J Bioenerg Biomembr (2015) 47:217–222

DOI 10.1007/s10863-015-9603-2

Characterization of mitochondrial bioenergetics

in neonatal anoxic model of rats
Puneet K. Samaiya & Sairam Krishnamurthy

Received: 20 September 2014 / Accepted: 19 January 2015 / Published online: 31 January 2015
# Springer Science+Business Media New York 2015

Abstract Neonatal anoxia at the time of birth can lead to Introduction

mitochondrial dysfunction and further neurodevelopmental
abnormalities. The present study investigated the mitochon- Perinatal asphyxia is a condition arising due to severe depri-
drial bioenergetics and associated sensorimotor changes in the vation of oxygen at the time of birth which may contribute to
anoxic neonatal rats. Rat pups after 30 h to birth (2 days) were permanent neurodevelopmental disability. It is considered as a
subjected to anoxia of two episodes (10 min in each) at a time global clinical problem. It has been implicated to cause motor
interval of 24 h by passing 100 % N2 into an enclosed cham- and cognitive alterations in neonates of variable severity like
ber. Brain mitochondrial respiration was measured using clark cerebral palsy, epilepsy, dystonia in the early age and attention
type oxygen electrode. A significant decrease in brain respi- deficit disorder, mental retardation and other neuropsychiatric
ratory control ratio (RCR; State III/IV respiration) at all-time syndromes at school age (Herrera-Marschitz et al. 2014; Strata
points, complex I (24 h) and complex II (30 min, 6 and 24 h) et al. 2004).
enzyme activities indicated loss of mitochondrial integrity and Brain being an active metabolizing tissue requires adequate
function A significant increase in levels of nitric oxide was provision of glucose and oxygen to maintain its normal func-
observed after second anoxic episode at all-time points. A tion. This critical requirement is compromised during anoxic
significant change in sensorimotor activity in terms of in- injury triggering cascade of biochemical events which lead to
creased reflex latency was observed 24 h after second episode excitotoxicity and influx of Ca2+ ions. This results in overpro-
in this model, which is an indication of loss of subcortical duction of nitric oxide (NO) apart from other reactive oxygen
maturation. All the above changes were observed after second species (ROS) within the mitochondria (Magistretti and
but not after the first anoxic exposure. Therefore, this anoxic Allaman 2013; Sun and Gilboe 1994; Herrera-Marschitz
model shows significant changes in mitochondrial bioenerget- et al. 2014). These mitochondrial events lead to opening of
ics, nitric oxide levels and sensorimotor effects after second membrane permeability transition pore and release of pro-
episode of anoxia. This model may be helpful to evaluate apoptotic proteins like cytochrome c which ultimately cause
mitochondrial targeted pharmacological intervention for the mitochondrial dysfunction and cell death (Morin et al. 2003).
treatment of anoxia. Brain injury as a result of asphyxia progressively appears in
two distinct phases, primarily as an initial phase which may
result due to necrotic cell death and secondarily the latter
Keywords Neonatal anoxia . Animal model . Mitochondrial phase due to apoptotic cell death. Thus, mitochondria may
bioenergetics . Nitric oxide . Sensorimotor activity be an important target for the progression of primary anoxic
injury into secondary injury. Therefore, energy deficiency due
P. K. Samaiya : S. Krishnamurthy (*)
to mitochondrial dysfunction plays a key role in post anoxic
Department of Pharmaceutics, Neurotherapeutics laboratory, Indian brain damage in newborns (Bolaños et al. 1998; Reddy et al.
Institute of Technology (Banaras Hindu University), 2011; Rosenberg et al. 1989).
Varanasi 221005, India In the present study, we have used the non-invasive two
e-mail: [email protected]
episodic model of anoxia in neonatal rats to study the role of
S. Krishnamurthy mitochondrial bioenergetics in asphyxia (Reddy et al. 2011).
e-mail: [email protected] This method is better optimized than other similar anoxic
218 J Bioenerg Biomembr (2015) 47:217–222

models as it uses two subsequent episodes of anoxia rather respiration and locomotor behavior for 5 min after removal
than a single prolonged episode. Two episodes increase the from the chamber, the animals were returned to the dams and
survival rate of neonates by decreasing the length of anoxic killed at different time points (30 min, 6 and 24 h respectively)
exposure. Several asphyxia models were evaluated for mito- following first and second anoxic exposure. The same experi-
chondrial functions but none of those have characterized mi- mental procedure was followed for the control group but the
tochondrial bioenergetics in terms of oxygen consumption in chamber contained air instead of nitrogen. The brains were
different states after anoxic exposure (Bolaños et al. 1998; rapidly dissected out and immediately used for mitochondrial
Puka-Sundvall et al. 2000; Rosenberg et al. 1989). We for isolation. Mitochondrial respiration studies were performed im-
the first time evaluate mitochondrial bioenergetics in the two mediately after mitochondrial isolation. Further, mitochondrial
episodic model of anoxia. pellets were stored at −80 ° C for nitrite estimation.
Thus, the present study characterizes the mitochondrial
bioenergetics in the two distinct episodes of anoxia. The re- Materials
sults of the current study may lead to precise understanding of
substrates involved in mitochondrial dysfunction and there- Reagents—Mannitol, sucrose, bovine serum albumin (BSA),
fore be helpful to evaluate mitochondrial targeted pharmaco- EGTA, HEPES potassium salt (St. Louis, MO, USA), potas-
logical intervention for the treatment of anoxia. sium phosphate monobasic anhydrous (KH2PO4), MgCl2,
malate, pyruvate, ADP, succinate, oligomycin, carbonyl cya-
nide 4-(trifluoromethoxy) phenylhydrazone (FCCP), Rote-
Material and methods none and Griess reagent were procured from Sigma-Aldrich.

Animals Behavioral parameter assessment

All experiments were conducted in accordance with the princi- Righting reflex: The assessment was done by placing the pups
ples of laboratory animal care (NIH publication number 85–23, on their back and the time taken to turn on to their belly. The
revised 1985) guidelines. The experimental procedures were ap- time for complete turn on to four limbs touching platform was
proved by the Institutional Animal Ethical Committee of BHU measured (Fan et al. 2005).
(Protocol No: Dean/11-12/CAEC/330). Charles Foster albino
pregnant rats (180–220 g) were procured from the Central An- Evaluation of mitochondrial bioenergetics
imal House, Institute of Medical Sciences, Banaras Hindu Uni-
versity (IMS-BHU). The animals were housed in polypropylene Mitochondrial isolation
cages under controlled environmental conditions of temperature
of 25±1 °C and 45–55 % relative humidity and a 12: 12 h light/ Isolation of mitochondria from the neonatal rat brain was done
dark cycle. Birth litter count ranged from 8 to 12 pups per rat of by the method of Berman and Hastings 1999 with some slight
approximately 30 h age and weighing 6–8 g, was used. Each modifications. Briefly, brains dissected at different time points
group has six animals with equal ratio of males and females. (30 min, 6 h and 24 h respectively) were homogenated in isola-
tion buffer (consisting of 215 mM mannitol, 75 mM sucrose,
Anoxic procedure 0.1 %w/v bovine serum albumin, 20 mM HEPES buffer and
1 mM of EGTA in 100 ml of distilled water and pH adjusted to
The anoxic procedure was performed as per Strata et al. with 7.2 with KOH) and first centrifuged at 1300 g for 5 m at 4 °C.
some slight modifications. Briefly the pups were kept in a an- Each supernatant was then topped off with isolation buffer with
oxic chamber (plexiglass with dimensions of 21 cm×18 cm× EGTA and centrifuged at 14,000×g for 10 min at 4 °C to get a
11 cm) contained a gas inlet and outlet with an airtight lid tighter mitochondrial pellet. Further a washing step was per-
equipped with a flowmeter and a nitrogen gas cylinder. The formed by suspending the pellets in isolation buffer without
chamber was partially immersed in warm water (35 and EGTA and again centrifuged at 14,000×g for 10 min to remove
37 °C) to avoid hypothermia (Takada et al. 2011). To induce EGTA from the pellets. Mitochondrial protein was estimated
anoxia, six pups (neonates) of approximate 30 h age were colorimetrically (Lowry et al. 1951) with a microplate reader
placed inside the chamber with continuous 100 % nitrogen (Biotek, USA) Fig. 1.
gas flow at 3 L/min at a pressure of approximately 101.7 kPa
into the chamber. Two anoxic episodes of 10 min each at an Measurement of mitochondrial function
interval of 24 h were used. The lid was removed immediately
after each episode and pups were exposed to atmosphere and Mitochondrial functionality was assessed using an Oxytherm
resuscitated by lying them on their back and spreading their Clark-type oxygen electrode (OXYT1/ED, Hansatech Instru-
limbs for recovery. After recovery, confirmed by skin color, ments, Norfolk, UK). Mitochondria (180–200 μg) were
J Bioenerg Biomembr (2015) 47:217–222 219

Fig. 1 Schematic representation

of experimental design. ‘+’
denotes performed and ‘-’denotes
not performed

placed in the sealed Oxytherm chamber containing respiration were no significant changes in different states of respiration
buffer (125 mMKCl, 0.1 % BSA, 20 mM HEPES, 2 mM post first anoxic exposure in state II [F (3, 15) = 2.803,
MgCl2, 2.5 mM KH2PO4, pH 7.2) and continuously stirred p>0.05], state III [F (3, 15)=2.375, p>0.05], state IV [F (3,
at 37 °C. The rate of oxygen consumption (Fig. 2) was defined 15)=1.872, p>0.05], state V complex-I [F (3, 15)=2.976,
as the slope of the response of isolated mitochondria to the p>0.05] and State V complex-II [F (3, 15)=2.293, p>0.05]
consecutive administrations of oxidative substrates as previ- (Fig. 2c) respiration at different time points. Whereas, a sig-
ously described (Gilmer et al. 2010). nificant difference was observed in state III [F (3, 15)=18.97,
p<0.05] and state IV respiration [F (3, 15)=5.021, p<0.05]
Assessment of brain mitochondrial nitrite level after second anoxic exposure. Post hoc analysis showed sig-
nificant (p<0.05) decrease in state-III respiration at all the
Nitrite levels were determined by a colorimetric assay using time points. However, decrease in state-IV respiration was
Greiss reagent (0.1 % at 540 nm) as described by Green et al. observed after 30 min compared to control and 6 h compared
1982. Equal volumes of supernatant and Greiss reagent were to 30 min groups. There was a marked decrease in state V
mixed, and this mixture was incubated for 10 min at room complex-I [F (3, 15)=4.442, p<0.05] and state V complex-
temperature in the dark. Absorbance at 540 nm was measured II respiration [F (3, 15)=4.300, p<0.05] at all the time points.
with microplate reader (Biotek, USA). The nitrite amount was Post hoc analysis revealed a significant (p<0.05) (Fig. 2d)
calculated in comparison to the standard nitrite curve and the change in complex-I driven state-V respiration (24 h) and
results were expressed as nanomoles of nitrite formed per complex-II mediated state-V respiration (30 min, 6 h and
milligram of protein. 24 h) as compared to control group animals.

Statistical analysis Changes in mitochondrial RCR (State III/State IV respiration)

after first and second anoxic episode
The data are expressed as means±SEM. For the statistical
analysis of mitochondrial bioenergetics, mitochondrial RCR There were no significant effect of first episode anoxic expo-
and brain nitrite level a one-way ANOVA followed by sure on RCR [F (3, 15)=3.230, p>0.05] (Fig. 2e). However, a
Newman- Keuls test was used. An unpaired t- test was used significant differences in RCR at different time points follow-
to compare the sensorimotor changes 24 h post first and sec- ing second episode of anoxic injury [F (3, 15) = 12.94,
ond exposure to anoxia. p<0.05] was observed. Post hoc analysis revealed that there
was a significant decrease (p<0.05) (Fig. 2f) in RCR at all-
time points post second exposure compared to control group
Results animals.

Changes in mitochondrial respiration after first and second Changes in brain mitochondrial nitrite levels after first
episodes of anoxia and second anoxic episode

A representative trace for the oxygen consumption of different There was no significant changes in nitrite level after first
groups and time points after first and second anoxic episode is anoxic exposure at different time points [F (3, 15)=0.7716,
shown in Fig. 2a and b. One-way ANOVA revealed that there p>0.05] (Fig. 3a). Whereas, significant changes in nitrite level
220 J Bioenerg Biomembr (2015) 47:217–222

Fig. 2 a A typical respiratory trace shows no significant changes in FCCP. This represents the maximum rate of respiration, causing
mitochondria bioenergetics at three different time points after first uncoupling of the ETC to ATP synthesis, and allows protons to rush back
anoxic exposure. Mitochondrial oxygen consumption was measured into the matrix. Rotenone was then added to shut down complex I–driven
using a Clark-type electrode in a continuously stirred sealed chamber respiration. State V (succinate) was determined by addition of succinate.
(Oxygraph System; Hansatech Instruments Ltd.). Purified mitochondrial This is the maximum rate of respiration via complex II, since FCCP is
protein was suspended in respiration buffer in a final volume of 250 μL. present in the system. b Mitochondria isolated at all-time points after
State II respiration was initiated by addition of P/M, with basal rate of second anoxic exposure showed a compromised mitochondrial bioener-
respiration. State III respiration was initiated by addition of ADP; the high getics. The effect of first and second episodes of anoxia on oxygen con-
level of oxygen utilization indicates that ADP is getting converted into sumption in different states of mitochondrial respiration (c and d respec-
ATP. State IV was measured by addition of oligomycin. The respiration tively) and respiratory control ratio (RCR; e and f respectively) in whole
returns to basal rate since the ATP synthase is shut down and no electrons brain mitochondria after 30 min, 6 h and 24 h. Bars represent group
are allowed to return to the matrix. The electron transport chain (ETC) means±SEM (n=4 in each group). *P<0.05 compared to control and
#
continues only to maintain mitochondrial membrane potential due to loss P<0.05 compared to anoxic 30 min post exposure groups respectively
of protons back into the matrix. State V was measured by addition of [One-way ANOVA followed by Student–Newman–Keuls test]

at all-time points following the second episode anoxic ex- nitrite levels at all- time points compared to control group
posure [F (3, 15)=16.75, p<0.05] (Fig. 3b). Further anal- and 6 h compared to 24 h group animals after second
ysis of data showed a significant (p< 0.05) elevation of episode post exposure.
J Bioenerg Biomembr (2015) 47:217–222 221

Fig. 3 The effect to first and second episodes of anoxia on whole brain compared to 6 h post exposure groups respectively [One-way ANOVA
nitrite level (a and b respectively). Bars represent group means±SEM followed by Student–Newman–Keuls test]
(n = 4 in each group).* P < 0.05 compared to control and #P < 0.05

Sensorimotor changes 24 h after first and second anoxic 2013). Previous studies targeting mitochondrial dysfunction
exposure have shown a decrease in state III respiration following anoxia
which is in agreement to our finding (Rosenberg et al. 1989).
An unpaired t test revealed no significant difference in the We found an increase in state IV respiration by using
reflex latency of control and anoxic group [t6 = 0.7313, oligomycin (ATP-synthase inhibitor) after second anoxic epi-
p>0.05] (Fig. 4a) 24 h after first episode of anoxia. Whereas, sode. Similar finding has been reported in focal traumatic
a significant difference was observed [t6 =6.708, p<0.05] brain injury model (Singh et al. 2006). The increase in state
(Fig. 4b) 24 h after second episode. IV respiration may be due to damage of the inner mitochon-
drial membrane which causes more proton to leak into the
matrix. This results in loss of maximal proton gradient which
Discussion is required to couple with ATP production. As State IV respi-
ration denotes the extent of coupling of proton motive force
In this present study, we characterized for the first time the with ATP production versus maintaining the basal metabolic
mitochondrial bioenergetics and associated sensorimotor rate, increase in oxygen utilization denotes uncoupling of the
changes using two subsequent episode model of perinatal an- ETC to ATP production.
oxia in rats. The changes in mitochondrial respiration and Subsequent addition of a mild uncoupler FCCP (state V)
sensorimotor deficit were observed after the second episode detaches the ETC from oxidative phosphorylation. This deter-
of anoxic injury which started as early as 30 min and contin- mines the maximum respiration capabilities of the ETC in its
ued up to 24 h post exposure. attempts to restore the dissipated proton gradient (Benz and
The state II respiration depicts the consumption of substrate McLaughlin 1983). As the mitochondria were not able to
Pyruvate/Malate to fuel the mitochondrial electron transport overcome this effect and maintain respiration due to loss in
chain (ETC). No difference in state II respiration was observed function of complex I at 24 h time point, the oxygen consump-
following first and second episode of anoxia between any tion at state V (complex I) was significantly reduced (Gilmer
experimental groups. State III respiration was initiated by ad- et al. 2010). Following the addition of rotenone, which acts as
dition of ADP. In this study, state III respiration significantly a competitive inhibitor to block complex I driven respiration,
declined at different time points. The reason for decline in succinate was added to determine if complex II driven respi-
state III respiration may be due to deficiency in supply of ration is affected by anoxic exposure. The significant loss of
respiratory substrates. This may occur due to compromise of complex-II state V respiration observed at all-time points post
ETC components and oxygen to produce ATP (Borutaite et al. second anoxic exposure indicates that susceptibility of

Fig. 4 Changes in reflex latency

after a first and b second episode
of anoxic exposure. Bars
represents group means±SEM
(n=6 in each group). *P<0.05
compared to control [two tailed
unpaired Student-t test]
222 J Bioenerg Biomembr (2015) 47:217–222

complex-II to anoxia. Previous experimental studies have Borutaite V, Toleikis A, Brown GC (2013) In the eye of the storm: mito-
chondrial damage during heart and brain ischaemia. FEBS J 280:
shown compromised complex-II after anoxic exposure
4999–5014
(Bolaños et al. 1998; Reddy et al. 2011). RCR is the ratio of Fan L-W, Lin S, Pang Y, Lei M, Zhang F, Rhodes PG, Cai Z (2005)
state III to state IV respiration and is a measure of mitochon- Hypoxia-ischemia induced neurological dysfunction and brain inju-
drial integrity (Gilmer et al. 2010). There was significant re- ry in the neonatal rat. Behav Brain Res 165:80–90
duction in RCR as early as 30 min and up to 24 h post second Gilmer LK, Ansari MA, Roberts KN, Scheff SW (2010) Age-related
changes in mitochondrial respiration and oxidative damage in the
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drial RCR was decreased immediately after hypoxia/anoxia 143
(Rosenberg et al. 1989). The results shows loss of mitochon- Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS,
drial integrity after second episode but not after first episode of Tannenbaum SR (1982) Analysis of nitrate, nitrite, and
anoxia. There was significant increase in nitric oxide in terms [15 N]nitrate in biological fluids. Anal Biochem 126:131–138
Herrera-Marschitz M (2014) Perinatal asphyxia: CNS development and
of nitrite as early as 30 min upto 6 h. The increased generation deficits with delayed onset. Front Neurosci 8:47
of nitric oxide continued up to 24 h post anoxic exposure. Kaizaki A et al (2013) Celecoxib reduces brain dopaminergic neuronal
Nitric oxide forms peroxinitrite (ONOO−) with superoxide dysfunction, and improves sensorimotor behavioral performance in
(O·−2), which causes mitochondrial oxidative stress and im- neonatal rats exposed to systemic lipopolysaccharide. J
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pairs mitochondrial respiration (Singh et al. 2007).
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein mea-
Anoxic insult in the neonatal rats delayed the appearance of surement with the Folin phenol reagent. J Biol Chem 193:265–275
righting reflex at 24 h time point post second exposure. This Magistretti PJ, Allaman I (2013) Brain energy metabolism. In:
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brain (Fan et al. 2005; Kaizaki et al. 2013). The loss in senso- Puka-Sundvall M et al (2000) Impairment of mitochondrial respiration
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condition mitochondrial bioenergetics plays an important role Reddy NR, Krishnamurthy S, Chourasia TK, Kumar A, Joy KP (2011)
Glutamate antagonism fails to reverse mitochondrial dysfunction in
in development of sensorimotor functions (Kaizaki et al. 2013). late phase of experimental neonatal asphyxia in rats. Neurochem Int
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sensorimotor changes post second anoxic exposure. The mod- Mitochondrial function after asphyxia in newborn lambs Stroke. J
Cereb Circ 20:674–679
el will be helpful to evaluate mitochondrial targeted pharma-
Singh IN, Sullivan PG, Deng Y, Mbye LH, Hall ED (2006) Time
cological interventions to treat neonatal anoxic conditions. course of post-traumatic mitochondrial oxidative damage and
dysfunction in a mouse model of focal traumatic brain injury:
Acknowledgments SK is thankful to Department of Biotechnology implications for neuroprotective therapy. J Cereb Blood Flow
(DBT), New Delhi, India for assistance in terms of research grant [102/ Metab: Off J Int Soc Cereb Blood Flow Metab 26:1407–
IFD/SAN/4654/2011-2012]. 1418
Singh IN, Sullivan PG, Hall ED (2007) Peroxynitrite-mediated oxidative
damage to brain mitochondria: protective effects of peroxynitrite
scavengers. J Neurosci Res 85:2216–2223
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