tetrazolium (NBT) tablets andhydrogen peroxide were purchased from Sigma, St. Louis, MO.2-Thiobarbituric acid (TBA) and potassium chloride wereobtained fromMerck, Darmstadt, Germany. All other reagentswere obtained from Sigma Chemicals (St. Louis, MO).

5.2. Tools

Centrifuge Denley BS400 (Denel, England), UV mini 1240 UV–VIS spectrophotometer (Shimadzu), and ultrasonic processor,Sonics (Vibra Cell), were used.

5.3. Animals

Twenty pregnant rats and their 40 offsprings were included inthis study. Pregnant rats were housed in stainless steel, wire-bottomed cages. They were maintained under standardconditions (12-h light/12-h dark, temperature 22–24 °C room);food andwaterwere given ad libitum. Ratswere obtained fromthe Research andAppliedHealth Center of Dicle University. Allanimal procedures were approved by the Ethics Committee ofExperimental Animals of Dicle University (DEHEK 15.02.2006/04 approval).

5.4. Diets

The three diets were isocaloric, consisting of a 24% proteindiet, an experimental middle-protein diet of 12% protein, andan experimental low-protein diet of 4% protein. The threediets were based on the AIN-93G standard diet recom-mended to support growth (King et al., 2002). The threediets (Dyets, Bethlehem, PA) were isocaloric; thus, the onlydietary variable altered was protein (Table 2). Food con-sumption and spillage were measured to the nearest 0.1 gusing a Tefal Scientific Model Gourmet 7986502/261-0304electronic scale.

5.5. Experimental models

5.5.1. Malnutrition modelTwenty pregnant rats, weighing 220–230 g, were divided into 4groups, each consisting of 5 pregnant rats. At the beginning ofpregnancy, rats were fed with different isocaloric diets,containing 24%, 12% or 4% protein (casein) (Table 2).

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The study groups were divided as follows:

Group 1: Control protein diet (CPD) (24% protein diet; adoptedas normal diet).

Group 2: Middle protein restriction (MPR) (12% protein).Group 3: Severe protein restriction (SPR) (4% protein).Group 4: Intrauterine growth restriction (IUGR).

During the neonatal period, in each study group, thenewborn pups were housed by their mothers. The newbornrats were weaned on postnatal day 22 by separating themfrom their mothers and placed in hanging basket cages.Each rat was housed in a separate cage so that foodconsumption and body weight could be measured daily.Body weights were measured to the nearest 0.1 g using aTefal Scientific Model Gourmet 7986502/261-0304 electronicscale.

From each study group, 10 male offsprings were taken byrandom sampling method, and were divided into 4 studygroups. From weaning until experimental day, all offspringswere fed with the same food as the mothers as mentionedabove. They were maintained under standard conditions(12-h light/12-h dark, temperature 22–24 °C room); food andwater were given ad libitum until they reached experimentalage.

The highest perinatal mortality rate was found in the IUGRgroup, while the highest neonatal mortality rate was detectedin severe protein restricted group.

5.5.2. Intrauterine growth restriction (IUGR) modelIUGR was induced by ligation of the bilateral uterine artery onday 17 of gestation according to a modified method ofWigglesworth (Wigglesworth, 1964). Maternal uterine arteryligation is one of the most frequently used methods to studythe consequences of uteroplacental insufficiency (Huizinga etal., 2004; Joshi et al., 2003; Lane et al., 2001; Levine et al., 1990).Since its activity has been demonstrated, we did not make asham group with the pregnant surgery.

5.6. Measurement of body and brain weights

The body weights of the newborn pups were measured at the2nd and 60th postnatal day, whereas brain weights weremeasured at the 60th postnatal day (experimental day) using aTefal Scientific Model Gourmet 7986502/261-0304 electronicscale.

5.7. Tissue preparation

5.7.1. Blood samplesOn the 60th postnatal day, offsprings were taken andanesthetized by amixture of ketamine (50mg/kg) and xylazine(10 mg/kg). The blood samples were obtained via intracardiacpuncture and kept at −20 °C.

5.7.2. Tissue samplesOffspring rats were sacrificed after taking blood samples byusing a lethal dose of ketamine and xylazine mixture. Thecerebral cortices, cerebellar hemispheres, and spinal cordregions were immediately removed for biochemical analyses.

The tissue samples were instantaneously placed at −20 °Cuntil biochemical measurements. Then they were homoge-nized in 10 vol. of ice-cold phosphate buffer (0.1 M, pH 7.4)containing 140 mM of KCl, 1 mM of ethylenediaminetetraace-tic acid, and 1 mM of phenyl methyl sulfonyl fluoride. Thehomogenate was centrifuged at 960×g for 10 min, and thesupernatant was used. All steps were carried out at 4 °C.

5.8. Tissue analyses

5.8.1. Malondialdehyde (MDA) determination in plasmasamplesLipid peroxidation was assessed in serum by thiobarbituricacid-reactive substances (TBARS) method (Levine et al., 1990).Briefly, 0.5 ml each of plasma was mixed with 2–5 ml of 20%trichloroacetic acid (Sigma, Germany) and centrifuged at3000 RPM for 10 min. The supernatant was decanted, andthe precipitate was washed once with 0.05 M sulfuric acid.Then, 3 ml of 0.2 g/dl TBA was added to the precipitate. Themixture was heated in boiling water for 30 min. After coolingon ice, the resulting chromogen was extracted with 4 ml of n-butyl alcohol. The organic phase was separated by centrifuga-tion at 3000 RPM for 10 min and absorbance was recorded atwavelength of 530 nm. MDA solution was made freshly by thehydrolysis of 1,1,3,3-TMP used as the standard.

5.8.2. Thiobarbituric acid-reactive substance (TBARS)As an index of lipid peroxidation, we used the formation ofTBARS during an acid-heating reaction. Briefly, the sampleswere mixed with 1 ml of trichloroacetic acid 10% and 1 ml ofTBA 0.67%, and then heated in a boiling water bath for 15 min.MDA an intermediate product of lipoperoxidation, was deter-mined by absorbance at 535 nm (Draper and Hadley, 1990).

5.8.3. Measurement of protein carbonylsThe oxidative damage to proteins was measured by thequantification of carbonyl groups based on the reaction withDNPH. In brief, proteins were precipitated by the addition of20% trichloroacetic acid and redissolved in DNPH and theabsorbance read at 370 nm (Levine et al., 1990).

5.8.4. Measurement of catalase (CAT) activityEnzyme assays were performed in tissue extracts obtained asfollows. To determine CAT activity, each tissue sample wassonicated in 50mMphosphate buffer (pH 7.0) and the resultingsuspension was centrifuged at 3000×g for 10 min. The super-natantwas used for enzyme assay. CAT activity wasmeasuredby the rate of decrease in hydrogen peroxide absorbance at240 nm (Dal-Pizzol et al., 2003).

5.8.5. Measurement of superoxide dismutase (SOD) activityThe determination of SOD activity was based on SOD'sinhibition of the reaction of superoxide anion (O2), fromxanthine by xanthine oxidase and the reduction of NBT(Podezasy and Wei, 1998).

5.8.6. Protein assayTotal protein concentration was determined according to themethod described by Lowry et al. (1951) with bovine serumalbumin as the standard.

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5.9. Statistical analysis

Results were expressed as mean±standard deviation of themean, and comparison among groups was made using one-way ANOVA followed by Tukey's post hoc test for multiplecomparisons. In all comparisons, Pb0.05 was considered toindicate statistical significance.

Acknowledgments

We thank Professor Omer Satici from the Department ofBiostatistics, School of Medicine University of Dicle, for hiswork in statistical analyses.

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