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Neurochemistry International 58 (2011) 582590

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Neurochemistry International
journal homepage: www.elsevier.com/locate/neuint

Glutamate antagonism fails to reverse mitochondrial dysfunction in late phase of experimental neonatal asphyxia in rats
Nagannathahalli Ranga Reddy a, Sairam Krishnamurthy a,*, Tapan Kumar Chourasia b, Ashok Kumar c, Keerikkattil Paily Joy b
a b c

Neurotherapeutics Lab, Department of Pharmaceutics, Institute of Technology, Banaras Hindu University, Varanasi 221005, U.P., India Department of Zoology, Centre of Advanced Study, Faculty of Science, Banaras Hindu University, Varanasi 221005, U.P., India 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

A B S T R A C T

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

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 benecial in the treatment of asphyxia. 2011 Elsevier Ltd. All rights reserved.

1. Introduction Birth asphyxia is the failure to initiate and sustain breathing at birth and as per the WHO estimates, there are about 49 million cases of birth asphyxia every year with a mortality rate of 1.2 million children. The incidence rate is even higher in the developing countries due to high prevalence of risk factors in mother, lack of antenatal care and higher incidence of preterm babies (Batool and Zulqar, 2006; Kolatat et al., 2000). As per Indian 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 of decient oxygen supply and this occurring at the time of birth causes severe damage especially to the brain besides other major organs. The injury occurs due to lack of perfusion, energy failure, glutamate excitotoxicity and also subsequent reperfusion (Suresh et al., 2007). The physiological outcomes of anoxia or hypoxia are collectively termed as asphyxia (Roy and Stefan, 2008). Asphyxia is considered to be an important cause of neonatal brain damage (Luca et al., 2006; Jeffrey, 2006) leading to neurodevelopmental abnormalities like mental retardation, learning decits, epilepsy and cerebral palsy in adult life (Jacques et al., 2008; Van-de et al., 2000). These survivors may even require long-term rehabilitation which is expensive and complex (Blomgren et al., 2003). Treatment of neonatal asphyxia involves use of non-pharmacological and pharmacological interventions. Non-pharmacological

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interventions include cleaning of air ways, resuscitation and hypothermia. Mild-to-moderate hypothermia is said to be protective by decreasing metabolic rate, excitatory amino acid release and also decreasing toxic free radical production (Batool and Zulqar, 2006). Pharmacological interventions include use of modulators of glutamate, monoamines (dopamine, nor epinephrine and serotonin), GABA, adenosine and growth factors (Suresh et al., 2007). Ca2+ overload inhibitors and anti-oxidants have also been considered as treatment options for asphyxia (Suresh et al., 2007). Of all the pharmacological measures available, over the recent past glutamate, antagonism has generated much interest as excitotoxicity is considered to be one of the major pathways leading to neuronal damage due to asphyxia (Keith, 2006). Non-competitive NMDA receptor antagonists such as MK 801 and ketamine were effective as neuroprotectants in reducing the brain damage in experimental models of asphyxia (Eric and Henrik, 1997; Foster et al., 1988; Alkan et al., 2001). Further studies with selective glutamate receptor antagonists like mGluR1 and mGluR5 proved to be even more effective (Domenico et al., 1999; Dorota et al., 2006). Surprisingly, in clinical trials these drugs were ineffective and showed severe side effects limiting their use in the treatment of asphyxia (James, 1992; Chen and Lipton, 2006). Brain injury due to asphyxia appears to progress in two distinct phases, 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 by apoptosis (delayed cell death) late after the initial insult (Elisabetta et al., 1997; Kenneth et al., 2000; Rajiv et al., 2001). However, most of the studies have focused exhaustively on acute injury. The studies of acute injury concerning the role and moderation of glutamate and other associated factors such as MPTP (mitochondrial membrane permeability transition pore), calcium overload and oxidative stress have been found to be effective in treatment of asphyxia in the experimental models (Evangelia et al., 1999; Thomas et al., 2002; Akihito et al., 2004;). Clinical studies have shown that severe perinatal anoxic injury is associated with delayed development of cerebral energy failure from 6 to 15 h after birth asphyxic insult (Alistair and Peter, 1998; Vannucci et al., 2004). Apparent recovery after an episode of transient ischemia is often followed by cell death after 24 h (Takaaki, 1982; Hori and Carpenter, 1994; Carol et al., 1997; Vannucci et al., 2004). These ndings suggest concomitant energy failure and metabolic derangement late after asphyxic insult. Hence, treatment may have to be continued throughout the recovery phase to get a benecial response. Consistent with the above observations delayed treatment with glutamate antagonist MK-801 after post ischemic hypothermia was found to rescue normal CA1 neurons in the hippocampus (Dietrich et al., 1995). However, very few studies have evaluated the delayed or chronic consequences of asphyxia in in vivo models which is considered to be the phase for pharmacological intervention (Ze et al., 2003; Gunn et al., 2005). Hence, evaluation of glutamate antagonism extending into the secondary injury process may reveal the causes for failure of glutamate treatment and provide further clues for effective treatment. In asphyxic injury, mitochondrial alterations are brought about by 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 cell death due to the production of free radicals, MPTP formation, calcium inux and energy failure (David and Samantha, 1998; Tobaben et al., 2011). As the growth and regeneration of neurons require mitochondrial energy (Robert and Peter, 1993; Zheng et al., 2004), possible treatment reversing mitochondrial dysfunction associated with asphyxia may prove to be effective. The long-term reciprocal relationship between glutamate release and mitochon-

drial damage in asphyxia has not been studied extensively. However, many studies have evaluated the role of mitochondria in cell death mediated through glutamate in acute process either in animal models or on neuronal cultures (Juan et al., 1998; Angeles et al., 1998; Tobaben et al., 2011). The present study evaluates the role of tissue glutamate and associated factors with mitochondrial function both during primary and secondary processes of asphyxic injury to understand the dynamic relationship between them. The modulation of glutamate activity and its role on mitochondrial function in both acute and delayed neuronal deaths were evaluated using ketamine a non-competitive glutamate antagonist in a rat model of neonatal anoxia.
2. Materials and methods 2.1. Animals Charles Foster albino pregnant rats (180220 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 8C and 4555% 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 8C till further experimentation. 2.2. Anoxic procedure A slightly modied 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 ow 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 SigmaAldrich (St. Louis, MO, USA). Thiobarbituric acid (TBA), ethylene glycol tetra-acetic acid (EGTA), 2-[4-(2hydroxyethyl)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 electrochemical 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

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were individually ltered through a 0.22 mm syringe lter 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 ow 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). Briey, 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 HEPESKOH, pH 7.2). Homogenates 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 HEPESKOH, pH 7.2, and again centrifuged at 14,000 g/10 min. All centrifugation procedures were performed at 4 8C. The nal 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 modied by Sunderman et al. (1985). The chromophore MDATBA formed in the reaction was extracted into organic layer and the absorbance was measured at 532 nm. The MDA concentrations 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 NADHphenazine methosulphatenitro 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 NBTdfz 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 uorimetrically as described by Shu-Gui (2002). Hitachi uorescence spectrophotometer (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 8C temperature and any unbound TMRM was removed by frequent washings (four times). Then the buffer was added to make up the nal volume and orescence emission was read at an excitation l 535 10 nm and emission l of 580 10 nm using slit no. 10. The peak uorescence intensity recorded was around 570 5 nm. The results are expressed as uorescence 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

3. Results 3.1. Effects of anoxia and ketamine on the brain glutamate concentrations Fig. 1A depicts the tissue glutamate concentration due to asphyxia and treatment with ketamine. On day 1 representing the primary process, One-way ANOVA showed that there was signicant differences in glutamate levels among groups in cerebral-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 signicantly increased glutamate levels in cerebral-cortex, extra cortical and cerebellar regions. Ketamine treatment signicantly reversed anoxia-induced increase in glutamate level in all the brain regions. Similarly on the 7th day (Fig. 1B) after anoxia representing the secondary process, One-way ANOVA showed that there was signicant differences in glutamate levels among groups in cerebrum [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 signicantly increased glutamate levels in cerebral-cortex, extra-cortex and cerebellum. However, ketamine reversed the anoxia-induced

Fig. 1. Effects of anoxia and ketamine on glutamate levels in cerebral-cortex (Cerebcortex), 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 (Oneway ANOVA followed by StudentNewmanKeuls test).

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elevated glutamate levels only in the cerebral cortex but not in either extra-cortex or cerebellum. 3.2. Effects of anoxia and ketamine on brain NO levels NO production is thought to be enhanced due to glutamate mediated calcium inux during asphyxia. This NO reacts with superoxide radicals to form peroxynitrite which leads to cellular damage. The results of NO (Fig. 2A) levels in primary process analyzed by One-way ANOVA show signicant differences in the 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 signicantly increased NO levels compared to control group and treatment with ketamine reversed these changes in all the three brain regions. Fig. 2B depicts the changes in NO levels during the secondary process. 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] and cerebellum [F (2, 15) = 31.95, p < 0.05] there was a signicant difference in NO levels among groups. Post hoc analysis revealed a signicant increase in NO levels in anoxic group compared to control in all the brain regions. Ketamine treatment reversed anoxic-induced increase in NO levels and interestingly, attenuation of NO levels by ketamine was signicantly lower than the control group.

Table 1 Acute effect of perinatal asphyxia on neonatal brain mitochondrial MDA, SOD and SDH levels in the rat pups. Control Anoxic Ketamine treated 72.69 24.4# 65.55 21.5*,# 65.04 23.3*,# 0.0495 0.012*,# 0.0358 0.006* 0.0196 0.003 3.31 0.5 2.56 0.4*,# 4.16 0.6*,#

MDA levels (mM MDA/mg protein) Cerebral-cortex 135.65 27.1 219.20 20.0* Extra-cortex 267.40 48.8 275.06 50.4 Cerebellum 205.45 54.4 224.47 37.9 SOD activity (units/min/mg protein) Cerebral-cortex 0.0757 0.013 0.0899 0.006 Extra-cortex 0.0587 0.007 0.0388 0.006* Cerebellum 0.0346 0.005 0.0303 0.005 SDH activity formazan produced (mM/min/mg protein) Cerebral-cortex 4.31 0.7 4.01 0.4 Extra-cortex 10.39 1.4 8.43 1.3* Cerebellum 8.49 1.1 7.97 1.4*

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.

3.3. Effects of anoxia and ketamine on mitochondrial MDA levels Mitochondrial MDA levels were estimated as an index of lipid peroxidation. From Table 1 representing the acute phase, One-way ANOVA 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 signicant differences in MDA levels among groups (Table 1). Post hoc analysis showed that anoxia signicantly increased MDA levels compared to control. Ketamine treatment reversed anoxia-induced MDA levels in all the brain regions. In chronic study represented in Table 2, One-way ANOVA showed that in cerebral-cortex [F (2, 15) = 3.793, p < 0.05], extracortex [F (2, 15) = 5.637, p < 0.05] and cerebellum [F (2, 15) = 4.879, p < 0.05] there was signicant differences in MDA levels among groups. Post hoc analysis revealed that anoxia signicantly increased MDA levels in the cerebellum, but decreased the levels in the cerebral cortex. However, there were no signicant differences between MDA levels in the extra cortex compared to control. Ketamine treatment increased MDA levels compared to anoxia in the extra cortex but did not signicantly change the MDA levels in other brain regions. 3.4. Effect on the mitochondrial SOD activity The changes in mitochondrial SOD activity as a measure of mitochondrial anti-oxidant function during the primary process are represented in Table 1. Analysis by One-way ANOVA showed
Table 2 Effect of perinatal asphyxia on day 7 on neonatal brain mitochondrial MDA, SOD and SDH levels in the rat pups. Control Anoxic Ketamine treated 20.916 3.77 28.16 3.94*,# 34.71 6.01* 0.005 0.001 0.006 0.001 0.008 0.001 1.02 0.19* 1.46 0.23*,# 2.08 0.21*

MDA levels (mM MDA/mg protein) Cereb-cortex 28.23 3.67 15.25 2.41* Extra-cortex 19.29 1.80 15.61 1.83 Cerebellum 20.69 1.89 39.94 4.61* SOD activity (units/min/mg protein) Cereb-cortex 0.006 0.001 0.005 0.001 Extra-cortex 0.008 0.001 0.008 0.002 Cerebellum 0.008 0.002 0.008 0.002 SDH activity formazan produced (mM/min/mg protein) Cereb-cortex 2.10 0.10 1.49 0.24* Extra-cortex 3.08 0.27 2.24 0.27* Cerebellum 3.83 0.20 2.91 0.44* 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 StudentNewmanKeuls test).

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.

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that there was signicant difference in the SOD activity in cerebralcortex [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] among groups. Post hoc analysis showed that during anoxia there was signicant fall in SOD activity in the extra-cortex region compared to control. Ketamine administration signicantly decreased SOD activity in cerebellar cortex and extra-cortex compared to control groups. Further, in cerebral cortex it showed a signicant decrease in SOD activity compared to anoxic groups. Analysis of SOD activity on day 7 after anoxia (Table 2) by Oneway ANOVA showed that there was no signicant difference among groups in cerebral-cortex [F (2, 15) = 1.031, p > 0.05] in extra 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-oxidant enzyme 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 anoxic phase are depicted in Table 1. One-way ANOVA showed that there was signicant differences in SDH activity among groups in extra cortex [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 activity in extra cortex and cerebellum regions revealed that SDH activity was signicantly compromised by anoxia compared to control. Ketamine administration could not reverse the changes in extra-

cortex and cerebellum but in fact further signicantly aggravated loss 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 signicant difference in 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 hoc analysis revealed that both anoxia and ketamine signicantly reduced SDH activity compared to control group in all the brain regions. In extra-cortex ketamine treatment further deteriorated anoxia-induced impairment of SDH activity. 3.6. MMP in anoxia and ketamine treated groups The changes in MMP values during the primary process are shown in Fig. 3A. One-way ANOVA showed that there was no signicant differences in uorescence emissions as a measure of MMP among groups in cortex [F (2, 15) = 0.9308, p > 0.05], extracortex [F (2, 15) = 0.2863, p > 0.05] and cerebellum [F (2, 15) = 1.599, p > 0.05]. This indicates that neither anoxia nor ketamine had any effect on MMP. However in secondary process (Fig. 3B), One-way ANOVA showed that there was signicant differences in mitochondrial membrane potential between groups in cerebral-cortex [F (2, 15) = 9.012, p < 0.05], extra-cortex [F (2, 15) = 10.76, p < 0.05] and cerebellum [F (2, 15) = 8.832, p < 0.05]. In contrast to the acute study, post hoc analysis showed that anoxia signicantly decreased MMP in all the brain regions compared to control. Ketamine treatment did not reverse anoxia-induced decline in MMP in any of the brain regions under investigation. Interestingly, in extra-cortex, ketamine further aggravated the loss in anoxiainduced MMP compared to control. 4. Discussion The present study was undertaken to investigate the dynamic relationship between glutamate concentration and the mitochondrial function in the progression of anoxia-induced neuronal injury. Salient ndings of the study are the presence of elevated levels of glutamate and nitric oxide both during primary and secondary anoxic injury. Interestingly, we also observed discernable loss of mitochondrial function and integrity in terms of decrease in SDH activity and MMP respectively after 7 days of injury. Further, chronic ketamine treatment did not reverse anoxia-induced mitochondrial dysfunction. Another interesting nding was that the anoxiainduced oxidative damage in terms of MDA and SOD had temporal and brain region specic effects. We presume that these may be important factors in determining the progress of secondary injury and therapeutic effects of glutamate antagonists. Three patterns of asphyxia are clinically observed based on the time of exposure of the fetus to asphyxia. They are prenatal (exposure to asphyxia in the womb), post-natal (exposure outside the womb i.e. after delivery) and perinatal asphyxia (i.e. around the time of delivery, it is preferably described as the exposure to insult 5 months before delivery to 1 month after delivery). The experimental anoxia induced in the present study represents post-natal asphyxia. However, the patterns of asphyxic injury in all the three forms showed that the nature of injury depends not on the time of insult but rather on type of hypoxic-ischemia (Sie et al., 2000). 4.1. Acute neonatal asphyxia raises tissue glutamate, NO and alters mitochondrial oxidative status

Fig. 3. Effects of anoxia and ketamine on MMP levels in cerebral-cortex, extracortex 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 StudentNewmanKeuls test).

We found that anoxic groups within 24 h (representing primary process) of anoxia showed a signicant increase in glutamate and NO levels in the cerebral cortex, extra cortex and cerebellum. This

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is in consistent with earlier reports that glutamate and NO levels increase acutely after neonatal asphyxia (Puka-Sundval et al., 1996; Tomris et al., 2005). Excitotoxicity is one of the important initial events leading to acute necrotic cell death after asphyxia (Maria et al., 1995). Depletion of oxygen and energy deciency due to anoxia leads to membrane trans-cellular ion pump failure resulting in intracellular calcium accumulation (Lina and Jeffrey, 2004). Calcium accumulation causes neuronal depolarization and release of glutamate at nerve terminals (Jeffrey, 2006). Further, there is inhibition of glial uptake of released extracellular glutamate and also reversal of glutamate transport mechanism into the glial cells due to energy deciency (Phillisa et al., 2000; Ursula et al., 2002). NMDA receptor mediated intracellular Ca2+ accumulations trigger the formation of NO, reactive oxidative species (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 to cause neuronal death by lipid peroxidation, protein nitrosylation, and energy depletion by inhibiting mitochondrial complex IIIII (Juan et al., 1998, 1997; Angeles et al., 1998). Predicatively, in the present study, there was an increase in the brain mitochondrial oxidative damage in terms of increased MDA levels shortly after anoxic injury. However, it is of interest to note that oxidative damage was observed only in cerebral cortex and not in extra cortex or cerebellum. Even though the MDA levels increased shortly after anoxia there was no concomitant increase in SOD and it was further decreased in extra-cortex region. Dismutation of superoxide into H2O2 which is then catalyzed to H2O and O2 is mediated by SOD and catalase respectively (Julio, 2003). The imbalance between the formation of ROS and antioxidant defense mechanisms has been reported to lead to oxidative stress (Julio, 2003). Hence, decrease in SOD shows that the anti-oxidant mechanism is compromised in anoxia which can lead to further oxidative damage and necrotic cell death (Pak, 2001). These results conjure well with the mechanistic pathway wherein the primary phase related rise in glutamate concentration causes excess intracellular Ca2+ inuxes leading to the activation of cytoplasmic 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 free radicals damage the cellular and mitochondrial membranes leading to metabolic derangement, necrotic cell death and release of apoptotic factors. Blockade of glutamate receptors can limit this damage 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 is associated with compromised mitochondrial function (Basavaraju et al., 2005). In the present study, increase in cortical mitochondrial oxidative stress during the primary phase translated into deranged mitochondrial function in terms of SDH activity. SDH forms a part of the complex-II mitochondrial membrane bound enzymes playing a central role in neuronal energy metabolism as a part of respiratory chain and also in the tricarboxylic acid (TCA) cycle (James and Timothy, 1995; Bora et al., 2001). Decreased neuronal mitochondrial SDH activity has been reported with the use of excitotoxin, kainic acid. This action was reported to be mediated through kainic acid-induced ROS production which inhibits the catalytic function of SDH (Federica et al., 2001). We observed no profound alteration in MMP due to anoxia during the rst 24 h. MMP is the proton gradient formed due to electron ow through a series of complex enzymes in the inner mitochondrial membrane during the generation of ATP (Guido et al., 2007). It regulates important events in the mitochondria such

as ATP synthesis, Ca2+ accumulation in the matrix and ROS generation (Tomohiro et al., 2005). Decline in MMP indicates loss of intactness of mitochondrial structure and cellular energy production. This decline is observed in necrotic or apoptotic cells (Gottlieb et al., 2003; Takehiko, 2006). Hence, decline in SDH activity but not MMP acutely after anoxia, indicates that though mitochondrial function was deranged, the membrane integrity was still maintained during this period. Hence, we can assume that acute anoxia-induced glutamate and NO had mild detrimental effect on the mitochondrial function. 4.3. Delayed process shows rise in tissue glutamate and NO along with mitochondrial dysfunction In the study related to progression of anoxic-injury by secondary process, we found that glutamate and NO levels remained elevated even after 7 days of initial anoxic insult. However, in contrast to acute study, there was a decrease in MMP besides the loss of SDH activity indicating loss of mitochondrial structural integrity and function. Some studies have even reported that cells which survive primary insult recover their mitochondrial function including MMP and may proceed to secondary injuryinduced cell death (Maria et al., 1995). It is interesting to note that cerebellar MDA values which were normal immediately after anoxic insult signicantly increased in about a week. This shows that although damage in the cerebral cortex was contained by endogenous antioxidant system, cerebellum is prone to long term damage by free radicals. There were no signicant changes in SOD levels in any of the three regions. These results indicate the normalization of anti-oxidant system after the anoxic stress. It is also interesting to note that in contrast to acute injury process, secondary increase in glutamate and NO was associated with loss of mitochondrial functional and structural derangement. We presume that the loss of mitochondrial function in anoxic injury may be due to sustained increase in glutamate concentration. On the other hand, mitochondrial function also appears to modulate neuronal excitotoxicity. It is reported that the viable energy dependent cellular mechanisms are necessary to generate a resting potential sufcient to maintain the voltage-dependent Mg2+ block of the NMDA receptor channel. In case of compromised energy production there is relief from the Mg2+ block which enables persistent activity of excitatory amino acids at the NMDA receptor, resulting in the opening of ion channels and subsequent neuronal damage (Novelli et al., 1988). Since the energy production is compromised in the secondary process, there may be further neuronal membrane depolarization and this coupled with lack of glutamate metabolism may cause sustained rise in glutamate levels. It has been reported that in a model of rodent perinatal anoxia, glutamate concentrations were consistently high up to 3 months post asphyxia (Christina et al., 1999). It was also observed that most of the neuronal death due to asphyxia occurred during secondary injury causing permanent alteration in the distinct brain regions (Elisabetta et al., 1997). It is possible that this reciprocal feed forward cycle involving prolonged glutamate-induced mitochondrial dysfunction and the mitochondrial dysfunction-induced glutamate toxicity may be responsible for sustenance of anoxiainduced 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 extra cellular space even though high concentration is available in the brain. As no extra cellular metabolism appears available for glutamate, regulation of glutamate concentration is through

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reuptake by transporters into the glia (Douglas et al., 1999; Gabriel et al., 2006). The extracellular glutamate in the glia is then metabolized to glutamine. This glutamine is transported back to neurons to be converted to glutamate, thereby completing the glutamateglutamine cycle (Douglas et al., 1999). NMDA antagonists such as ketamine do not appear to directly affect excitatory amino acid transporters such as EAAT2 or EAAT3 (Young et al., 2003; Jung et al., 2006). More recently, low dose ketamine administration in healthy volunteers was associated with increased glutamine levels in the prefrontal cortex indicating increased glutaminergic transmission (Laura et al., 2005). As ketamine on the contrary in the present study acutely decreased glutamate, it is possible that it specically interfered with anoxic mechanisms leading to excitotoxicity. It was found that ketamine suppressed release of glutamate by hippocampal neurons in rat (Murata et al., 2000). Taking these facts together, it can be assumed that the decrease in glutamate by ketamine during anoxia may be related to its effect on neuronal polarity. Incidentally, it has also been reported that in an animal model of schizophrenia, ketamine decreased glutamate and other monoaminergic levels in the dentate gyrus and LY379268, an mGluR2/3 agonist selectively reversed glutamate levels but not other monoamines (Gabor et al., 2006). However, during the secondary process ketamine selectively decreased anoxia-induced glutamate levels only in the cerebral cortex. The NO levels were decreased by ketamine in both primary and secondary processes in all the brain regions. The decrease in NO by ketamine can be attributed to inhibition of NOS activity due to decrease in NMDA-induced intracellular Ca2+ accumulations (Ruei et al., 2005). Ketamine was reported to inhibit NO production in a model of cerebral ischemia by blocking NMDA receptors (Shinn et al., 1996). The mitochondrial oxidative stress in terms of MDA was decreased by ketamine in acute process, but however was increased in extra-cortex and cerebellum over 7 days of administration. This perhaps shows that ketamine effect on oxidative stress is rather an indirect effect secondary to other factors. Ketamine decreased SOD activity during primary process but did not alter its activity in the secondary process of anoxia indicating lack of any signicant effect on the mitochondrial antioxidant system. Ketamine profoundly decreased mitochondrial SDH activity during the primary process in extra-cortex and cerebellum but no discernible change in MMP was observed. This shows that even though ketamine altered mitochondrial function in specic brain regions it did not translate into loss of mitochondrial integrity. Consistent with our observations, ketamine did not decrease MMP of neuronal cultures exposed to glutamate for 8 min (Sabine et al., 2000). However, during secondary process ketamine induced loss of SDH activity in all the regions and had region specic effects on MMP in different brain regions. Even though during the secondary process ketamine decreased glutamate levels in the cortex it did not translate into reversal of anoxia-induced loss of mitochondrial function or integrity. In the extra cortex, ketamine further aggravated loss of MMP. This shows that glutamate antagonism alone would not be able to protect mitochondrial dysfunction during the secondary process. In essence, ketamine did not have any benecial effect on mitochondrial function or integrity in either primary or secondary injury. Thus, ketamine acutely attenuated anoxia-induced increase in glutamate, NO and MDA levels in all brain regions indicating moderation of glutamate concentration and anti-oxidant effect. Repeated ketamine treatment similar to acute effect decreased NO levels, but did not modulate SOD levels and interestingly showed region specic effects on glutamate, MDA, SDH and MMP. It has been reported that over stimulation of glutamate leads to oxidative

stress mediated mitochondrial membrane damage which leads to MPTP formation and loss of MMP (Anna et al., 2000; Bernd et al., 1998). This membrane damage leads to release of cytochrome-C and other apoptotic factors which are responsible for delayed cell death (Alejandro et al., 1996; Nickolay et al., 2002; Bennet et al., 2006). Loss of ATP supply and mitochondrial damage is reported to be the primary cause of cell death by acute process in neuronal cultures exposed to glutamate (Maria et al., 1995). In neuronal culture studies use of glutamate antagonists in acute exposure showed to rescue mitochondrial function by inhibition of glutamate mediated excess calcium inux and generation of ROS and thereby cell death (Francesc et al., 1996; Atlante et al., 1997). However, in the present study ketamine decreased tissue glutamate but was unable to rescue mitochondrial damage. This may be due to the fact that physiological responses to acute anoxia are limited in cell culture studies. It is also interesting to nd that use of ketamine itself was detrimental to mitochondrial function. These results show that during the anoxic primary injury process the mitochondrial function was preserved in spite of excitotoxicity. In contrast, during the anoxic secondary injury the rise in tissue glutamate concentration was associated with mitochondrial dysfunction and ketamine administration was not able to rescue this process. Though, preclinical studies on NMDA antagonist showed them to be promising agents for treatment of anoxia and ischemia (Flint, 1992; Dorota et al., 2006), however they were not successful in clinical trials (Michelle and Michael, 2003). It is also interesting to note that increase in NO was decreased by ketamine in both acute and prolonged processes indicating decrease in oxidative stress. However, this did not translate into salvaging mitochondrial function contrary to previous studies. We assume that concomitant preservation of mitochondrial function and integrity along with glutamate antagonism may enhance the success of glutamate therapies in anoxic injury. Thus, we hypothesize from the present results that disruption of the bi-directional feed forward cycle between excess glutamate and mitochondrial dysfunction by use of drugs which preserve mitochondrial function can improve the treatment of asphyxic injury. Acknowledgement NRR is thankful to University Grant Commission (UGC), New Delhi, India, for the student fellowship. References
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