Neurochemistry International 58 (2011) 582–590

Glutamate antagonism fails to reverse mitochondrial dysfunction in late phaseof experimental neonatal asphyxia in rats

Nagannathahalli Ranga Reddy a, Sairam Krishnamurthy a,*, Tapan Kumar Chourasia b,Ashok Kumar c, Keerikkattil Paily Joy b

a Neurotherapeutics Lab, Department of Pharmaceutics, Institute of Technology, Banaras Hindu University, Varanasi 221005, U.P., Indiab Department of Zoology, Centre of Advanced Study, Faculty of Science, Banaras Hindu University, Varanasi 221005, U.P., Indiac Department of Pediatrics, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, U.P., India

A R T I C L E I N F O

Article history:

Received 2 August 2010

Received in revised form 6 January 2011

Accepted 18 January 2011

Available online 12 February 2011

Keywords:

Anoxia

Glutamate

Nitric oxide

Mitochondrial function

Ketamine

A B S T R A C T

Neonatal asphyxia is a primary contributor to neonatal mortality and neuro-developmental disorders. It

progresses in two distinct phases, as initial primary process and latter as the secondary process. A

dynamic relationship exists between excitotoxicity and mitochondrial dysfunction during the

progression of asphyxic injury. Study of status of glutamate and mitochondrial function in tandem

during primary and secondary processes may give new leads to the treatment of asphyxia. Neonatal

asphyxia was induced in rat pups on the day of birth by subjecting them to two episodes (10 min each) of

anoxia, 24 h apart by passing 100% N2 into an enclosed chamber. The NMDA antagonist ketamine

(20 mg/kg/day) was administered either for 1 day or 7 days after anoxic exposure. Tissue glutamate and

nitric oxide were estimated in the cerebral cortex, extra-cortex and cerebellum. The mitochondria from

the above brain regions were used for the estimation of malondialdehyde, and activities of superoxide

dismutase and succinate dehydrogenase. Mitochondrial membrane potential was evaluated by using

Rhodamine dye. Anoxia during the primary process increased glutamate and nitric oxide levels; however

the mitochondrial function was unaltered in terms of succinate dehydrogenase and membrane potential.

Acute ketamine treatment reversed the increase in both glutamate and nitric oxide levels and partially

attenuated mitochondrial function in terms of succinate dehydrogenase activity. The elevated glutamate

and nitric oxide levels were maintained during the secondary process but however with concomitant

loss of mitochondrial function. Repeated ketamine administration reversed glutamate levels only in the

cerebral cortex, where as nitric oxide was decreased in all the brain regions. However, repeated ketamine

administration was unable to reverse anoxia-induced mitochondrial dysfunction. The failure of

glutamate antagonism in the treatment of asphyxia may be due to persistence of mitochondrial

dysfunction. Therefore, additionally targeting mitochondrial function may prove to be therapeutically

beneficial in the treatment of asphyxia.

� 2011 Elsevier Ltd. All rights reserved.

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1. Introduction

Birth asphyxia is ‘‘the failure to initiate and sustain breathing atbirth’’ and as per the WHO estimates, there are about 4–9 millioncases of birth asphyxia every year with a mortality rate of 1.2million children. The incidence rate is even higher in thedeveloping countries due to high prevalence of risk factors inmother, lack of antenatal care and higher incidence of pretermbabies (Batool and Zulfiqar, 2006; Kolatat et al., 2000). As perIndian Council of Medical Research, the incidence of birth asphyxia

* Corresponding author. Tel.: +91 9935509199.

E-mail addresses: saibliss@hotmail.com, ksairam.phe@itbhu.ac.in

(S. Krishnamurthy).

0197-0186/$ – see front matter � 2011 Elsevier Ltd. All rights reserved.

doi:10.1016/j.neuint.2011.01.021

in India is about 10% (Deorari et al., 2000). Anoxia is a condition ofdeficient oxygen supply and this occurring at the time of birthcauses severe damage especially to the brain besides other majororgans. The injury occurs due to lack of perfusion, energy failure,glutamate excitotoxicity and also subsequent reperfusion (Sureshet al., 2007). The physiological outcomes of anoxia or hypoxia arecollectively termed as asphyxia (Roy and Stefan, 2008). Asphyxia isconsidered to be an important cause of neonatal brain damage(Luca et al., 2006; Jeffrey, 2006) leading to neurodevelopmentalabnormalities like mental retardation, learning deficits, epilepsyand cerebral palsy in adult life (Jacques et al., 2008; Van-de et al.,2000). These survivors may even require long-term rehabilitationwhich is expensive and complex (Blomgren et al., 2003).

Treatment of neonatal asphyxia involves use of non-pharmaco-logical and pharmacological interventions. Non-pharmacological

N.R. Reddy et al. / Neurochemistry International 58 (2011) 582–590 583

interventions include cleaning of air ways, resuscitation andhypothermia. Mild-to-moderate hypothermia is said to be protec-tive by decreasing metabolic rate, excitatory amino acid release andalso decreasing toxic free radical production (Batool and Zulfiqar,2006). Pharmacological interventions include use of modulators ofglutamate, monoamines (dopamine, nor epinephrine and seroto-nin), GABA, adenosine and growth factors (Suresh et al., 2007). Ca2+

overload inhibitors and anti-oxidants have also been considered astreatment options for asphyxia (Suresh et al., 2007). Of all thepharmacological measures available, over the recent past glutamate,antagonism has generated much interest as excitotoxicity isconsidered to be one of the major pathways leading to neuronaldamage due to asphyxia (Keith, 2006). Non-competitive NMDAreceptor antagonists such as MK 801 and ketamine were effective asneuroprotectants in reducing the brain damage in experimentalmodels of asphyxia (Eric and Henrik, 1997; Foster et al., 1988; Alkanet al., 2001). Further studies with selective glutamate receptorantagonists like mGluR1 and mGluR5 proved to be even moreeffective (Domenico et al., 1999; Dorota et al., 2006). Surprisingly, inclinical trials these drugs were ineffective and showed severe sideeffects limiting their use in the treatment of asphyxia (James, 1992;Chen and Lipton, 2006).

Brain injury due to asphyxia appears to progress in two distinctphases, as an initial (primary) process and the latter (secondary)process. Primary injury process is due to necrosis (rapid cell death)during the hypoxic/ischemic insult and secondary process is byapoptosis (delayed cell death) late after the initial insult (Elisabettaet al., 1997; Kenneth et al., 2000; Rajiv et al., 2001). However, mostof the studies have focused exhaustively on acute injury. Thestudies of acute injury concerning the role and moderation ofglutamate and other associated factors such as MPTP (mitochon-drial membrane permeability transition pore), calcium overloadand oxidative stress have been found to be effective in treatment ofasphyxia in the experimental models (Evangelia et al., 1999;Thomas et al., 2002; Akihito et al., 2004;). Clinical studies haveshown that severe perinatal anoxic injury is associated withdelayed development of cerebral energy failure from 6 to 15 h afterbirth asphyxic insult (Alistair and Peter, 1998; Vannucci et al.,2004). Apparent recovery after an episode of transient ischemia isoften followed by cell death after 24 h (Takaaki, 1982; Hori andCarpenter, 1994; Carol et al., 1997; Vannucci et al., 2004). Thesefindings suggest concomitant energy failure and metabolicderangement late after asphyxic insult. Hence, treatment mayhave to be continued throughout the recovery phase to get abeneficial response. Consistent with the above observationsdelayed treatment with glutamate antagonist MK-801 after postischemic hypothermia was found to rescue normal CA1 neurons inthe hippocampus (Dietrich et al., 1995). However, very few studieshave evaluated the delayed or chronic consequences of asphyxia inin vivo models which is considered to be the phase forpharmacological intervention (Ze et al., 2003; Gunn et al., 2005).Hence, evaluation of glutamate antagonism extending into thesecondary injury process may reveal the causes for failure ofglutamate treatment and provide further clues for effectivetreatment.

In asphyxic injury, mitochondrial alterations are brought aboutby over stimulation of glutamate receptors by excess glutamate(Gordon et al., 2003; Henrik, 2004; Xiao-Jian et al., 2008).Mitochondria is said to play a prominent role in neuronal celldeath due to the production of free radicals, MPTP formation,calcium influx and energy failure (David and Samantha, 1998;Tobaben et al., 2011). As the growth and regeneration of neuronsrequire mitochondrial energy (Robert and Peter, 1993; Zheng et al.,2004), possible treatment reversing mitochondrial dysfunctionassociated with asphyxia may prove to be effective. The long-termreciprocal relationship between glutamate release and mitochon-

drial damage in asphyxia has not been studied extensively.However, many studies have evaluated the role of mitochondria incell death mediated through glutamate in acute process either inanimal models or on neuronal cultures (Juan et al., 1998; Angeleset al., 1998; Tobaben et al., 2011). The present study evaluates therole of tissue glutamate and associated factors with mitochondrialfunction both during primary and secondary processes of asphyxicinjury to understand the dynamic relationship between them. Themodulation of glutamate activity and its role on mitochondrialfunction in both acute and delayed neuronal deaths wereevaluated using ketamine a non-competitive glutamate antagonistin a rat model of neonatal anoxia.

2. Materials and methods

2.1. Animals

Charles Foster albino pregnant rats (180–220 g) were obtained from the Central

Animal House, Institute of Medical Sciences, Banaras Hindu University (B.H.U.). The

animals were housed in polypropylene cages at an ambient temperature of 25� 1 8Cand 45–55% RH, with a 12:12 h light/dark cycle. They had free access to commercial food

pellets (Doodh dhara Pashu Ahar, India) and water. Birth litter count ranged from 8 to 12

pups per rat. Experiments were conducted between 09:00 and 14:00 h. The experimental

procedures were approved by Institutional animal ethical committee, Banaras Hindu

University. ‘‘Principles of laboratory animal care’’ (NIH publication number 85-23,

revised 1985) guidelines were followed. All possible measures were taken to minimize

the animal suffering and to reduce the number of animals used. Animals were divided

into two sets, one for acute (primary process) study within 24 h of anoxia and another for

7 days study after anoxia (secondary process). Each set had 3 groups of 6 animals each

representing control, anoxia and ketamine treatment. Ketamine was given immediately

after recovery from anoxia in a dose of 20 mg/kg i.p. with the volume of a dose not more

than 0.1 ml/10 g body weight. In case of acute study, ketamine was given in 4 divided

doses (5 mg/kg i.p.) with an interval of 2 h (Evangelia et al., 1999) and for the 7 day study;

ketamine was administered as a single daily dose (20 mg/kg i.p.) for 7 days. The control

and anoxic pups received equal volume of normal saline in the same manner (Evangelia

et al., 1999). All rat pups were returned to their dams after anoxia and were decapitated

after 30 min of last dose of ketamine. The brain was micro-dissected into cerebral-cortex,

extra-cortex (the sub cortical regions) and, cerebellum and stored immediately at�80 8Ctill further experimentation.

2.2. Anoxic procedure

A slightly modified procedure of Strata et al. (2004) was followed. The anoxic

chamber was made of plexiglas with dimensions of 21 cm � 18 cm � 11 cm with an

airtight lid. There was a provision for an inlet and outlet of gases on the opposite

walls of the chamber. The flow rate of gas (3 l/min) into chamber was controlled by

using rotameters. Pups in groups of 6 were placed in an airtight plexiglas chamber

over a heating pad maintained at 37 8C temperature. The rat pups were subjected to

two episodes (10 min each) of anoxia on the day of birth and the following day with

an interval of 24 h. Anoxia was induced by passing nitrogen gas (100%) into the

closed chamber. After each episode of anoxia the lid was removed immediately and

pups were exposed to atmosphere. The pups were assisted to resuscitate by lying

them on their back and spreading their limbs.

2.3. Materials

Tetra methyl rhodamine methyl ester (TMRM), glutamate, O-phthalaldehyde

(OPA) and Griess reagent were procured from Sigma–Aldrich (St. Louis, MO, USA).

Thiobarbituric acid (TBA), ethylene glycol tetra-acetic acid (EGTA), 2-[4-(2-

hydroxyethyl)1-piperazinyl]ethane sulphonic acid (HEPES buffer, acid free) were

purchased from Hi media (Mumbai) and sodium succinate, sodium azide,

phenazine methane sulphonate (PMS) and nitro blue tetrazolium were purchased

from Merck (Daidrmstadt, Germany). Ketamine injection IP was procured from

Samarth life sciences Pvt. Ltd., India. All other chemicals and reagents were

procured from local suppliers and were of analytical grade.

2.4. Brain glutamate estimation in different brain regions

The tissue level of glutamate in the cerebral-cortex, extra-cortex and cerebellum

was estimated using high performance liquid chromatography with electrochemi-

cal detector (James and Brian, 1997). In brief, the brain tissue samples were

homogenized in 0.17 M perchloric acid by Polytron homogenizer. Homogenates

were then centrifuged at 33,000 � g (Biofuge Stratos, Heaureas, Germany) at 4 8C.

The supernatant samples containing glutamate were derivatized by combining

100 ml of sample with 20 ml of internal standard (3.3 mM homoglutamine) and

12 ml of (OPA)-SO3 solution (22 mg OPA, 0.5 ml 0.0313 M Na2SO3, 0.5 ml EtOH, and

9 ml 0.1 M Borax). During method development, the stock concentration of Na2SO3

ranged from 7.55 mM to 1.0 M. Samples, internal standards and (OPA)-SO3 reagent

Fig. 1. Effects of anoxia and ketamine on glutamate levels in cerebral-cortex (Cereb-

cortex), extra-cortex (Ext-cortex) and cerebellum on day 1 and day 7 are

represented in panel A and B respectively. Bars represent data as mean � S.E.M.,

n = 6, *p < 0.05 compared to control and #p < 0.05 compared to anoxic control (One-

way ANOVA followed by Student–Newman–Keuls test).

N.R. Reddy et al. / Neurochemistry International 58 (2011) 582–590584

were individually filtered through a 0.22 mm syringe filter prior to mixing. The

mixture was allowed to react for 15 min at 37 8C before 20 ml of the reaction

mixture was injected into the column (Spherisorb, RP C18, 5 mm particle size,

4.6 mm i.d. � 250 mm at 30 8C) connected to a electrochemical detector (Model

2465, Waters, Milford, MA, USA) at a potential of +0.8 V with glassy carbon working

electrode vs Ag/AgCl reference electrode. An ESA model 582 solvent delivery

module (ESA, Chelmsford, MA) pumped the mobile phase (0.1 M Na2HPO4, 25 mM

EDTA, 5% MeOH, adjusted to pH 5.2 with NaOH) at a consistent flow rate of 1.2 ml/

min. During method development the mobile phase pH ranged from 5.18 to 5.60.

Samples were injected into a 20 ml injection loop (model 7127, Rheodyne, Inc.,

Cotati, CA) which preceded the guard column. The glutamate concentration was

expressed as nanograms per milligram of protein.

2.5. Estimation of neuronal nitric oxide (NO)

The NO levels in tissue homogenate of cerebral-cortex, extra-cortex and

cerebellum were estimated by monitoring the amount of nitrite (NO2�) formed as a

metabolic product of NO as described by Griess (1879). Briefly, to the volume of

tissue homogenate, equivalent volume of Griess reagent (Sigma, USA), was added

and then allowed to incubate at 45 8C for 15 min and the color produced as a

function of NO was read at 540 nm using a Multiskan microplate reader (Thermo

Electron Corporation, USA). The nitrite amount was calculated in comparison to the

standard nitrite curve and the results were expressed as nanomoles of nitrite

formed per milligram of protein.

2.6. Evaluation of mitochondrial function and oxidative stress

2.6.1. Mitochondria isolation procedure

Mitochondria were isolated by standard differential centrifugation (Pedersen

et al., 1978). The brain regions were homogenized in (1:10, w/v) ice cold isolation

buffer (250 mM sucrose, 1 mM EGTA and 10 mM HEPES–KOH, pH 7.2). Homo-

genates were centrifuged at 600 � g/5 min and the resulting supernatant was

centrifuged at 10,000 � g/15 min and supernatant discarded. Pellets were next

suspended in medium (1 ml) consisting of 250 mM sucrose, 0.3 mM EGTA and

10 mM HEPES–KOH, pH 7.2, and again centrifuged at 14,000 � g/10 min. All

centrifugation procedures were performed at 4 8C. The final mitochondrial pellet

was resuspended in medium (1 ml) containing 250 mM sucrose and 10 mM HEPES–

KOH, pH 7.2 and used within 3 h. Mitochondrial protein content was estimated

using the method of Lowry et al. (1951).

2.6.2. Estimation of mitochondrial malondialdehyde (MDA)

Mitochondrial MDA content was measured based on the TBA reaction test using

the method described by Uchiyama and Mihara (1978) and modified by Sunderman

et al. (1985). The chromophore MDA–TBA formed in the reaction was extracted into

organic layer and the absorbance was measured at 532 nm. The MDA concentra-

tions are expressed as micromoles of MDA per milligram of protein.

2.6.3. Estimation of mitochondrial superoxide dismutase (SOD) activity

The activity of superoxide: superoxide oxidoreductase (EC 1.15.1.1) was assayed

by the method of Kakkar et al. (1984) based on the formation of NADH–phenazine

methosulphate–nitro blue tetrazolium formazan measured at 560 nm against

butanol as blank. A system devoid of enzyme served as the control. A single unit of

the enzyme was expressed as 50% inhibition of NBT reduction per minute per

microgram of protein under the assay conditions.

2.6.4. Estimation of mitochondrial succinate dehydrogenase activity (SDH)

The mitochondrial succinate: acceptor oxidoreductase (EC 1.3.99.1) was

determined by standard protocol (Sally and Margaret, 1989) based on the

progressive reduction of NBT to an insoluble colored compound (a diformazan

(dfz)) used as a reaction indicator. The reaction of NBT was mediated by H+ released

in the conversion of succinate to fumarate. The concentration of NBT–dfz produced

was measured at 570 nm. The mean SDH activity of each region was expressed as

micromole formazan produced per min per microgram of protein.

2.6.5. Estimation of mitochondrial membrane potential (MMP)

The Rhodamine dye taken up by healthy mitochondria was measured

fluorimetrically as described by Shu-Gui (2002). Hitachi fluorescence spectropho-

tometer (model F-2500, Japan) was used. In brief, the mitochondrial suspension was

mixed with TMRM solution. The mixture was then incubated for 5 min at 25 8Ctemperature and any unbound TMRM was removed by frequent washings (four

times). Then the buffer was added to make up the final volume and florescence

emission was read at an excitation l 535 � 10 nm and emission l of 580 � 10 nm

using slit no. 10. The peak fluorescence intensity recorded was around 570 � 5 nm. The

results are expressed as fluorescence intensity value per milligram of protein.

2.7. Statistical analysis

The data were analyzed with GraphPad Prism version 4 (San Diego, CA).

Statistical analysis of data was done by One-way ANOVA, followed by Newman–

Keuls test. Data are expressed as mean � S.E.M. A level of p < 0.05 was accepted as

statistically significant.

3. Results

3.1. Effects of anoxia and ketamine on the brain glutamate

concentrations

Fig. 1A depicts the tissue glutamate concentration due toasphyxia and treatment with ketamine. On day 1 representing theprimary process, One-way ANOVA showed that there wassignificant differences in glutamate levels among groups incerebral-cortex [F (2, 15) = 32.30, p < 0.05], extra-cortex [F (2,15) = 5.998, p < 0.05] and cerebellum [F (2, 15) = 30.05, p < 0.05].Post hoc analysis showed that anoxia significantly increasedglutamate levels in cerebral-cortex, extra cortical and cerebellarregions. Ketamine treatment significantly reversed anoxia-in-duced increase in glutamate level in all the brain regions.

Similarly on the 7th day (Fig. 1B) after anoxia representing thesecondary process, One-way ANOVA showed that there wassignificant differences in glutamate levels among groups incerebrum [F (2, 15) = 39.51, p < 0.05], extra-cortex [F (2,15) = 5.307, p > 0.05] and cerebellum [F (2, 15) = 35.04,p > 0.05]. Post hoc analysis showed that anoxia significantlyincreased glutamate levels in cerebral-cortex, extra-cortex andcerebellum. However, ketamine reversed the anoxia-induced

Table 1Acute effect of perinatal asphyxia on neonatal brain mitochondrial MDA, SOD and

SDH levels in the rat pups.

Control Anoxic Ketamine treated

MDA levels (mM MDA/mg protein)

Cerebral-cortex 135.65�27.1 219.20�20.0* 72.69�24.4#

Extra-cortex 267.40�48.8 275.06�50.4 65.55�21.5*,#

Cerebellum 205.45�54.4 224.47�37.9 65.04�23.3*,#

SOD activity (units/min/mg protein)

Cerebral-cortex 0.0757�0.013 0.0899�0.006 0.0495� 0.012*,#

Extra-cortex 0.0587�0.007 0.0388�0.006* 0.0358� 0.006*

Cerebellum 0.0346�0.005 0.0303�0.005 0.0196� 0.003

SDH activity formazan produced (mM/min/mg protein)

Cerebral-cortex 4.31�0.7 4.01�0.4 3.31� 0.5

Extra-cortex 10.39�1.4 8.43�1.3* 2.56� 0.4*,#

Cerebellum 8.49�1.1 7.97�1.4* 4.16� 0.6*,#

Results are mean� S.E.M. values for effect of anoxia and ketamine on mitochondrial

MDA, SOD and SDH in cerebral-cortex, extra-cortex and cerebellum on day 1.* p<0.05 compared to control.# p<0.05 compared to anoxic control.

N.R. Reddy et al. / Neurochemistry International 58 (2011) 582–590 585

elevated glutamate levels only in the cerebral cortex but not ineither extra-cortex or cerebellum.

3.2. Effects of anoxia and ketamine on brain NO levels

NO production is thought to be enhanced due to glutamatemediated calcium influx during asphyxia. This NO reacts withsuperoxide radicals to form peroxynitrite which leads to cellulardamage. The results of NO (Fig. 2A) levels in primary processanalyzed by One-way ANOVA show significant differences inthe cerebral-cortex [F (2, 15) = 4.951, p < 0.05], extra-cortex [F

(2, 15) = 15.45, p < 0.05] and cerebellum [F (2, 15) = 58.84,p < 0.05]. Post hoc analysis revealed that anoxia significantlyincreased NO levels compared to control group and treatmentwith ketamine reversed these changes in all the three brainregions.

Fig. 2B depicts the changes in NO levels during the secondaryprocess. One-way ANOVA showed that in the cerebral-cortex [F (2,15) = 150.8, p < 0.05], extra-cortex [F (2, 15) = 42.30, p < 0.05] andcerebellum [F (2, 15) = 31.95, p < 0.05] there was a significantdifference in NO levels among groups. Post hoc analysis revealed asignificant increase in NO levels in anoxic group compared tocontrol in all the brain regions. Ketamine treatment reversedanoxic-induced increase in NO levels and interestingly, attenua-tion of NO levels by ketamine was significantly lower than thecontrol group.

Fig. 2. Effects of anoxia and ketamine on NO levels on day 1 and 7 are represented in

panel A and B respectively in cerebral-cortex, extra-cortex and cerebellum. Bars

represent data as mean� S.E.M., n = 6, *p < 0.05 compared to control and #p < 0.05

compared to anoxic control (One-way ANOVA followed by Student–Newman–Keuls test).

3.3. Effects of anoxia and ketamine on mitochondrial MDA levels

Mitochondrial MDA levels were estimated as an index of lipidperoxidation. From Table 1 representing the acute phase, One-wayANOVA showed that in cerebral-cortex [F (2, 15) = 9.353, p < 0.05],extra-cortex [F (2, 15) = 7.855, p < 0.05] and cerebellum [F (2,15) = 4.609, p < 0.05] there was significant differences in MDAlevels among groups (Table 1). Post hoc analysis showed thatanoxia significantly increased MDA levels compared to control.Ketamine treatment reversed anoxia-induced MDA levels in all thebrain regions.

In chronic study represented in Table 2, One-way ANOVAshowed that in cerebral-cortex [F (2, 15) = 3.793, p < 0.05], extra-cortex [F (2, 15) = 5.637, p < 0.05] and cerebellum [F (2,15) = 4.879, p < 0.05] there was significant differences in MDAlevels among groups. Post hoc analysis revealed that anoxiasignificantly increased MDA levels in the cerebellum, butdecreased the levels in the cerebral cortex. However, there wereno significant differences between MDA levels in the extra cortexcompared to control. Ketamine treatment increased MDA levelscompared to anoxia in the extra cortex but did not significantlychange the MDA levels in other brain regions.

3.4. Effect on the mitochondrial SOD activity

The changes in mitochondrial SOD activity as a measure ofmitochondrial anti-oxidant function during the primary processare represented in Table 1. Analysis by One-way ANOVA showed

Table 2Effect of perinatal asphyxia on day 7 on neonatal brain mitochondrial MDA, SOD

and SDH levels in the rat pups.

Control Anoxic Ketamine treated

MDA levels (mM MDA/mg protein)

Cereb-cortex 28.23�3.67 15.25�2.41* 20.916�3.77

Extra-cortex 19.29�1.80 15.61�1.83 28.16�3.94*,#

Cerebellum 20.69�1.89 39.94�4.61* 34.71�6.01*

SOD activity (units/min/mg protein)

Cereb-cortex 0.006� 0.001 0.005� 0.001 0.005� 0.001

Extra-cortex 0.008� 0.001 0.008� 0.002 0.006� 0.001

Cerebellum 0.008� 0.002 0.008� 0.002 0.008� 0.001

SDH activity formazan produced (mM/min/mg protein)

Cereb-cortex 2.10� 0.10 1.49� 0.24* 1.02� 0.19*

Extra-cortex 3.08� 0.27 2.24� 0.27* 1.46� 0.23*,#

Cerebellum 3.83� 0.20 2.91� 0.44* 2.08� 0.21*

Results are mean� S.E.M. values for effect of anoxia and ketamine on mitochondrial

MDA, SOD and SDH in cerebral-cortex, extra-cortex and cerebellum on day 7.* p<0.05 compared to control.# p<0.05 compared to anoxic control.

N.R. Reddy et al. / Neurochemistry International 58 (2011) 582–590586

that there was significant difference in the SOD activity in cerebral-cortex [F (2, 15) = 17.02, p < 0.05], extra cortex [F (2, 15) = 4.056,p < 0.05] but not in cerebellum [F (2, 15) = 2.804, p > 0.05] amonggroups. Post hoc analysis showed that during anoxia there wassignificant fall in SOD activity in the extra-cortex region comparedto control. Ketamine administration significantly decreased SODactivity in cerebellar cortex and extra-cortex compared to controlgroups. Further, in cerebral cortex it showed a significant decreasein SOD activity compared to anoxic groups.

Analysis of SOD activity on day 7 after anoxia (Table 2) by One-way ANOVA showed that there was no significant differenceamong groups in cerebral-cortex [F (2, 15) = 1.031, p > 0.05] inextra cortex [F (2, 15) = 1.789, p > 0.05] and in cerebellum [F (2,15) = 0.02355, p > 0.05]. This indicates that the anti-oxidantenzyme system remained unaltered during the secondary process.

3.5. Mitochondrial SDH activity in anoxia and ketamine treated

groups

The results of changes in SDH activity during the primary anoxicphase are depicted in Table 1. One-way ANOVA showed that therewas significant differences in SDH activity among groups in extracortex [F (2, 15) = 14.21, p < 0.05] and cerebellum [F (2,15) = 14.10, p < 0.05] but not in the cerebral-cortex [F (2,15) = 0.8371, p > 0.05]. Further, post hoc analysis of SDH activityin extra cortex and cerebellum regions revealed that SDH activitywas significantly compromised by anoxia compared to control.Ketamine administration could not reverse the changes in extra-

Fig. 3. Effects of anoxia and ketamine on MMP levels in cerebral-cortex, extra-

cortex and cerebellum on day 1 and day 7 are represented in panel A and B

respectively. Bars represent data as mean � S.E.M., n = 6, *p < 0.05 compared to

control and #p < 0.05 compared to anoxic control (One-way ANOVA followed by

Student–Newman–Keuls test).

cortex and cerebellum but in fact further significantly aggravatedloss of SDH activity compared to control

Analysis of SDH activity data on the 7th day of anoxia (Table 2)by One-way ANOVA showed that there was significant differencein activity among groups in cerebral-cortex [F (2, 15) = 8.418,p < 0.05], extra cortex [F (2, 15) = 10.09, p < 0.05] and cerebellum[F (2, 15) = 8.265, p < 0.05]. In contrast to the acute study, post hocanalysis revealed that both anoxia and ketamine significantlyreduced SDH activity compared to control group in all the brainregions. In extra-cortex ketamine treatment further deterioratedanoxia-induced impairment of SDH activity.

3.6. MMP in anoxia and ketamine treated groups

The changes in MMP values during the primary process areshown in Fig. 3A. One-way ANOVA showed that there was nosignificant differences in fluorescence emissions as a measure ofMMP among groups in cortex [F (2, 15) = 0.9308, p > 0.05], extra-cortex [F (2, 15) = 0.2863, p > 0.05] and cerebellum [F (2,15) = 1.599, p > 0.05]. This indicates that neither anoxia norketamine had any effect on MMP.

However in secondary process (Fig. 3B), One-way ANOVAshowed that there was significant differences in mitochondrialmembrane potential between groups in cerebral-cortex [F (2,15) = 9.012, p < 0.05], extra-cortex [F (2, 15) = 10.76, p < 0.05] andcerebellum [F (2, 15) = 8.832, p < 0.05]. In contrast to the acutestudy, post hoc analysis showed that anoxia significantlydecreased MMP in all the brain regions compared to control.Ketamine treatment did not reverse anoxia-induced decline inMMP in any of the brain regions under investigation. Interestingly,in extra-cortex, ketamine further aggravated the loss in anoxia-induced MMP compared to control.

4. Discussion

The present study was undertaken to investigate the dynamicrelationship between glutamate concentration and the mitochon-drial function in the progression of anoxia-induced neuronal injury.Salient findings of the study are the presence of elevated levels ofglutamate and nitric oxide both during primary and secondaryanoxic injury. Interestingly, we also observed discernable loss ofmitochondrial function and integrity in terms of decrease in SDHactivity and MMP respectively after 7 days of injury. Further, chronicketamine treatment did not reverse anoxia-induced mitochondrialdysfunction. Another interesting finding was that the anoxia-induced oxidative damage in terms of MDA and SOD had temporaland brain region specific effects. We presume that these may beimportant factors in determining the progress of secondary injuryand therapeutic effects of glutamate antagonists.

Three patterns of asphyxia are clinically observed based on thetime of exposure of the fetus to asphyxia. They are prenatal(exposure to asphyxia in the womb), post-natal (exposure outsidethe womb i.e. after delivery) and perinatal asphyxia (i.e. around thetime of delivery, it is preferably described as the exposure to insult 5months before delivery to 1 month after delivery). The experimentalanoxia induced in the present study represents post-natal asphyxia.However, the patterns of asphyxic injury in all the three formsshowed that the nature of injury depends not on the time of insultbut rather on type of hypoxic-ischemia (Sie et al., 2000).

4.1. Acute neonatal asphyxia raises tissue glutamate, NO and alters

mitochondrial oxidative status

We found that anoxic groups within 24 h (representing primaryprocess) of anoxia showed a significant increase in glutamate andNO levels in the cerebral cortex, extra cortex and cerebellum. This

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is in consistent with earlier reports that glutamate and NO levelsincrease acutely after neonatal asphyxia (Puka-Sundval et al.,1996; Tomris et al., 2005). Excitotoxicity is one of the importantinitial events leading to acute necrotic cell death after asphyxia(Maria et al., 1995). Depletion of oxygen and energy deficiency dueto anoxia leads to membrane trans-cellular ion pump failureresulting in intracellular calcium accumulation (Lina and Jeffrey,2004). Calcium accumulation causes neuronal depolarization andrelease of glutamate at nerve terminals (Jeffrey, 2006). Further,there is inhibition of glial uptake of released extracellularglutamate and also reversal of glutamate transport mechanisminto the glial cells due to energy deficiency (Phillisa et al., 2000;Ursula et al., 2002). NMDA receptor mediated intracellular Ca2+

accumulations trigger the formation of NO, reactive oxidativespecies (ROS) and other apoptotic factors’ formation (Valina et al.,1991; Leighton et al., 2001; Cacha et al., 2002; Tobaben et al.,2011). NO forms peroxynitrite in combination with free radicals tocause neuronal death by lipid peroxidation, protein nitrosylation,and energy depletion by inhibiting mitochondrial complex II–III(Juan et al., 1998, 1997; Angeles et al., 1998).

Predicatively, in the present study, there was an increase in thebrain mitochondrial oxidative damage in terms of increased MDAlevels shortly after anoxic injury. However, it is of interest to notethat oxidative damage was observed only in cerebral cortex andnot in extra cortex or cerebellum. Even though the MDA levelsincreased shortly after anoxia there was no concomitant increasein SOD and it was further decreased in extra-cortex region.Dismutation of superoxide into H2O2 which is then catalyzed toH2O and O2 is mediated by SOD and catalase respectively (Julio,2003). The imbalance between the formation of ROS andantioxidant defense mechanisms has been reported to lead tooxidative stress (Julio, 2003). Hence, decrease in SOD shows thatthe anti-oxidant mechanism is compromised in anoxia which canlead to further oxidative damage and necrotic cell death (Pak,2001). These results conjure well with the mechanistic pathwaywherein the primary phase related rise in glutamate concentrationcauses excess intracellular Ca2+ influxes leading to the activation ofcytoplasmic enzymes like phosphokinases, nitric oxide synthases.This then causes membrane lipid lysis, production of MDA,oxidative free radicals and NO (Cacha et al., 2002). These freeradicals damage the cellular and mitochondrial membranesleading to metabolic derangement, necrotic cell death and releaseof apoptotic factors. Blockade of glutamate receptors can limit thisdamage by inhibition of elevation in intracellular Ca2+ levels.

4.2. Acute phase shows alteration in mitochondrial SDH activity but

MMP remains intact

It is reported that in asphyxia the increase in oxidative stress isassociated with compromised mitochondrial function (Basavarajuet al., 2005). In the present study, increase in cortical mitochondrialoxidative stress during the primary phase translated into derangedmitochondrial function in terms of SDH activity. SDH forms a partof the complex-II mitochondrial membrane bound enzymesplaying a central role in neuronal energy metabolism as a partof respiratory chain and also in the tricarboxylic acid (TCA) cycle(James and Timothy, 1995; Bora et al., 2001). Decreased neuronalmitochondrial SDH activity has been reported with the use ofexcitotoxin, kainic acid. This action was reported to be mediatedthrough kainic acid-induced ROS production which inhibits thecatalytic function of SDH (Federica et al., 2001).

We observed no profound alteration in MMP due to anoxiaduring the first 24 h. MMP is the proton gradient formed due toelectron flow through a series of complex enzymes in the innermitochondrial membrane during the generation of ATP (Guidoet al., 2007). It regulates important events in the mitochondria such

as ATP synthesis, Ca2+ accumulation in the matrix and ROSgeneration (Tomohiro et al., 2005). Decline in MMP indicates lossof intactness of mitochondrial structure and cellular energyproduction. This decline is observed in necrotic or apoptotic cells(Gottlieb et al., 2003; Takehiko, 2006).

Hence, decline in SDH activity but not MMP acutely afteranoxia, indicates that though mitochondrial function was de-ranged, the membrane integrity was still maintained during thisperiod. Hence, we can assume that acute anoxia-induced gluta-mate and NO had mild detrimental effect on the mitochondrialfunction.

4.3. Delayed process shows rise in tissue glutamate and NO along with

mitochondrial dysfunction

In the study related to progression of anoxic-injury bysecondary process, we found that glutamate and NO levelsremained elevated even after 7 days of initial anoxic insult.However, in contrast to acute study, there was a decrease in MMPbesides the loss of SDH activity indicating loss of mitochondrialstructural integrity and function. Some studies have even reportedthat cells which survive primary insult recover their mitochondrialfunction including MMP and may proceed to secondary injury-induced cell death (Maria et al., 1995). It is interesting to note thatcerebellar MDA values which were normal immediately afteranoxic insult significantly increased in about a week. This showsthat although damage in the cerebral cortex was contained byendogenous antioxidant system, cerebellum is prone to long termdamage by free radicals. There were no significant changes in SODlevels in any of the three regions. These results indicate thenormalization of anti-oxidant system after the anoxic stress. It isalso interesting to note that in contrast to acute injury process,secondary increase in glutamate and NO was associated with lossof mitochondrial functional and structural derangement. Wepresume that the loss of mitochondrial function in anoxic injurymay be due to sustained increase in glutamate concentration.

On the other hand, mitochondrial function also appears tomodulate neuronal excitotoxicity. It is reported that the viableenergy dependent cellular mechanisms are necessary to generate aresting potential sufficient to maintain the voltage-dependentMg2+ block of the NMDA receptor channel. In case of compromisedenergy production there is relief from the Mg2+ block whichenables persistent activity of excitatory amino acids at the NMDAreceptor, resulting in the opening of ion channels and subsequentneuronal damage (Novelli et al., 1988). Since the energy productionis compromised in the secondary process, there may be furtherneuronal membrane depolarization and this coupled with lack ofglutamate metabolism may cause sustained rise in glutamatelevels. It has been reported that in a model of rodent perinatalanoxia, glutamate concentrations were consistently high up to 3months post asphyxia (Christina et al., 1999). It was also observedthat most of the neuronal death due to asphyxia occurred duringsecondary injury causing permanent alteration in the distinct brainregions (Elisabetta et al., 1997). It is possible that this reciprocalfeed forward cycle involving prolonged glutamate-induced mito-chondrial dysfunction and the mitochondrial dysfunction-inducedglutamate toxicity may be responsible for sustenance of anoxia-induced secondary damage.

4.4. Ketamine attenuates glutamate toxicity, but has no protective

effects on mitochondrial function in both acute and delayed processes

Only very small fraction of glutamate is present in the extracellular space even though high concentration is available in thebrain. As no extra cellular metabolism appears available forglutamate, regulation of glutamate concentration is through

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reuptake by transporters into the glia (Douglas et al., 1999; Gabrielet al., 2006). The extracellular glutamate in the glia is thenmetabolized to glutamine. This glutamine is transported back toneurons to be converted to glutamate, thereby completing theglutamate–glutamine cycle (Douglas et al., 1999). NMDA antago-nists such as ketamine do not appear to directly affect excitatoryamino acid transporters such as EAAT2 or EAAT3 (Young et al.,2003; Jung et al., 2006). More recently, low dose ketamineadministration in healthy volunteers was associated with in-creased glutamine levels in the prefrontal cortex indicatingincreased glutaminergic transmission (Laura et al., 2005). Asketamine on the contrary in the present study acutely decreasedglutamate, it is possible that it specifically interfered with anoxicmechanisms leading to excitotoxicity. It was found that ketaminesuppressed release of glutamate by hippocampal neurons in rat(Murata et al., 2000). Taking these facts together, it can be assumedthat the decrease in glutamate by ketamine during anoxia may berelated to its effect on neuronal polarity. Incidentally, it has alsobeen reported that in an animal model of schizophrenia, ketaminedecreased glutamate and other monoaminergic levels in thedentate gyrus and LY379268, an mGluR2/3 agonist selectivelyreversed glutamate levels but not other monoamines (Gabor et al.,2006). However, during the secondary process ketamine selec-tively decreased anoxia-induced glutamate levels only in thecerebral cortex.

The NO levels were decreased by ketamine in both primary andsecondary processes in all the brain regions. The decrease in NO byketamine can be attributed to inhibition of NOS activity due todecrease in NMDA-induced intracellular Ca2+ accumulations (Rueiet al., 2005). Ketamine was reported to inhibit NO production in amodel of cerebral ischemia by blocking NMDA receptors (Shinnet al., 1996).

The mitochondrial oxidative stress in terms of MDA wasdecreased by ketamine in acute process, but however wasincreased in extra-cortex and cerebellum over 7 days ofadministration. This perhaps shows that ketamine effect onoxidative stress is rather an indirect effect secondary to otherfactors. Ketamine decreased SOD activity during primary processbut did not alter its activity in the secondary process of anoxiaindicating lack of any significant effect on the mitochondrial anti-oxidant system. Ketamine profoundly decreased mitochondrialSDH activity during the primary process in extra-cortex andcerebellum but no discernible change in MMP was observed. Thisshows that even though ketamine altered mitochondrial functionin specific brain regions it did not translate into loss ofmitochondrial integrity. Consistent with our observations, keta-mine did not decrease MMP of neuronal cultures exposed toglutamate for 8 min (Sabine et al., 2000). However, duringsecondary process ketamine induced loss of SDH activity in allthe regions and had region specific effects on MMP in differentbrain regions. Even though during the secondary process ketaminedecreased glutamate levels in the cortex it did not translate intoreversal of anoxia-induced loss of mitochondrial function orintegrity. In the extra cortex, ketamine further aggravated loss ofMMP. This shows that glutamate antagonism alone would not beable to protect mitochondrial dysfunction during the secondaryprocess. In essence, ketamine did not have any beneficial effect onmitochondrial function or integrity in either primary or secondaryinjury.

Thus, ketamine acutely attenuated anoxia-induced increase inglutamate, NO and MDA levels in all brain regions indicatingmoderation of glutamate concentration and anti-oxidant effect.Repeated ketamine treatment similar to acute effect decreased NOlevels, but did not modulate SOD levels and interestingly showedregion specific effects on glutamate, MDA, SDH and MMP. It hasbeen reported that over stimulation of glutamate leads to oxidative

stress mediated mitochondrial membrane damage which leads toMPTP formation and loss of MMP (Anna et al., 2000; Bernd et al.,1998). This membrane damage leads to release of cytochrome-Cand other apoptotic factors which are responsible for delayed celldeath (Alejandro et al., 1996; Nickolay et al., 2002; Bennet et al.,2006). Loss of ATP supply and mitochondrial damage is reported tobe the primary cause of cell death by acute process in neuronalcultures exposed to glutamate (Maria et al., 1995). In neuronalculture studies use of glutamate antagonists in acute exposureshowed to rescue mitochondrial function by inhibition ofglutamate mediated excess calcium influx and generation ofROS and thereby cell death (Francesc et al., 1996; Atlante et al.,1997). However, in the present study ketamine decreased tissueglutamate but was unable to rescue mitochondrial damage. Thismay be due to the fact that physiological responses to acute anoxiaare limited in cell culture studies. It is also interesting to find thatuse of ketamine itself was detrimental to mitochondrial function.

These results show that during the anoxic primary injuryprocess the mitochondrial function was preserved in spite ofexcitotoxicity. In contrast, during the anoxic secondary injury therise in tissue glutamate concentration was associated withmitochondrial dysfunction and ketamine administration was notable to rescue this process. Though, preclinical studies on NMDAantagonist showed them to be promising agents for treatment ofanoxia and ischemia (Flint, 1992; Dorota et al., 2006), howeverthey were not successful in clinical trials (Michelle and Michael,2003). It is also interesting to note that increase in NO wasdecreased by ketamine in both acute and prolonged processesindicating decrease in oxidative stress. However, this did nottranslate into salvaging mitochondrial function contrary toprevious studies. We assume that concomitant preservation ofmitochondrial function and integrity along with glutamateantagonism may enhance the success of glutamate therapies inanoxic injury. Thus, we hypothesize from the present results thatdisruption of the bi-directional feed forward cycle between excessglutamate and mitochondrial dysfunction by use of drugs whichpreserve mitochondrial function can improve the treatment ofasphyxic injury.

Acknowledgement

NRR is thankful to University Grant Commission (UGC), NewDelhi, India, for the student fellowship.

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